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Introduction Most agronomic and horticultural crop species are angiosperms. Angiosperms are vascular plants that produce their seeds enclosed in a matured ovary, a fruit; the fruit arises from a flower. In contrast, some tree crops—such as pine or spruce—are gymnosperms, which are vascular plants possessing “naked seeds” that are not enclosed within fruit structures. There are two groups of angiosperms: monocots and dicots. Although all angiosperms have some reproductive features in common, species vary in their mode of reproduction. A species’ reproductive mode is fundamental to the methods applied to develop improved cultivars. Learning Objectives • Review hereditary mechanisms and flower anatomy. • Understand the sexual reproduction processes of pollination, fertilization, and seed development. • Become familiar with asexual reproduction. • Comprehend the implications of reproductive mode for crop breeding strategies. Hereditary Mechanisms Heredity, Genotypes, and Phenotypes Plant breeders take advantage of the mechanisms of heredity to develop and maintain cultivars. The observable characteristics and performance of cultivar (of a plant), its phenotype, is the result of the cultivar’s genotype, as influenced by the environment. In other words, phenotype is a function of both genotype and environment, plus the interaction between genotype and environment. $P=G+E+(G \times E)$ where • $P= \text{Phenotype}$ • $G= \text{Genotype}$ • $E= \text{Environment}$ Fundamental to effective and efficient plant breeding is an understanding of the hereditary mechanisms that affect genotype: • Nuclear division and chromosomes • Modes of reproduction Deoxyribonucleic Acid (DNA) And Chromosomes Every cell nucleus contains the genetic material of the cell, deoxyribonucleic acid (DNA), located in chromosomes. Each chromosome is a single DNA molecule. Associated with the DNA are special proteins called histones (see the round yellow shapes in Figure 2 around which the DNA is wound like “beads on a string” along the chromosome) that are related to the organization of the DNA, as well as enzymes involved in replication of the DNA strand. In plant cells, DNA and associated genetic information are mainly located in nuclear chromosomes. However, some additional DNA and genetic information is located in specialized cell structures that are “extra-nuclear,” meaning found outside of the cell nucleus. Nuclear vs. Organellar DNA Although by far the main portion of DNA and genetic information is located on nuclear chromosomes, additional DNA (and genetic information) is located in two types of organelles in plant cells: plastids and mitochondria. Each plant cell usually contains multiple plastids and mitochondria, which contain multiple copies of circular DNA each. Different from the inheritance of nuclear DNA, organellar DNA is maternally inherited in most plant species and not undergoing meiosis. Mitochondrial DNA is the small circular chromosome found inside mitochondria, a type of organelle found in cells, and that are the sites of energy production.Illustration by NIH-NHGRI. Chromosomes And Genomes A genome is the basic set of chromosomes inherited as a unit from one parent. Somatic cells (non-germ cells) of diploid species contain two sets (2n) of the basic genome (haploid) (1n) number of chromosomes. Among species, the number of chromosomes varies. Within a species, the chromosome number (2n in somatic cells and 1n in germ cells) is ordinarily constant. However, crop species include both diploids and polyploids, which are plants with more than two sets of chromosomes in their cells. Crop Chromosome Number Crop species include a wide range of chromosome numbers. The genomic formula of crop species (with 2n representing the somatic chromosome number, n, the haploid number, and x, the basic chromosome number) can reveal whether or not the crop is a polyploid. For example, vanilla, coconut, pecans, alfalfa, leek, and sour cherry all have the same number of chromosomes, but the first three are diploids (2n=2x=32), and the last three are polyploids (2n=4x=32). Polyploids can be classified as either autopolyploids (see crop examples labeled as “auto” in the table where their status is known) or allopolyploids (“allo” in the table). Autopolyploidsare polyploids with multiple chromosome sets derived from a single species, whereas allopolyploids are polyploids with genomes derived from different species. Interactive Crop Chromosome Number Click each of the crop groups below to see additional information. Mitosis and Meiosis Mitosis is one phase of the cell cycle. Mitosis is divided into four arbitrary stages. Progress between stages, however, is gradual and continuous. During cell division, DNA is duplicated and distributed to daughter nuclei. The number of chromosomes in the daughter nuclei depends on the process of nuclear division. There are two nuclear division processes by which new cells are formed. Mitosis Let’s follow mitosis, the process by which somatic cells, non-germ cells, are reproduced. We’ll begin with interphase of the cell cycle. This illustration represents a diploid, or 2n, somatic nucleus. It has two sets of chromosomes, shown here as a blue set and a red set. Each chromosome is duplicated at the end of interphase during the synthesis period of the cell cycle that precedes mitosis. Mitosis is a process of nuclear division. It has four stages: • Prophase • Metaphase • Anaphase • Telophase Let’s see what happens during each of these stages. Query $2$ FYI: Cell Cycle • G2-Gap This period occurs after DNA replication is complete, but before mitosis begins. • M-Mitosis The nucleus divides, distributing a complete set of chromosomes to each daughter nucleus. The cell subsequently undergoes cytokinesis, cytoplasmic division, completing the formation of the two daughter cells; each repeats a new cell cycle. Of the cell cycle phases, mitosis is the shortest, typically lasting only 1 to 3 hours. • G1-Interphase Occurs after the completion of mitosis and precedes DNA replication. • S-Synthesis DNA replication occurs. This phase can be identified because it is the only phase during which the cell can incorporate radioactive thymidine into nuclear DNA. Thymidine is related to one of the purine bases of DNA, thymine. Meiosis In contrast to mitosis, meiosis is the process through which germ cells, microspores and megaspores, are derived. Meiosis is similar to mitosis except in two important aspects: • Meiosis involves two successive divisions. • Homologous chromosomes replicate only once during the two divisions. Thus, the diploid microspore and mega spore mother cells are meiotically reduced to the haploid, or 1n, chromosome number of the gametes. In meiosis, there are two successive divisions, called meiosis I and meiosis II. Each of these is divided into four phases analogous to those of mitosis. Like mitosis, these stages progress in a gradual and continuous manner. Similarities to Mitosis Meiosis I resembles mitosis in that: • The division results in the production of two daughter cells. • Cells are derived from a microspore or megaspore mother cell. • Replication of homologous chromosomes precedes it. Review Now review these two processes: mitosis and meiosis. Again, pay attention to commonalities and differences. You should be able to identify key features of each process and stage. Process Divisions Equational division One equational division, one reductional division Results in Two 2n daughter cells Four 1n daughter cells Stages Four: • Prophase • Metaphase • Anaphase • Telophase Eight or nine: • Prophase I • Metaphase I • Anaphase I • Telophase I • Interkinesis (sometimes) • Prophase II • Metaphase II • Anaphase II • Telophase II Study Question 1 Identify the cell division process that best fits each statement by dragging the label to the box next to it. Query $4$ Study Question 2 Barley has a diploid chromosome number of 14. Identify the number of chromosomes in various cell or tissue types. Sexual Reproduction Reproduction enables the propagation of new individuals. Reproduction in crop species may occur sexually, asexually, or both. • Sexual reproduction: Requires the fusion of egg and sperm cells (known as gametes) to obtain the next generation. The life cycle of a typical angiosperm involves sexual reproduction based on the process of meiosis, in which the chromosome number of cells in the female and male reproductive organs is reduced by half to form female and male gametes. Meiosis is the process responsible for the genetic segregation observed in progeny of heterozygous individuals. • Asexual reproduction: Propagation occurs without the fusion of male and female gametes. Asexual reproduction is based on the multiplication of cells by mitosis and results in two new cells that are genetically identical to each other and to the cell from which they originated. Synopsis of the Life Cycle of the Angiosperm Plant In the animal kingdom, the production of gametes follows immediately after the meiotic divisions. Normally, therefore, the gametes are the only haploid (1n) representatives of the animal life cycle. In plants, however, almost invariably (and without exception in higher plants) the immediate products of the meiotic divisions are not gametes but spores. The higher plants (angiosperms) we recognize, e.g., the oak tree, turfgrass, clover, wheat, are the diploid (2n) or sporophytic stage of the plant’s life cycle. In these plants the haploid vegetative or gametophytic stage is short-lived and quite inconspicuous. The sporophyte produces spores as a result of sporogenesis or meiosis. Spores undergo a few nuclear divisions in a process known as gametogenesis to form mature gametophytes. Gametes develop within the gametophytes. All higher plants produce two types of spores, microspores and megaspores. Corresponding to these two types of spores are the two different modes of their development, microgametogenesis and megagametogenesis, which culminate respectively in two dissimilar and relatively simple plants, the mature microgametophytes and megagametophytes. Male Spore Formation Each of many 2n microsporocytes (pollen mother cells) within the anther undergoes meiosis with the result that four haploid (1n) microspores are produced within the anther for each original microsporocyte. FYI: Microsporogenesis Microsporogenesis is the process by which male gametes (pollen grains) are formed. This process can be divided into three parts: 1. Meiosis I 2. Meiosis II 3. Endomitosis Each of these is subdivided into several stages. The stages of microsporogenesis are transitory. The sequence of stages is shown in the following photos (from Chang and Neuffer, 1989). Each photo represents a momentary expression, which may not be a good representation of the complete event. The event in each photo is indicated by an arrow. Microgametogenesis The single nucleus of each microspore divides once by mitosis and one of the two daughter nuclei draws about itself a mass of deeply staining cytoplasm. This nucleus is known as the generative nucleus while the other is the tube nucleus. The generative nucleus undergoes a single mitotic division to form two male gametes, or sperm cells. (In some plants this division does not occur until pollination has taken place and the generative nucleus is moving through the pollen tube.) This constitutes a pollen grain or a mature microgametophyte. Female Spore and Gamete Formation Megasporogenesis A 2n megasporocyte (megaspore mother cell) in each ovule undergoes meiosis. Four megaspores result, each with a haploid chromosome number. Three of these disintegrate; the fourth develops into the mature female gametophyte. Megagametogenesis (development of the female gametophytes and gametes) The surviving megaspore enlarges greatly to form the embryo sac. Three successive mitotic divisions, starting with the original nucleus of this megaspore, produce eight haploid daughter nuclei within the embryo sac. These orient themselves as follows: • The two polar nuclei lie together in the middle of the sac. • Three nuclei are located at the end of the sac where the sperm will enter, the center one becoming the female gamete or egg, and the two flanking ones the synergids. • The remaining three, the antipodal cells, come to lie at the opposite end of the sac. The number of antipodal cells, however varies greatly from zero in Oenothera species to more than 100 in some grass species. The embryo sac with the eight haploid nuclei thus arranged is the mature megagametophyte the female gamete or egg, and the two flanking ones the synergids; the remaining three, the antipodal cells, come to lie at the opposite end of the sac. The number of antipodal cells, however varies greatly from zero in Oenothera species to more than 100 in some grass species. The embryo sac with the eight haploid nuclei thus arranged is the mature megagametophyte. Double Fertilization Pollen grains are freed by opening of the anther wall and are carried to the stigma of the same or other plants. Each pollen grain soon sends out a small thin pollen tube (generated by the tube nucleus) which penetrates the tissues of the stigma and digests its way through these and the stylar tissues down to one of the ovules. The sperm cells pass down the pollen tube behind the tube nucleus. Once the pollen tube reaches the ovule it penetrates the embryo sac, the tube nucleus disintegrates, and the two sperm cells enter. One of the sperms fuses with the haploid egg to produce the 2n zygote, while the other fuses with the two polar nuclei to give a 3n (triploid) product, the endosperm nucleus. Further Development The triploid endosperm tissue grows more rapidly than the embryo at first. Later on, the embryo, which develops from the zygote, grows at the expense of the endosperm. Depending on the species, endosperm tissue may or may not persist at the time the seed has completed growth. With the resumption of growth (seed germination) the embryo continues development until it reaches the mature sporophyte stage, at which time microspores and megaspores are again produced. Study Question 3 For Your Information Endomitosis Interphase occurs between meiosis II and endomitosis. Chromosomes replicate during interphase. Endomitosis is a process of cell division resulting in the production of the pollen grains. Endomitosis is also divided into phases. Figures 23 through 36 show these phases, as well as the gradually increasing accumulation of starch granules in the cell. The starch grains progressively obscure the visibility or resolvability of the cellular structures of the male gametophyte. Interphase The cell nucleus is round, condensed and nondifferentiated . Chromosomes replicate during this period. First Prophase The chromosomes condense into short thick threads surrounding the nucleolus. Middle First Prophase The chromosomes continue to condense into short thick threads which allow identification of individual chromosomes. The nucleolus and the nucleolus-organizing region of chromosome six are visible. Late First Prophase The chromosomes further condense to become short thick rods. The nucleolus and the nucleolus-organizing regions with chromosome six are clearly seen. First Metaphase The nucleolus disappears and the ten chromosomes are arranged in one plane close to one another. First Anaphase The sister chromatids are now separated and moving towards the opposite poles. Late First Anaphase The separated chromosomes have reached the opposite poles and formed two chromosome clusters. First Telophase The chromosomes at each pole are now extended and surround the nucleolus. Binucleate Two nuclei are formed at the opposite poles. The generative nucleus (bottom) is usually located near the germ pole. It will further divide to form two sperm. Second Prophase The generative nucleus underneath the surface of the intine wall becomes cup-shaped and will proceed to the second nuclear division. The vegetative nucleus is not at resting stage and appears to continue its metabolic activities. Second Metaphase The nucleolus disappears, the ten chromosomes are arranged in one single plane. The vegetative nucleus is dark-stained. Second Anaphase The sister chromatids are now separated and moving towards the opposite poles . The vegetative nucleus remains large and clear. Second Telophase The chromosomes at each pole are now extended and surround the nucleolus. The vegetative nucleus remains large and clear. Mature Pollen The mature pollen grain now has three nuclei. The top two condensed, crescent-shaped nuclei, surrounding the germ pore are the sperms. The large one at the bottom is the vegetative nucleus. Meiosis I Meiosis I is a reductional division-the number of chromosomes in the nucleus is reduced to the haploid number. Meiosis I has four phases: prophase I, metaphase I, anaphase I, and telophase I. Premeiotic Interphase The irregularly shaped pollen mother cell has dense protoplasm, no vacuoles, no clear cell wall structure and an undifferentiated nucleus. Prophase I Prophase I is the longest phase of Meiosis I. During Prophase I, the nuclear membrane breaks down, the chromosomes contract, and the spindle forms. Prophase I has several substages: lepotene, zyotene, pachytene, diplotene, and diakinesis. Leptotene Cell becomes round with dense protoplasm. The chromatin threads are greatly extended and coiled around the nucleolus. Synapsis is initiated. Single and double strand configuration is evident. The chromomeres are visible. Late Zygotene-Early Pachytene The pairing of the homologous chromosomes is complete. The condensed chromosomes show details of hetero-chromatin and knobs. The nucleolus and nucleolar-organizing region of chromosome six are visible. Pachytene The paired chromosomes are further condensed to become a very thick thread. Individual chromosomes can be identified by their relative lengths, distinctive chromomere patterns, position of knobs, and other recognizable characteristics. The nucleolar-organizing region of chromosome six is clearly attached to the nucleolus. The chromosomes continue to condense into short, thick threads. The paired chromosomes appear to be repulsing one another, except regions where an actual crossover took place. The chiasmata are frequently seen as X-shaped and looped chromosome configurations. Late Diplotene The chiasmata are terminalized and the very short condensed chromosome pairs are separated from each other. The X-shaped and looped chromosome configurations are still shown. The nucleolar- organizing region of chromosome six is firmly attached to the nucleolus. Diakinesis The condensed chromosome pairs are separated from each other. The chiasmata , the X-shaped and looped configurations are still seen. Late Diakinesis The chromosome pairs are dark, round bodies and the nucleolus starts to disappear. Metaphase I During metaphase I, chromosomes migrate to the spindle equator. Metaphase I (side view) The nucleolus has disappeared. The paired chromosomes lie at the equatorial plate of the spindle structure. The chiasmata have moved to the ends of the paired chromosome. Metaphase I (polar view) The paired chromosomes appear as dense bodies scattered on a single plane of the protoplast. Anaphase I The paired chromosomes separate and move toward the opposite poles. The V-shaped configuration of the chromosome is due to movement of the centromere ahead of the arms. The number of chromosomes at each pole is now reduced to half the number possessed by the microspore mother cell. Telophase I The chromosomes at each pole are now extended. The nucleolus reappears and the cytoplasm divides (cytokinesis) to form two half-mooned cells. Meiosis II Meiosis II is an equational division during which sister chromatids separate and are distributed to daughter nuclei. Thus, each nucleus receives the haploid number of chromosomes. Meiosis II is divided into four phases: prophase II, metaphase II, anaphase II, and telophase II. These phases are analogous to the four phases of Meiosis I. Prophase II The chromosomes condense into short thick threads surrounding the small nucleolus. Metaphase II The chromosomes (each chromosome has two sister chromatids) lie at the equatorial plate of the spindle structure. Nucleoli have again disappeared. Anaphase II The two sister chromatids seen collectively as a dark staining mass, are now separated and have moved towards the opposite poles. Telophase II The chromosomes at each pole are extended, the nucleoli reappear and the cytoplasm divides to form four cone-shaped cells. Four cone-shaped microspores are formed and are enclosed inside the maternal wall, which is being digested and will thus release the four microspores. The newly released free microspores are undifferentiated, cone-shaped, and appear to have no distinct cell walls. Early Uninucleate Cell The shape of the microspores are round with dense cytoplasm. The nucleus is located near the center and the cells are undifferentiated with no vacuoles and no clear wall structure. Late Early-Uninucleate Cell The microspores start to differentiate. The exine and intine structures are being formed. The cytoplasm remains dense, but many small vacuoles are being formed. The nucleus is still near the center of the protoplast. Middle Uninucleate Cell A large vacuole is forming in the protoplast, pushing the nucleus to one side. Late Uninucleate Cell The differentiation of exine and intine, germ pore and annulus are complete. Creases seen are due to pressure of coverslip on rigid spherical pollen wall. Cell volume increases four to six times.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.01%3A_Reproduction_in_Crop_Plants.txt

Sexual Reproduction Kinds of Flowers Inflorescence type influences the techniques that are used to control pollination in developing cultivars and in maintaining the genetic purity of cultivars. Inflorescence types can also be used to identify plants. Flowers are classified into a couple of categories. Flowers are either complete or incomplete and either perfect or imperfect. A flower having all of the main floral parts (sepals, petals, pistils, and stamens) is said to be complete, whereas a flower lacking one or more of these structures is said to be incomplete. The stamen (male part) and pistil (female part) are not always present together in a single flower. When both are present, the flower is said to be perfect (or bisexual). Imperfect flowers are those that are unisexual, either male or female. Table 1 Examples of plants with complete and incomplete flowers. Complete flowers Incomplete flowers Soybean Alfalfa Clovers Common bean Vetches Cotton Tomato Rapeseed Sunflower Tomato Cabbage Tobacco Maize Sorghum Oat Barley Wheat Sugar beet Fig Date palm Forage grasses Turf grasses Rice Spinach Notice that plants in the legume family (Leguminosae or Fabaceae) have complete flowers, whereas plants belonging to the grass family (Gramineae or Poaceae) have incomplete flowers. Flower Dissection Dissect a complete and incomplete flower. Think about how the presence or absence of a floral structure might influence the pollination process, and thus, the methods that can be used to develop improved cultivars or to maintain the genetic purity of the cultivar. Grass Floret Complete Soybean Flower Dissection 1. Standard petals: Collectively, petals are called the corolla. Petals are typically large and conpicuous and are not required for reproduction. Soybean has five petals: one standard petal, two wing petals and two keel petals 2. Wing petals: The dissected view of the two wing petals. 3. Keel: The keel is composed of two united petals. The keel encloses the stamina column. Stamens are the pollen-bearing organs of the flower. Stamens are composed of slender stalks (filaments) that support anthers. Pollen grains are produced in the anthers. The pistil is the seed-bearing organ of the flower. It consists of stigma, style, and ovaries. The stigma is the part that is receptive to pollen. Following pollination and fertilization, seed form in ovaries. 1. Sepals: Like the petals, sepals are not neccessary for reproduction. Sepals are small and inconspicuous. They enclose and protect the flower while still a bud. Collectively they form the calyx. 2. Pedicel: The pedicel is the stalk of the flower, attaching to the plant Wheat Spike Dissection Grass Floret Study Question 4 Query \(3\) Study Question 5 Select the floral part or parts necessary for reproduction: Query \(4\) Table 2 Examples of crops and different floral systems. Adapted from Lersten (1980). Flower Characteristics Terms Examples Male and female expression in INDIVIDUAL FLOWERS Male and female in ONE flower bisexual, hermaphroditic, monoclinous, perfect Wheat, peach 1. Pollen shed before stigma is receptive protandry (prevent self-pollination) Carrot, walnut 1. Stigma matures and ceases to be receptive before pollen is shed protogyny (prevent self-pollination) Pearl millet, pecan 1. Stigma receptive, and pollen shed, after flower opens chasmogamy (promote self-pollination) Violet, rye 1. Stigma receptive, and pollen shed, in closed flower cleistogamy (ensure self-pollination) Oat, peanut Perfect flowers of TWO types on SAME plant heterostyly Buckwheat, flax 1. Long styles and short stamens pin flower 1. Short styles and long stamens thrum flower Male and female in SEPARATE flowers unisexual, diclinous, imperfect Cucumber, hemp 1. Male flower male, staminate 1. Female flower female, pistillate, carpellate Flower DISTRIBUTION on PLANTS Male and female flowers on one plant monoecious Maize, oak Male and female flowers on separate plants dioecious Yams, asparagus • Male, female, and perfect flowers mixed, polygamous Red maple, papaya 1. On same plants polygamomonoecious Coconut, mango 1. On separate plants polygamodioecious Strawberry, holly Perfect and Imperfect Flowers Perfect flowers have both staminate and pistillate structures in the same flower. Imperfect flowers are either staminate or pistillate. An imperfect flower is staminate if it possesses stamen. Conversely, an imperfect flower is pistillate if it bears a pistil. Staminate flowers are considered “male” because they produce pollen, whereas pistillate flowers are “female” because they possess ovules. Staminate and pistillate flowers may occur on the same or different plants of the same species. Species having such specializations are either: • monoecious — staminate and pistillate flowers are separate but occur on the same plant; or • dioecious — staminate and pistillate flowers are on separate plants. Analogous to the separate sexes in animals, a dioecious plant must have a partner of the opposite type to complete its life cycle. Usually, about half of all individuals of a dioecious species are of each type, staminate or pistillate. Thus, the dioecious condition is reproductively expensive in that only about half of the species’ plants can produce seed. Table 3 Examples of monoecious and dioecious plants. Monoecious Dioecious Maize Walnut Oil palm Squash Cassava Wile rice Castor bean White pine Hemp Hops Spinach Yam Date palm Cottonwood Asparagus Nutmeg The “mono-” prefix indicates one and the “di-” prefix indicates two. The “-oecious” part of the word translates to “house.” Thus, an easy way to remember the distinction between these terms is to remember that in monoecious species, the staminate and pistillate flowers reside in the same house or plant, whereas in dioecious species, these flowers reside in two different houses or plants. Study Question 6 Pollination and Fertilization Pollination occurs when a pollen grain (from the staminate flower) is placed on a receptive stigma (of the pistillate flower), either naturally or artificially. Fertilization requires that a male gamete and a female gamete fuse to form a zygote. These gametes may be from the same or different plants. There are two kinds of pollination processes in sexual reproduction. • Self-pollination — seeds develop from the union of male and female gametes produced on the same plant or clone. The development of seed by self-pollination is also referred to as autogamy. • Cross-pollination — seeds develop from the fusion of gametes produced on different plants. The development of seed by cross-pollination is known as allogamy. Pollination and Fertilization Self-Pollination Several floral mechanisms enforce self-pollination. • Flowers do not open, preventing external pollen from reaching the stigma. • Anthesis occurs before the flower opens. • Stigma elongates through the staminal column (filaments and anthers) immediately after anthesis. • Floral organs may obscure the stigma after the flower opens. Although these mechanisms usually enforce self-pollination, a low frequency of cross-pollination may occur. The frequency of cross-pollination in normally self-pollinating species generally depends on the species and environmental conditions. Soybean is an example of a species that is normally self-pollinated. Before the flower opens, the anthers burst and pollen grains fall out of the anthers on to the receptive stigma contained in the same flower: self-pollination occurs. Cross-Pollination Floral Mechanisms of Promotion Several floral mechanisms promote cross-pollination. • Emergence or maturity of the staminate and pistillate flowers is asynchronous. • Protandry — anthesis occurs before stigma are receptive. • Protogyny — pistillate flower matures before the staminate flower. • Flowers are monoecious or dioecious. Mechanical obstruction between the staminate and pistillate flowers in the same individual prevents self-pollination. Gametes produced on the same plant or clone are unable to effect fertilization. • Mechanical obstruction between the staminate and pistillate flowers in the same individual prevents self-pollination. Alfalfa flowers, for example, have a membrane over the stigma that precludes self-pollination. When a bee lands on the flower, the keel is tripped, rupturing the membrane and exposing the stigma to pollen carried by the bee from other plants it has visited, effecting cross-pollination. • Gametes produced on the same plant or clone are unable to effect fertilization. • Self-sterility — gametes from same individual cannot successfully fuse to form a zygote. Sterility can be caused by lack of function of pollen (male gametes) or ovules (female gametes). • Male sterility — either genetic or cytoplasmic, occurs because the pollen is not viable. Female sterility occurs when the ovule is defective or seed development is inhibited. • Self-incompatibility — self-pollination may occur, but fertilization and seed set fail. Pollen Transportation Pollen is transported from the staminate flower to the pistillate flower by wind, insects, or animals. Occasionally pollen is transported to receptive stigma of the same individual and self-pollination may occur. For example, pollen from the tassel of a maize plant may land on and pollinate silks on the same plant, effecting self-pollination. Sunflower is ordinarily cross-pollinated. Bees often carry pollen from one plant and deposit it on other plants. Classification Plants are classified as either self- or cross-pollinated based on which of these processes most frequently produces its seed. Click each category for more information. Query \(7\) Study Question 7 You encounter an unfamiliar flowering plant. What key floral feature(s) would you check to determine the plant’s likely mode of pollination, self or cross-pollinating? Query \(8\) Study Question 8 For each of the following types, indicate the probable mode of pollination by clicking on the appropriate button. Assume no male sterility or self-incompatibility. Asexual Reproduction Some species can be propagated without a gametophytic stage. The fusion of gametes (fertilization) is omitted from the life cycle. Reduction in chromosome number (meiosis) and seed production may or may not occur. Asexual reproduction produces individuals genetically identical to the maternal parent. There are several mechanisms of asexual reproduction. • Vegetative Propagation • Tissue Culture • Apomixis Vegetative Propagation In some species, new individuals can arise from a group of differentiated or undifferentiated cells of the parent plant; no embryo or seed is produced. Because such new individuals develop asexually from a single parent, they are genetically identical to that parent. These progeny are clones. Numerous tissues and organs may asexually produce progeny. • Rhizomes – Rhizomes are specialized underground stems that can branch at nodes to produce new plants. Banana, bromegrass, hops, and johnsongrass can be reproduced from rhizomes. • Stolons – These “runners” or horizontal-growing, above-ground stems develop adventitious roots whose axillary buds can become independent plants. Strawberry is an example of a crop that can be reproduced from stolons. • Bulbs and bulbils – These short underground stems have thickened or fleshy scales (modified leaves) that can form buds. These buds detach and form “offsets” or new individuals. Onions and garlic are commonly propagated from bulbs. • Tubers – Tubers are also short, enlarged stem tissue, containing food reserves. Nodes or “eyes” in such tissue can give rise to adventitious roots and separate plants. Potatoes are commonly propagated from eyes cut from tubers. • Suckers – Suckers arising as lateral shoots from the base of stems can separate and form new plants. Pineapple, sweet potato, and date palm are examples. Suckers may also derive from adventitious buds on the roots. Roses, poplars, and some other woody species can be propagated from such root cuttings or rootstocks. • Corms – A corm is an underground, tuber-like base of a vertical stem that can also produce a separate plant. Taro, an important starch crop in Southeast Asia and the Pacific Islands, is propagated from corms. Banana also can be propagated from corms. • Stem cuttings – When placed in moist soil, cuttings from aerial stems of some species, such as sugarcane, pineapple, and cassava, can give rise to new plants from the nodes and lateral buds. The usual mode of reproduction of some species is vegetative. However, other species that reproduce sexually are more commonly propagated vegetatively to maintain genetic purity, including some forage cultivars and many horticultural species. Vegetative reproduction does not usually provide opportunity for selection of genetic variants. Tissue Culture Tissue culture is a specialized type of asexual propagation. Tissue culture usually involves excision of undifferentiated cells or meristematic pieces of a plant and growing these in vitro on sterile nutrient agar medium; cell division is by mitosis. By manipulating the components of the medium, the tissue can be prompted to develop roots or shoots. Eventually, new individuals may be separated and transplanted to soil. Tissue culturing takes advantage of the totipotency of somatic cells. That is, these cells contain the plant’s entire genome and have the potential to develop into whole plants. Some species that cannot normally be reproduced vegetatively may be reproduced by tissue culture. Tissue culture is of interest to plant breeders as a technique to • maintain and propagate genetically identical plants that otherwise can only be reproduced sexually; • provide disease-free plants of species that often transmit pathogens to progeny when propagated by conventional vegetative means; and • create novel genetic variation within which selections can be made. Under some conditions, tissue culturing can promote genetic changes. Apomixis Process Apomixis differs from other forms of asexual reproduction in that seed is produced. Unlike sexual reproduction, however, apomictic seed is developed from sexual organs or related structures without fertilization. Pollination is also usually omitted. Agamospermy Apomixis generally involves forms of agamospermy, which is a process through which seeds develop without fertilization. There are two different degrees of agamospermy. • Obligate — Seed produced arises from asexual reproduction. • Advantages: Preserves genotype, including heterozygotic genotypes • Disadvantages: Precludes genetic recombination and variation for selection of improved cultivars • Facultative — Although most of the seed generated is asexually produced, sexual reproduction occurs regularly. • Advantages: Permits development of genetic variation for selection of improved cultivars • Disadvantages: Cultivars may be genetically unstable, making it difficult to maintain the desired genotype Each of these degrees of agamospermy provides advantages and disadvantages from the plant breeding perspective. There are also two general types of agamospermy. • Autonomous — Endosperm forms without pollination or fertilization. • Pseudogamous — Although fertilization (the fusion of gametes) does not occur, pollination is apparently required to stimulate apomictic embryo or embryo sac development to produce seed. Pollination adds no genetic material. Mechanisms of Cause The mechanisms that cause apomixis differ by the cell that undergoes mitosis to produce the embryo of the seed. • Adventitious embryony — The embryo develops directly from diploid sporophytic tissue, skipping the gametophytic stage. This is the simplest form of agamospermy. • Apospory — Nucellus or integument cells, which are somatic cells, undergo mitosis to produce a diploid embryo sac. • Apospory is the most common form of apomixis in angiosperms. • Diplospory — The embryo and endosperm derive from the diploid megaspore mother cell. The megaspore mother cell’s nucleus divides by mitosis, rather than meiosis, resulting in a diploid embryo sac. • Parthenogenesis — The egg cell divides mitotically to form the embryo without fertilization. • Androgenesis — A haploid embryo develops from a male sperm nucleus after it enters the embryo sac. The individual that develops from the seed is haploid and has the genotype of the sperm from which it is derived. Study Question 9 Apomictic embryos may form from reduced (haploid) or unreduced (diploid) cells. For each situation, select the button according to whether the resulting embryo could be homozygous, heterozygous, neither, or both. Answer both parts of this question and then check your answer. Discussion Crops can be self-pollinated, cross-pollinated, or vegetatively propagated. Discuss the breeding consequences of these three different methods of propagation. In addition: previously, a student suggested that with today’s technologies, plants can simply be converted into self- or cross-pollinated or into vegetatively propagated species. Do you agree? Provide arguments in favor or against this statement, and examples, in case you are aware of any. Finally, if it was possible, which type of crops would be your favorite, and why? Study Question 10 For each of the following terms, identify whether the term is associated with sexual, asexual, or both manners of reproduction by clicking on the appropriate button.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.02%3A_Flower_Morphology_and_Distribution.txt

Introduction Most crop species require seed production for their propagation. Some species, however, possess mechanisms that regulate fertility. Such mechanisms can reduce or prevent seed set, and affect self- or cross-pollination. These fertility-regulating mechanisms may be an obstacle or a benefit to the plant breeder. In this module, we’ll explore these mechanisms and their utility. Learning Objectives • Mechanisms and utility of incompatibility systems. • Modes of sex inheritance in plants and their application in plant breeding. • Male sterility systems and their applications in plant breeding. Self-incompatibility Genetic-based Self-incompatibility Self-incompatibility is the inability of a plant to set seed when self-pollinated, even though it produces viable pollen. In contrast, cross-pollination generally results in seed set in self-incompatible species. Many plant families include species with self-incompatibility systems, such as Fabaceae (Leguminosae), Poaceae (Gramineae), Solanaceae, Brassicaceae (Cruciferae), and Asteraceae (Compositae). Self-incompatibility may be caused by genetic interactions between pistil and pollen, or by physical obstacles that hinder self-fertilization. Self-incompatibility may be caused by genetic interactions between the pistil and pollen-producing physiological factors that interfere with fertilization of female gametes by male gametes produced on the same plant or on a closely-related plant. Typically one or few self-incompatibility (SI) genes are involved in this self/non-self discrimination process, depending on the plant species. Pollen can be rendered ineffectual at several points in the pollination process: 1. Sperm enters the embryo sac but does not fuse with the egg. 2. Pollen tube penetrates the stigma but it grows too slowly in the style to reach the ovary while the ovule is still receptive. 3. Pollen germinates but the tube is unable to penetrate the stigma. 4. Pollen germination on the stigma is inhibited. Pistil receptivity to a particular pollen grain depends on the SI alleles carried by the pistil. The phenotype of the pollen (its capacity to fertilize the female gamete) is determined by either the pollen’s alleles (gametophytic incompatibility) or by the alleles of the plant that produced the pollen (sporophytic incompatibility). There are two self-incompatibility systems that result from genetic interactions: • Gametophytic Self-incompatibility • Sporophytic Self-incompatibility Both incompatibility types influence the rate of pollen tube growth, but their genetic controls and location of effect differ. Both types involve multiple alleles. Gametophytic Self-incompatibility • Gametophytic self-incompatibility involves the allele possessed by the pollen grain. The incompatibility effect occurs in the style. However, in some species with gametophytic SI, incompatibility is expressed on the surface of stigma (in grasses). • Gametophytic self-incompatibility is controlled by a series of alleles. The rate of pollen tube growth responds to the allelic interaction of both the style and the pollen. • If both the stylar tissue and pollen possess identical alleles, pollen tube growth is inhibited. • If stylar and pollen alleles differ, tube growth occurs at normal rates. FYI: Homozygous Flowers What happens if the pistillate flower is homozygous for the S allele? The same rules apply: if the pollen carries an allele that matches the one possessed by the pistil, fertilization will not occur; if they differ, seed can form. However, it should be noted that plants homozygous for a self-incompatibility allele in the gametophytic system are rare. Why are plants homozygous for gametophytic self-incompatibility rare? Homozygotes are unusual because the probability of a pollen grain carrying the same allele as the pistil successfully overcoming the incompatibility of their matching alleles is small—but such events do occur occasionally. Thus, a population may have a few individuals homozygous for an S allele. In Fig 2A, all pollen grains are incompatible with the pistil as they fail to germinate beyond the stigmatic surface. In Fig 2B, nearly all pollen grains are compatible with the pistil as they germinated and pollen tubes grew through the stylar tissues and eventually entered the ovule for fertilization. Sporophytic Self-incompatibility Sporophytic self-incompatibility involves dominance and depends on the allelic composition of the plant that produced the pollen. Incompatibility is expressed at the surface of stigma. In sporophytic self-incompatibility systems, the rate of pollen tube growth depends on the presence or absence of a dominant allele at the SI locus (will be called S locus in the following) carried by the pollen-producing plant. In the pollen, any S allele can exhibit dominance-that dominance is determined by the sporophyte, the plant that produces the pollen (thus, this system of self-incompatibility is termed “sporophytic”). Two important points need to be emphasized about sporophytic self-incompatibility. The genotype of the pollen-producing plant determines self-incompatibility, not the allelic composition of the pollen itself. In other words, every male gamete has the same ability to fertilize a female as every other male gamete, irrespective of the pollen’s individual genotype. There is no dominance in the female stigmatic tissue. How does the genotype of the pollen parent transmit the influence of the dominant allele to the pollen? Although we don’t yet understand this, we describe it as ‘imprinting.’ That is, the pollen “remembers” the genetic environment in which it developed and is conditioned to behave in accordance with that environment. Cell walls of pollen grains are consisted of at least two layers, the intine or inner layer and the exine or the outer layer. The exine is made up of a highly durable organic polymer, sporopollenin. The exine is believed to be derived from the somatic tissues of the pollen-producing parent and likely play a role in pollen-pistil interaction. This could explain why the compatibility response is determined by pollen-producing parent, instead of the pollen itself. Here’s an example of sporophytic self-incompatibility. Assume the species is diploid and that there are four possible self-incompatibility alleles: S1, S2, S3, S4. Let S1 be a dominant allele. Let’s see what happens. (Color indicates the source of the allele, from the female or the male parent.) Why are no offspring produced? The S1 allele in the plant producing the pollen is dominant. Hence, both the gamete types produced by that plant, S1 and S3, will behave as if they were both dominant alleles (S1). As a result, neither pollen type will be able to effect fertilization of this female—neither type of pollen tube will be able to penetrate the style because their growth will be impeded on the S1S2 stigma. 1. In sporophytic systems, hindrance of pollen tube growth is localized on the surface of the stigma. However, an exception is gametophytic self-incompatibility in several grass species such as rye and ryegrass, where pollen tube growth is inhibited on the surface of the stigma. 2. In gametophytic systems, growth is impeded in the style. Pollen tube growth inhibition A feature that distinguishes sporophytic from gametophytic self-incompatibility is the location of pollen tube growth inhibition. When there are multiple S alleles in sporophytic systems, genetic segregation ratios become complex. The presence of a dominant allele in the pollen-producing plant conditions incompatibility if the female carries that same dominant allele. Assume a diploid species and dominance: S1 > S2 > S3 > S4. Female Genotype Male Genotype Pollen Genotypes Offspring Explanation No Matching alleles so incompatible No S2 is imprinted on S3 pollen grains. Yes 1/4 S1S2: 1/4 S1S3: 1/4 S2S3: 1/4 S3S3 S2 is imprinted on S3 pollen grains. Self-incompatibility Systems Many crops have self-incompatibility systems. Why might these systems have evolved? Self-incompatibility is common among naturally cross-pollinated species. Self-incompatibility prevents or limits self-fertilization and promotes out-crossing. Out-crossing maintains heterozygosity and heterogeneity in a population, which often improves plant vigor and productivity. In some species, homozygosity can severely reduce vigor, a phenomenon referred to as ‘inbreeding depression.’ Table 1 Examples of crops with self-incompatibility. Gametophytic Self-incompatibility Sporophytic Self-incompatibility Alsike clover Sunflower (wild populations) Red clover Buckwheat Tall fescue Cacao Potato Brassica species Rye (a two-loci system) Cabbage Sugar beet (a four-loci system) Broccoli Alfafa Kale Tobacco Brussel sprouts Not found in monocots! Techniques to Overcome Self-incompatibility In order to self-pollinate or mate closely-related plants that are normally self-incompatible, plant breeders can employ various techniques that bypass self-incompatibility mechanisms. The technique used depends on the type of self-incompatibility. Uses of Self-incompatibility Genes Some systems have been developed or proposed for utilizing self-incompatibility genes to control pollination and produce F1 hybrid varieties. Gametophytic System Cross-pollination of vegetatively propagated, self-incompatible clones. Using this approach, seed can be obtained from species that are normally self-incompatible, and thus propagated vegetatively. An example is the production of a hybrid variety of bahiagrass, Tifhi. Sporophytic System Bud-pollination can be used in Brassicaceae for self-pollination and thus inbreeding, resulting in inbred lines or families homozygous for an allele at the self-incompatibility (SI) locus (only one SI locus is present in, e.g., cabbage). Hybrids can subsequently be produced by planting two such lines with different fixed SI alleles side by side. Any seed produced is expected to be hybrid seed, since self-pollination within parental lines is prevented by SI. The resulting hybrid will be heterozygous for the SI locus and thus self-incompatible. However, this is not critical since the economic product does not require pollination and is vegetative, e.g., cabbage and kale. Pseudo-Compatibility Produce inbreds in environments that promote pseudo-compatibility and their hybrids in environments that prevent self-fertilization. Sugar beet is normally self-incompatible. However, when grown at high elevations, plants are self-compatible. Low Elevation: Sugar beet inbreds will behave as normal self-incompatible lines at low elevations — self-pollination is genetically eliminated. All seed produced at the lower elevation will be the result of out-crossing among inbred lines, and thus will be hybrid. High Elevation: Self-fertilized seed can be obtained via elevation-induced pseudo-self compatibility. Sufficient quantities of selfed seed can be produced for subsequent hybrid generation. Gametophytic System • Pseudo-compatibility: Expose the plant(s) to lower or higher temperatures, elevated CO2 concentration, or electric shock to induce a pseudo-compatibility response. • Sf alleles: Self-fertility (Sf) alleles, reported to be present in some species can be transferred into a population using conventional breeding methods. The presence of an Sf allele allows self-fertilization. These alleles can be present at self-incompatibility loci, but can also be present at distinct self-fertility loci. Sporophytic System • Removal of stigmatic surfaces: Mechanical (e.g., rupturing) or chemical removal of the stigma surface eliminates the inhibiting factors [believed to be specialized proteins or enzymes, (Barrett, 1998)] that inhibit pollen tube penetration of the style. With those factors absent, pollen tube growth can proceed normally and fertilization can be achieved. • Bud pollination: Pollen is placed on an immature stigma before the inhibiting factor is formed. An example of a gametophytic system is the production of a hybrid variety of bahiagrass, Tifhi. Male Sterility Male sterility is due to the failure of a plant to produce functional anthers or pollen; usually, its female gametes are normal. Thus, male sterility prevents self-pollination and can be used to ensure cross-pollination without emasculation. Male sterility can have genetic causes or be induced chemically. Female sterility, the failure of a plant to produce functional ovaries or eggs, can also occur, but is of little use to plant breeders and will not be discussed. Genetic-based Male sterility Male-sterile gene expression may be complete or partial, and may vary with environment. Breeders desire complete expression that is stable regardless of environment. The extent of sterility is measured by the percentage of viable pollen produced or percentage of seed set. Male sterility is used by breeders to eliminate the necessity of emasculation to control pollination — male-sterile plants cannot self-pollinate. Viable Pollen Measurement The percentage of viable pollen can be estimated using a microscope. Fresh pollen from a plant or group of plants is placed on a microscope slide and viewed at low magnification, 10X. In the field, a hand-held magnifier is often adequate for scoring the percentage of viable pollen. Pollen grains can also be stained and viewed under a microscope in the lab. Pollen viability can also be tested in bioassays. Male-sterility is controlled by nuclear genes, the cytoplasm, or by genetic interaction between the cytoplasm and nucleus. Controlled by the action of specific gene(s) in the nucleus. Usually, the recessive allele(s), designated ms, conditions inhibition of normal anthers or pollen development. Thus, the male sterility phenotype is expressed in plants homozygous for the ms allele. • ms ms = male sterile (therefore, is functionally a female plant) • Ms _ = male fertile (normal) Maintenance of sterility genes in a population is challenging. Uses of Genetic Male Sterility Numerous crops have genes causing male sterility in their gene pools. There are two major uses of genetic male sterility: • Eliminate hand emasculation in making crosses • Increase natural cross-pollination in populations of self-pollinated crops These uses necessitate the transfer of the ms allele into the population being worked with; then the ms allele is maintained in the population through selection for sterility in each subsequent generation. Because genetic male sterility is controlled by a recessive gene(s), it is not possible to get a true-breeding or homozygous ms population. However, the recessive ms allele can be maintained at a high frequency in the population. Genetics of Male Sterility Interactive A male sterile line can be created and maintained by pollination of a line with an identical genotype except for the dominant allele for male fertility. To make the hybrid, male and female rows are planted in a specific pattern and selected plants are allowed to randomly mate with homozygous fertile plants. Query \(2\) Study Question 1: Sterile and Fertile Phenotypes Cytoplasmic Male-sterility The genetic composition of the cytoplasm determines male sterility. The genetic makeup of the cytoplasm results from genes located in mitochondria. Cytoplasm is inherited entirely through the female line. The cytoplasm can be • Normal (N) — normal development of anthers and pollen = male fertile; or • Sterile (S or CMS) — anthers or pollen are non-functional = male sterile Female Line In all organisms, the cytoplasm contains genetic material in the mitochondria. Unlike the genetic material in the nucleus, however, cytoplasmic genetic material is not subject to recombination. Cytoplasm is inherited strictly through the female parent because the male parent does not contribute cytoplasm to the zygote. Recall that the zygote is formed when the sperm nucleus and the egg nucleus fuse; since the zygote’s nucleus is contained in the cytoplasm of the egg and the sperm has no cytoplasm, the zygote inherits its cytoplasm only from its female parent. This fact is used by evolutionary biologists to trace family lines, avoiding the complexity resulting from genetic recombination and segregation that occur with nuclear genetic material. In some systems, the expression of the male sterility phenotype depends on the interaction between cytoplasmic and nuclear genes. In these systems, a plant having sterile cytoplasm, but a nuclear dominant fertility-restorer gene (Rf _), will express a fertile phenotype. The particular combination of cytoplasm and nuclear genes determines the phenotype. Table 2 Phenotype of cytoplasm and nucleus genotype combinations. Cytoplasm Type Nucleus Genotype Rf_ rf rf CMS Male Fertile Male Sterile N Male Fertile Male Fertile Plant breeders must pay close attention to both the cytoplasm type and nuclear genes of the parents used in crosses to generate and maintain cytoplasmic male sterility. Sorghum Example Several crops have cytoplasmic male sterility types available, including sunflower, millet, wheat, maize, and sorghum. Let’s examine the maintenance and use of cytoplasmic male sterility in sorghum. This system involves interaction between the cytoplasm and a nuclear gene. There are two types of cytoplasm possible: S = sterile; N = normal. There are two possible alleles at the restorer locus in the nucleus, Rf (dominant) and rf (recessive). In this example, we’ll look at three types of inbreds. Table 3 Genotypes and phenotypes of sorghum inbred line types. Inbred Type Cytoplasm Type Nuclear Genotype † Male Phenotype A line S rf rf Sterile B line N rf rf Fertile (normal) R line N, or Rf Rf Fertile (normal) S Rf Rf Fertile (normal) † Although we use rf and Rf in this example, sorghum breeders indicate the dominant and recessive nuclear fertility restorer alleles with different symbols: msc = rf and Msc = Rf. How are these inbred (homozygous) lines maintained? A line Assume that the cultivar ‘Martin’ has two versions, one with normal cytoplasm and one that differs only in that it has a sterile type cytoplasm (‘Martin S’). The normal version is a B inbred type and Martin S is an A type. Mate the sterile version as the female with the normal, fertile version. Notice that there is no blending or segregation of the cytoplasm. The F1 always has the cytoplasmic type of its female parent. B line B and R lines can be maintained simply by self-pollination. This is usually accomplished by growing each line in an isolated block and allowing the plants to intermate. Since the line is homozygous and fertile, the seed produced will also be homozygous and fertile. R line Like the B line, the R line can be maintained simply by self-pollination. This is usually accomplished by growing each line in an isolated block and allowing the plants to intermate. Since the line is homozygous and fertile, the seed produced will also be homozygous and fertile. How is single-cross hybrid seed produced using cytoplasmic male sterility? Cross a male sterile line (e.g., A line) by a fertile line (e.g., R line). ‘Caprock’ is an R line. Try This: Cytoplasmic Male Sterility You’re planning to create a set of hybrids carrying various combinations of cytoplasms and fertility restorer alleles. A, B and R lines are available for generating the hybrids.For the cross shown below, predict the type of offspring, if any, will be produced. Engineered Genetic Male Sterility Different systems of male sterility and fertility restoration are being worked on by various companies. All of these involve molecular techniques (genetic engineering) and the development of transgenic plants. One of the first to be utilized was the development of male sterile plant by transforming plant cells with a bacterial gene. This male sterility is dominant, since when the gene is present the plants are male sterile. The normal state for the plants is that the male sterile gene is not present and that gives normal, male fertility. As with the naturally occurring genetic male sterility we discussed earlier, it is impossible to maintain a population of completely male sterile plants as we can in cytoplasmic male sterility; so to identify the male sterile plants from the male fertile ones and to be able to eliminate male fertile plants, an herbicide-resistance gene has been linked to the male sterility gene. This resistance is also dominant, since when present it confers resistance and when absent (the normal state of the untransformed plants) plants are susceptible to the herbicide. System: Linking herbicide-resistant gene to the male sterility gene A plant is transformed to contain linked male sterility and herbicide resistance genes Ms R (The transformed plant will have only one homologue containing both the Ms and R genes, thus will neither be homozygous nor heterozygous, but rather what we call hemizygous for the two genes.) We now must incorporate this linked pair into an elite inbred line by backcrossing. (The symbol “-” indicates that there is no allele for this gene present.) The elite inbred will be developed and then maintained by crossing the male sterile plants with the normal inbred to give 50% sterile and 50% fertile plants. Discussion for Further Thought For the diploid, self-pollinated species tomato (Solanum lycopersicum), discuss from a genetic perspective the strengths and weaknesses of different hybridization systems that use either hand emasculation, male sterility, or genetic engineering, and come to a consensus, on which one you prefer. Chemically-induced Genetic Male Sterility In species that lack genetic male sterility or cytoplasmic male sterility, or in species in which these are difficult to work with, breeders can use chemicals to induce male sterility. These chemicals have at one time or another been called by the following names: • Gametocides • Pollen suppressants • Hybridizing agents These inhibit the production of viable pollen or prevent pollen shed, but do not damage the pistillate flower or interfere with seed development. There are both advantages and disadvantages to chemically-induced male sterility. Advantages • No need to develop and maintain cytoplasmic or genetic male sterility systems • Applied to normally self-pollinated species, the chemicals prevent self-pollination and facilitate cross-pollination without the necessity of hand emasculation. This approach has been used in cotton, maize, sorghum, and various vegetable crops. It was also tested for hybrid wheat production. Disadvantages • Incomplete pollen sterility can occur, resulting in some selfing. Several factors can account for incomplete sterility. • differential genotypic reactions to the chemical agents • timing and dosage of application is critical and varies with genotype • environmental effects on chemical and interaction with genotype • long periods of flowering cause difficulties in maintaining optimum dosage in plants • Does not provide a means of pollen transfer to produce hybrid seed, and so additional methods must still be employed. Hybrid Wheat Since wheat has perfect flowers and is normally self-pollinating, the production of hybrid varieties was impractical without the use of male gametocides. Gametocides were applied to the female plants prior to flowering to kill their pollen and prevent their self-pollination; the treatment was not applied to adjacent rows containing the intended male parent. Thus, seed produced by the female plants resulted from cross-pollination with the desired males. Although hybrid seed could be produced in this manner, the approach has been largely abandoned by most commercial seed companies—farmers did not buy the hybrid seed because the hybrids did not yield sufficiently better than conventional wheat to justify the additional seed costs. Sex Inheritance Description The ultimate biological mechanism to prevent self-pollination, and thus inbreeding, is to have distinct male and female flowers on different plants. In such “dioecious” plant species, which are common in the plant kingdom, male and female plants occur usually at a 1:1 ratio. The decision on whether a plant becomes male or female is genetically determined, and can in some species be influenced by environmental factors. As a consequence, development of inbred lines can be accomplished either by crosses of male and female sister plants, or by employing environmental factors to induce plants with both sexes to allow self-pollination. In addition to dioecious and hermaphrodite plant species, there are various intermediate variants realized in plants (Table 4). Table 4 Sex systems in flowering plants. Data from Charlesworth 2002, Heredity 88, 94-101. Plant Term Definition of Plant Term Sexually monomorphic Hermaphrodite Flowers have both male and female organs (Tomato, potato, sugarcane) Monoecious Separate sex flowers on the same plant (Cassava, maize, banana) Gynomonoecious Individuals have both female and hermaphrodite flowers Andromonoecious Individuals have both male and hermaphrodite flowers Sexually polymorphic Dioecious Male and female plants (spinach, asparagus) Gynodioecious Individual either female or hermaphrodite (papaya) Androdioecious Individuals either male or hermaphrodite Genetics of Sex Inheritance Distinct male and female flowers can be of practical importance, if present on the same plant. Best example is maize, where spatial separation of female and male flowers facilitates both elimination of male flowers to produce “female plants” for hybrid seed production, and self-pollination for inbred development. However, the majority of plants carry either flowers containing both male and female organs, or male and female flowers on distinct plants. In the latter case, pollination will be mediated by wind or insects. Female plants will set seed only if a male plant is located in the vicinity. There are two genetic mechanisms of sex inheritance: • Sex chromosomes — Like in humans, the sex of an individual is determined by specific chromosomes. In the case of humans, carriers of two X-chromosomes are females, whereas males have one copy each of the X- and Y-chromosomes. Since X- and Y-chromosomes differ cytologically, their pairing in meiosis might be incomplete. • Autosomal inheritance — Genes affecting sex inheritance are located on “regular” chromosomes (autosomes), forming bivalents in meiosis. Depending on the plant species, sex determination can be due to one or a limited number of genes (mono-, or oligogenic inheritance). Sex Inheritance Systems Application and Challenges Application Like self-incompatibility or male sterility, sex inheritance can be used for controlled crosses between any pair of genotypes in hybrid seed production schemes. One genotype would be used as female and seed parent, the other parent as pollen donor. Challenges in using sex inheritance for hybrid seed production As plants with only one sex cannot be sexually maintained, it is either required to use environmental or chemical factors to induce the other sex to allow self-pollination (if available), or to develop male and female sister lines for each hybrid parent, comparable to male sterile and maintainer lines when using male sterility. This is described in more detail in the following cucumber and asparagus examples. Cucumber Cucumber (Cucumis sativus) has a wide range of floral types. Staminate (male), pistillate (female), and hermaphrodite flowers can occur in different arrangements. Generally, embryonic flower buds possess both staminate and pistillate initials, and thus the potential to develop into any of the above-mentioned flower types. The cucumber phenotype with regard to floral types depends on autosomal genes and their interaction with environmental factors. Sex inheritance is controlled by a minimum of three major loci: • m+, m — controls the tendency to form hermaphrodite versus male or female flowers. mm homozygotes develop hermaphrodite flowers. • F+, F — controls the female tendency, with F allele being dominant and favoring female flowers. This locus is subject to strong environmental influence. Among environmental factors, in particular photoperiodic conditions and temperature affect flower formation. • a+, a — Homozygotes for a allele intensify male tendency. The effects of a are dependent on the allelic composition at the ‘F’ locus. Male tendency is only pronounced in ‘F+F+‘ homozygotes. Although other loci such as ‘de’ and ‘cp’ have been shown to affect sex types in cucumber, the main sex types are determined by the above mentioned loci m, F, and a (Table 5). Table 5. Phenotypes and Genotypes of basic sex types in cucumber.Note: “-/-” means that any allele can be present at this locus Genotype, locus Phenotype m F a Androecious (male) -/- F/F a/a Monoecious m+/m+ F/F -/- Hermaphroditic m/m F/F -/- Gynoecious (female) m+/m+ F/F -/- CHEMICAL REGULATION OF SEX EXPRESSION Phytohormones can be applied to alter flower phenotypes. Whereas auxin and ethylene promote female flowers, gibberellin promotes male flowers. Moreover, silver nitrate and silver thiosulfate induce male flowers even in strongly gynoecious genotypes. These treatments have become invaluable in cucumber breeding, as they allow efficient development of gynoecious inbreds to be used as female hybrid parents, and even the production of hybrids from crosses of two gynoecious lines. Asparagus Sex inheritance in Asparagus officinalis, is similar to humans, based on sex chromosomes. XX chromosome carriers are female, XY carriers are male. Males and females occur at about equal frequencies in natural populations. Males have been shown to be more productive. For this reasons varieties with only male plants are preferred. At a low frequency and for unknown reasons from a genetic perspective, andromonoecious XY plants occur. These plants can be self pollinated, and will result in a 1:2:1 ratio with regard to the sex chromosomes in resulting offspring. One quarter of those will thus carry two copies of the “male” Y chromosome, and are thus called supermales. As Asparagus can be vegetatively propagated, those YY plants are potential variety candidates. Alternatively, they can be used as male parent in hybrid breeding schemes to be crossed with female XX genotypes, and result in 100% male XY offspring. In contrast, if regular XY males would be crossed to XX females, the offspring would segregate 1:1 into XY and XX plants, and XX plants would need to be eliminated by producers. Controlling Hybridization Knowledge of the breeding system of a crop species is essential to take advantage of the types of gene action that give the most useful cultivars. Self-incompatibility systems are important in many natural species for forcing outcrossing and thus maintaining vigor through heterozygosity. As we have seen, this system can be adapted to help produce F1 hybrid cultivars in domesticated species containing self-incompatibility loci. Similarly, sex inheritance can be employed in controlled F1 hybrid seed production schemes. Male sterility is a rather unimportant method of enforcing outcrossing in natural plant species. It has, however, become such an important tool in the production of hybrid cultivars that it is utilized in many species of both cross- and self-pollinated crops.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.03%3A_Controlled_Hybridization-_Self-incompatibility_Male-sterility_and_Sex-inheritance.txt

Introduction Plant breeders take advantage of the variation that occurs within a population to develop improved cultivars. Ordinarily, the goal of the plant breeder is to combine the favorable characteristics of one plant or cultivar with the desirable traits of another plant or cultivar to obtain a new combination that has the best of both. Understanding the genetics of desired, as well as undesirable, characteristics enhances the efficiency of the plant improvement process. Learning Objectives • Understand the molecular basis of genes and chromosomes. • Understand the basic principles of transcription and translation. • Understand Mendelian mechanisms and patterns of inheritance. • Be able to differentiate among different types of gene action. • Determine genotypic and phenotypic consequences of independently inherited genes through generations of self-pollination. • Know how epistasis occurs through interaction of genes and alteration of expected phenotypic ratios. Overview of Genetics The Science of Genetics Genetics is one of the principal sciences that underlie plant breeding. Genetics is the study of heredity, genes, chromosomes, and variation in biological organisms. The science of genetics is often divided into four major subdisciplines: • Transmission genetics (also called classical or Mendelian genetics) • Quantitative genetics • Population genetics, and • Molecular genetics Transmission genetics deals with how genes and genetic traits are transmitted from generation to generation and how genes recombine. The foundation of modern genetics is recognized to have occurred in the mid-1800s when Gregor Mendel analyzed the results of crosses he made among garden pea plants. Mendel concluded that inherited characteristics (now called traits or phenotypes) are determined by factors (now known as genes) that he observed. He also realized that each organism contained two copies of each “factor” (gene), one inherited from its mother and one from its father. Mendel discovered the principles of heredity when he noticed how inherited traits (e.g., seed shape round vs. wrinkled; pod color yellow vs. green; flower position axial vs. terminal; or plant height tall vs. short) are passed from parents to offspring. Transmission (Mendelian) genetics is the focus of this module. Genetic Subdisciplines Quantitative genetics focuses on the study of inheritance when phenotypes exhibit continuous variation or distribution. In particular, it considers the effects of many genes that could be simultaneously influencing such traits, as well as the relative contributions of the environment and the interaction between genotype and environment. Quantitative genetics is the focus of the module on Inheritance of Quantitative Traits. Population genetics entails a study of heredity in groups of individuals for traits that are usually determined by one or only a few genes. It deals with gene distribution and genetic diversity within and among populations and subpopulations. Population genetics includes assessment and prediction of response to selection. It describes relationships between allele and genotype frequencies due to four main evolutionary forces: natural selection, genetic drift, mutation, and gene flow. Population genetics is the focus of the module on Inbreeding and Heterosis. Molecular genetics is concerned with the molecular structure and function of genes. It includes the study of DNA structure and replication and deals with gene expression and regulation. Gene Structure Genes and Chromosomes To understand inheritance, it is essential to understand gene structure and action. Let’s review key terminologies and principles. For a more in-depth review, please refer to biology or genetic textbooks, for example, From Genes to Genomes (Hartwell et al. 2011), Genetics: A Conceptual Approach (Pierce 2012), or iGenetics: A Molecular Approach (Russell 2010). Genes are encoded with DNA. Most of the DNA in plants is located in the nucleus of cells and arranged in groups of genes along multiple, linearly-shaped, chromosomes. Nuclear DNA is subject to Mendelian inheritance, which will be discussed later in this module. In addition to its occurrence in chromosomes in the nucleus, DNA is also located in organelles present in the cytoplasm of plant cells. FYI: Cytoplasmic DNA In plants, DNA is not just present in the nucleus of cells. It is also located in other membrane-bound, specialized subunits known as organelles that are found within the cytoplasm, or cell fluid. Two plant cell organelles that contain DNA are chloroplasts (which are plastids or organelles that carry pigments-specifically green chlorophyll) and mitochondria (singular, mitochondrion; organelles that break down complex carbohydrates and sugars into usable forms, and thus supply energy for the plant). The non-nuclear, organellar DNA located in plants follows cytoplasmic inheritance and is not subject to Mendelian inheritance. Cytoplasmic inheritance is also known as extrachromosomal or extranuclear inheritance, and is of significance in certain types of male sterility where the genes for those traits are present in the mitochondria, not in nuclear chromosomes. Molecular Basis of Chromosomes Chromosome – Each chromosome contains a single DNA molecule (Figure 2). DNA DNA (deoxyribonucleic acid) is composed of two chains of polynucleotides. Polynucleotides are also called nucleic acids, and consist of linear polymers that are macromolecules formed by the chemical joining of many identical or similar units called nucleotides. Every nucleotide in each chain consists of a nitrogen-containing base, deoxyribose (a sugar), and a phosphate group. Nucleotides within each chain are held together by sugar-phosphate (phospho-diester) bonds (Figure 3). Nitrogen-containing bases are purines (adenine, A, and guanine, G) and pyrimidines (cytosine, C, and thymine, T). Pairing occurs between one purine and one pyrimidine and is specific. Sequences of consecutive nucleotides constitute genes (Figure 4). C always pairs with G T always pairs with A DNA replication is semiconservative. The process of DNA replication is not yet fully understood. Basically, there are three steps. 1. Two strands of DNA unwind and pull apart. 2. Free (unbound) nucleotides bind to complementary bases on an original strand of DNA. 3. One newly formed strand and a template DNA strand re-coil to form a double helix. This process is semiconservative because each resulting double-stranded DNA molecule is composed of a newly synthesized strand and a template strand (Figure 4). Since one strand of each DNA molecule is an original strand, there is less probability of error occurring during replication. Genes Many genes are present in each chromosome. Each specific gene occurs at a defined point on a chromosome, the gene locus, on each of the two homologous chromosomes. More than one form of a particular gene, alleles, may occupy the same locus on homologous chromosomes. Alleles Alleles are variants that differ slightly in their DNA sequence. Diploid plant species have two sets of chromosomes, each of which can possess a different allele for a particular gene. For example, a gene for seed color might have the two alleles, A and a. Allele A causes one phenotype (e.g., brown seed color) and allele a causes a different phenotype (e.g., white seed color). For that gene, the genotype could be either AA, Aa, or aa. If one allele at a locus on a homologous chromosome partially or completely masks the expression of the other in influencing the phenotype, the allele that is expressed is termed dominant and the allele that is masked is termed recessive. By convention, we often write the dominant form with an uppercase letter, and the recessive form in lowercase. In the example above for seed color, allele A is the dominant allele. If the A allele is completely dominant to the a allele, individuals with either the AA or Aa genotypes would have the brown seed color phenotype, while aa individuals would have white seeds. An individual is heterozygous (Aa) when two different alleles are present at a locus and is homozygous—in this example, either homozygous dominant (AA) or homozygous recessive (aa)—when the same alleles are present on both chromosomes. Alleles at a locus can interact in several ways that are revealed by their phenotype, whether heterozygous or homozygous. FYI: Homozygosity and Heterozygosity For a given locus, an individual with a genotype of either AA or aa is homozygous for that gene and is known as a homozygote; the status of the gene is referred to as homozygosity. An individual with the genotype Aa is heterozygous for that gene and is called a heterozygote; the status is known as heterozygosity. In the case of polyploid individuals, those with the genotypes AAAA (tetraploid) or aaa (triploid) would be examples of homozygotes and those with genotypes of AAaa (tetraploid) or AAaaaa (hexaploid) would be examples of heterozygotes. The terms homozygous and heterozygous are used to describe the status of single genes or all gene loci within an individual, not within a population. There may be many different alleles of a gene present in a population of individuals, but for each diploid individual there are only two alleles per gene. For each individual, there is one allele from each parent and each allele per gene is present at corresponding loci on homologous chromosomes. With regard to populations, a homogeneous population would be one in which all individuals in the population would have the same genotype and possess the same alleles for one or more genes. In contrast, a heterogeneous population would be characterized by differing alleles at one or more loci. Note that a cross between two homozygous parents produces progeny that are homogeneous because all of the individual offspring are genetically identical. However, the offspring would be heterozygous for all loci for which different alleles occurred in the two parents. Terms and Definitions Gene Expression, Translation, and Transcription DNA, Protein, and Other Gene Products In order to have a better understanding of the concept of gene that will be the focus of this and the following lesson on linkage, it is critical to understand the chemical nature of DNA . Let’s review the pathways by which the genetic information in DNA is transferred from one DNA molecule to another (the process termed DNA replication) and from DNA to ribonucleic acid (RNA) molecules (called transcription), and then transferred from RNA to a protein (termed translation) by a code that specifies the amino acid sequence of the protein (see Figure 6). A gene is a stretch of DNA along a chromosome consisting of sequences of consecutive nucleotides. Recall that genetic information in DNA is coded in the sequence of four nucleotides that are abbreviated by the type of nitrogen-containing base that each contains—the purines A and G and the pyrimidines C and T. Through DNA replication, genetic information of an individual is transmitted from cell to cell during development and from generation to generation during reproduction. DNA Structure Examine the following for a better understanding of the chemical structure of the nucleotides that comprise the basic building blocks of DNA and the process of DNA replication: Review the chemical structure of DNA and what occurs during the process of DNA replication. DNA replication occurs within the synthesis phase of the cell cycle. Four types of chemical bases—A, G, C, T—in gene sequences carry the instructions for assembling a protein (Figure8). The base pairs are bonded together by H-bonds to form the “rungs of a DNA ladder” (Figure 8). Nucleotides Nucleotides are the basic building blocks of nucleic acids such as DNA and RNA, which are polymers made of long chains of nucleotides. DNA is double-stranded and RNA is single-stranded (Figure 9). Note that in RNA, the chemical base uracil (U) replaces thymine (T). Genes generally express their effect by coding for polypeptide chains, which are polymers consisting of ten or more aminoacids linked by peptide bonds. One or more polypeptides make up a protein. The DNA sequence of a gene is used as the basis for producing a specific protein sequence. Proteins are the complex molecules responsible for most biological functions in the cell. Gene Expression, RNA, Translation, and Transcription Amino acids are the building blocks of proteins. A protein is composed of one or more long chains of amino acids, the sequence of which corresponds to the DNA sequence of the gene that encodes it. The process of creating proteins from the genetic code in DNA is referred to as gene expression. The general process of gene expression in the cells of eukaryotes such as plants involves numerous steps, which are described below. FYI: Eukaryotes Plants are multicellular organisms known as eukaryotes, which are organisms possessing cells that contain DNA in a nucleus and other membrane-bound, specialized subunits known as organelles that are found within the cytoplasm, or cell fluid. Two plant cell organelles that contain DNA are chloroplasts (which are plastids or organelles that carry pigments—specifically green chlorophyll) and mitochondria (singular, mitochondrion; organelles that break down complex carbohydrates and sugars into usable forms, and thus supply energy for the plant). The non-nuclear, organellar DNA located in plants follows cytoplasmic inheritance and is not subject to Mendelian inheritance. Cytoplasmic inheritance is also known as extrachromosomal or extranuclear inheritance, and is of significance in certain types of male sterility where the genes for those traits are present in the mitochondria, not in nuclear chromosomes. In contrast to eukaryotes, prokaryotes such as bacteria are often unicellular and lack a cell nucleus and usually have their DNA in a single circular molecule. Transcription is a process in which the sequence of nucleotides in one DNA strand of a gene is copied into the nucleotides of an RNA molecule. The order of nucleic acids in RNA complements those on the DNA strand from which it is transcribed. In the RNA strand, however, uracil(U), rather than thymine (T), is the base that complements adenine (A). As the RNA transcript is formed, each base in the DNA is paired with a base in an RNA nucleotide, which is progressively added to the RNA strand as it grows. Transcription occurs in the nucleus of the cell (Figure 13). In a procedure known as RNA processing, intervening sequences or introns are removed from the RNA transcript by splicing. Introns are a special type of so-called non-coding DNA sequences that do not code for amino acids, but are located within genes until such sequences are removed during RNA processing. (Note that aside from intron sequences, most non-coding DNA found in chromosomes is located between (not within) gene loci along the chromosome.) The regions between the introns in the fully processed RNA are called exons, the sequences that code for proteins (Figure 10). The ends of the transcript are also modified. The fully processed RNA is referred to as mRNA (messenger RNA). mRNA is a single-stranded sequence of nucleic acid and it moves from the cell nucleus to the cytoplasm where proteins are made (Figure 10). Translation is the process through which mRNA directs the assembly of amino acids in the proper sequence to synthesize the particular protein. Ribosomes in the cell cytoplasm read the base sequence of the mRNA (Figure 10). In the translated part of the mRNA, each adjacent group of three nucleotides constitutes a coding group or codon. Each codon specifies an amino acid subunit in the polypeptide chain. Adapter molecules, tRNA (transfer RNA) are complexed with the specific amino acid corresponding to the base sequence of the given mRNA. tRNA molecules bring the amino acids specified by the mRNA to the ribosomes where they are added to the growing protein chain. When the polypeptide chain is complete, it is released from the mRNA and forms a protein molecule. The order of amino acids determines the structure of the protein which affects its action. Basic Steps of Transcription These are the basic steps of transcription and translation: 1. During transcription, a region of double-stranded DNA is momentarily pushed open, separating the two strands and allowing an enzyme known as RNA polymerase to build a strand of mRNA corresponding to that region of DNA. 2. The tRNA anticodon attaches to the mRNA codon. The tRNA has a region called the “anticodon” that complements the codon sequence of the mRNA (Figure 14). 3. The specific amino acid complexed with the tRNA is held in place while the tRNA-amino acid complex corresponding to the next codon moves into place. A peptide bond is formed between the adjacent amino acids, building the protein molecule. Inheritance and Gene Action Mechanisms Inheritance is based on the behavior of chromosomes and the genes that they carry. During meiosis and gametogenesis, homologous chromosomes separate. Each gamete receives one (haploid) set of chromosomes. The particular chromosome of a homologous pair that is distributed to a given gamete is random. When two gametes fuse during fertilization, the zygote receives from each parent one set of chromosomes, and the alleles that they each carry. The resulting combination of alleles in the zygote determines its genotype. Because the distribution of homologous chromosomes to gametes is random, the fusion of gametes to form the zygote may produce different genetic combinations. Thus, within a population, variation for specific traits or characters may be observed. If the variation for a given trait is due to contrasting alleles at one or more loci, rather than to responses to the environment, the variation is heritable and can be transmitted from parent to progeny. Plant breeders select plants that exhibit desirable characteristics and those plants carry the desired allele of the gene that encodes the characteristic of interest. Each gene or combination of genes and alleles, as influenced by the environment, determines the phenotype or observed expression of the particular trait. An individual’s allelic composition at corresponding loci on homologous chromosomes confers the expression of that gene. Alleles at corresponding loci interact. One allele may mask the presence of the other allele(s). Alleles at a locus can interact in different ways, including no dominance (also referred to as additive gene action), partial dominance, complete dominance, and over-dominance. Gene Action There are several general types of gene action. The type of gene action and the alleles present for a given gene affect the phenotype. Let’s consider the gene action as indicated by the phenotype of a diploid individual heterozygous at the given single locus compared to the phenotype of its parents. Addictive gene action (no dominance) The progeny’s phenotypic value is at the midpoint between both parents. Complete dominance The phenotype of the heterozygous progeny equals the phenotype of the homozygous dominant parent. Partial (incomplete) dominance The heterozygous progeny has a phenotypic value greater than that of the mid-parent value (MPV), but less than that of the homozygous dominant parent. Over-dominance The phenotype of the heterozygous progeny is greater than either parent. Study Questions 1: Parental and Progeny Value Comparison Two diploid plants having different phenotypes for characters A, B, and C are mated. The progeny are grown out and their phenotypes are evaluated. Assume that both parents are homozygous at each locus. Compare the parental and progeny values for each character. Select the gene action at each locus. A Locus Genotype Phenotype value Parent One AA 75 Parent Two aa 40 Progeny Aa 75 Query $2$ B Locus Genotype Phenotype value Parent One BB 60 Parent Two bb 20 Progeny Bb 55 Deviations from Expected Phenotypes Multiple Alleles With complete dominance of the type that we have been discussing, two different alleles exist for a trait, but only one of the alleles is observed in the phenotype. But it is important to understand that dominance does not affect the way in which genes are inherited. For some characters, there are reasons other than dominance among alleles at the same locus that explain deviations from expected phenotypes. Multiple alleles—rather than just two—can occur at a single locus. Examples of multiple alleles at a single locus include the ABO blood group system in humans or the S alleles that control self-incompatibility in plants. Multiple alleles at a locus are sometimes referred to as an allelic series. However, while there may be more than two alleles per gene present in a population, be aware that the genotype of any given individual diploid plant in the population possesses only two alleles. Penetrance is a measure of the percentage of individuals having a particular genotype that express the expected phenotype. Incomplete penetrance occurs when a genotype does not always produce the expected phenotype. Expressivity is a related concept that describes the degree to which a character is expressed. Incomplete Penetrance Incomplete penetrance and variable expressivity are due to effects of other genes or environmental factors that change the effect of a particular gene. For example, a phenotype produced by an enzyme encoded by a particular gene may be expressed only within a narrow temperature range. In barley, a recessive allele occurs that produces albino plants when they are grown at lower temperatures. The allele inhibits chlorophyll production. But if barley plants that are homozygous recessive for this allele are grown above a critical temperature, the effect is not present so the plants have normal chlorophyll and are green. Lethal alleles can change expected phenotypic ratios as well. Lethal alleles cause death when present, so that one or more genotypes will be missing from the offspring of a cross. Lethal alleles can be recessive (causing death only in homozygotes) or dominant (both homozygotes and heterozygotes with the allele will die). Dominant lethal alleles are rarely maintained in populations. Essential genes are genes that when mutated can result in a lethal phenotype. Study Questions 2: Deviations from Expected Phenotypes An example of a recessive lethal allele is one that controls chlorophyll production in the aurea strain of golden-leaved snapdragons. Aurea plants are heterozygous for the gene. A cross between two aurea plants produces progeny in the ratio of 2:1 golden to green. The expected phenotypic ratios in the progeny would be 1:2:1 white to golden to green. However, the white-leaved offspring die before germination or in the seedling stage due to a lack of ability to make chlorophyll. What are the genotypes for each of these leaf phenotypes in the progeny of a cross between aurea snapdragons? Mendelian Heredity Gregor Mendel analyzed the segregation of hereditary traits. We now know that the genotype is the genetic constitution of an organism and the phenotype is the observable characteristic or set of characteristics of an organism produced by interactions between its genotype and the environment. The phenotype is influenced by not only the genotype but also environmental effects and developmental events and by actions of other genes and their products. Therefore, individuals with the same genotype can have different phenotypes and conversely, individuals with the same phenotype can have different genotypes. Terminology The parental generation of a cross is often called the P generation. Using symbolism based on what is called the F Symbol, the progeny of the mating of two parents is typically called the F1 or first filial generation. The subsequent generation produced by either self-pollination or crossing among the F1 offspring (a type of mating called inbreeding) is referred to as the F2 generation, or the second filial generation. The progeny resulting from self-pollination of each consecutive generation following the F2 is referred to as F3, F4, F5, and so on. Another kind of symbolism is based on the S Symbol. The S symbol is used to describe the offspring of a single cross—specifically the cross between two homozygous parents. F and S symbolism have been developed to describe progeny developed by hybridization and self-pollination. F and S Symbolism It is important to note on pages 28-33 of Fehr’s textbook, plant breeders have developed a variety of systems using either the F or the S symbol to describe progeny developed by hybridization and self-pollination. What is challenging is that depending on the plant breeder, F and S symbols may be used in different, often contradictory, ways. The table below depicts examples of the particular system chosen and the way in which symbols are defined for use (Fehr, 1987, p. 28-33). Table 3 Symbol Description F1 Hybrids produced from the mating of homozygous parents. F2 = S0 First segregating generation produced from the cross of two or more parents F3 = S1 Offspring from self-pollination of F2 (or S0) plants F5 = S3 Offspring from self-pollination of F4 (or S2) plants Syn 1 Synthetic 1 = Offspring from random mating of an F2 population Syn 4 Synthetic 4 = Offspring from random mating of a Syn3 population F2:5 line F2-derived line in F5 = an F5 generation line available for planting that originated from an F2 generation S2:9 line S2 -derived line in S9 = an S9 generation line available fro planting that originated from an S2 generation Crosses A cross involving a single trait (e.g., seed color) is referred to as a monohybrid cross, while one involving two traits (e.g., seed color and plant height) is termed a dihybrid cross. Conventionally, in equations used to symbolize a cross, the female parent is listed first and the male parent second, as in this example involving a single locus in diploid individuals: AA x aa ⇒ Aa Crosses that are done both ways are referred to as reciprocal crosses. For example, the reciprocal cross of the one above would be: aa x AA ⇒ Aa Reciprocal crosses can be used to determine whether a trait is maternally inherited. If a trait is controlled by genes located in cytoplasmic DNA, the segregation ratios between reciprocal crosses would be different because cytoplasmic DNA is inherited only through the female parent. Predicting Segregation Ratios If the genetic basis of a trait is known, principles developed by Mendel can be used to predict the outcome of crosses. There are three common approaches used to analyze segregation results, two of which use the listing of all possible genotypes and phenotypes of zygotes and gametes by systematic enumeration and the other of which uses mathematical rules. • The Punnett Square Method is best for situations involving one or two genes. All possible gametes are written down in a square and then combined systematically to depict an array of genotypes of the offspring. • The Branching or Forked-Line Method [See Appendix C for some examples] also works well for situations involving one or two genes. It uses a tally system in a diagram of branching lines. • The Probability Method is based on two rules in mathematical probability theory—the Multiplicative Rule and the Additive Rule—and deals with the frequency of events. Punnett Square Examples Parental Monohybrid Cross Trait Seed color Alleles Y yellow y green Cross yellow seeds x green seeds YY x yy (homozygous dominant x homozygous recessive) Offspring called F1 generation Genotype all alike Yy (heterozygous) Phenotype all alike Yy (green) Pollen Egg   1/2y 1/2y 1/2Y 1/4Yy 1/4Yy 1/2Y 1/4Yy 1/4Yy F1 Monohybrid Cross Alleles Y yellow y green Cross yellow seeds x green seeds Yy x Yy (heterozygous x heterozygous) Offspring called F2 generation Genotypic ratio 1:2:1 YY (homozygous dominant): Yy (heterozygous): yy (homozygous recessive) Phenotypic ratio 3:1 Y_ (yellow): yy (green) Results: Pollen Egg   1/2Y 1/2y 1/2Y 1/4 YY 1/4 Yy 1/2y 1/4 Yy 1/4 yy Dihybrid Cross Trait Seed shape and seed color Alleles R round, r wrinkled, Y yellow, y green Cross Round, yellow seeds x round, yellow seeds RrYy x RrYy (heterozygous x heterozygous) Offspring called F3 generation Genotypic ratio 1:2:1:2:4:2:1:2:1 RRYY:RRYy:RRyy:RrYY:RrYy:Rryy:rrYY:rrYy:rryy Phenotypic ratio 9:3:3:1 R_Y_ (round, yellow): R_yy (round, green): rrY_ (wrinkled, yellow): rryy (wrinkled, green) Results: Pollen Egg   1/4 RY 1/4 Ry 1/4 rY 1/4 ry 1/4 RY 1/16 RRYY 1/16 RRYy 1/16 RrYY 1/16 RrYy 1/4 Ry 1/16 RRYy 1/16 RRyy 1/16 RrYy 1/16 Rryy 1/4 rY 1/16 RrYY 1/16 RrYy 1/16 rrYy 1/16 rrYy 1/4 ry 1/16 RrYy 1/16 Rryy 1/16 rrYy 1/16 rryy Branching or Forked-Line Method Below is an example of the forked-line or branch diagram method for determining the outcome of an intercross involving three independently assorting genes in peas. Traits Plant height, seed color and seed texture Alleles D tall / d dwarf G yellow / g green W round / w wrinkled Cross Tall plants with yellow, round seeds x dwarf plants with green, wrinkled seeds DDGGWW x ddggww (homozygous dominant x homozygous recessive) F1 DdGgWw Expected F2 phenotypes for each trait Segregation of gene for plant height Segregation of gene for seed color Segregation of gene for seed texture Combined phenotype of all three genes 3/4 D_(tall) 3/4 G_(yellow) 3/4 W_(round) 27/64 D_G_W(tall, yellow, round) 1/4 ww(winkled) 9/64 D_G_ww(tall, yellow, wrinkled) 1/4 gg(green) 3/4 W_(round) 9/64 D_ggW_(tall, green, round) 1/4 ww(wrinkled) 3/64 D_ggww(tall, green, wrinkled) 1/4 dd(dwarf) 3/4 G_(yellow) 3/4 W_(round) 9/64 ddG_W_(dwarf, yellow, round) 1/4 ww(wrinkled) 3/64 ddG_ww(dwarf, yellow, wrinkled) 1/4gg(green) 3/4 W_(round) 3/64 ddggW_(dwarf, green, round) 1/4 ww(wrinkled) 1/64 ddggww(dwarf, green, wrinkled) Rules of Probability Using probability theory can allow for accounting of the frequency of events, such as the chance of obtaining a head on a coin toss or obtaining a dominant homozygote (AA) from the mating between two heterozygotes (Aa). To figure out the probability of an event, all possible outcomes must be determined. For a coin toss, there are two possible events—heads or tails—each with a probability of ½ that it would occur. For the progeny produced by a heterozygote, the probability associated with each type of offspring is ¼ (AA), ½ (Aa) and ¼ (aa). The Multiplicative Rule states that if events X and Y are independent, the probability that they occur together (that is A and B), is the probability of A times the probability of B. It is denoted as: $P(A)\times P(B)$ The Additive Rule states that if events X and Y are independent, the probability that at least one of them occurs (that is A or B), is the probability of A plus the probability of B minus the probability that both A and B occur together. It is denoted as: $P(A) + P(B) - [P(A) \times P(B)]$ Mendel’s Principles Mendel’s analysis of monohybrid crosses identified three key principles: The Principle of Uniformity If both parents are homozygous, their F1 is genetically uniform. To the right is a Punnett Square showing an example of this phenomenon, depicting the genotypic and phenotypic ratios and chromosomes of the diploid parents, haploid gametes, and the F1 generation. The Principle of Segregation In a heterozygote, two different alleles of a gene locus segregate from each other in the formation of gametes. Below are two figures (one using a Punnett Square and the other the fork or branch diagram method) showing an example of Mendel’s law of segregation. The figures depict the genotypic and phenotypic ratios and chromosomes of the F1 heterozygote, haploid gametes, and the F2 generation. The Principle of Independent Assortment Alleles at different gene loci are transmitted independently of one another during the production of gametes. Below are two figures (one using a Punnett Square and the other the fork or branch diagram method) showing an example of Mendel’s law of independent assortment. The figures depict the genotypic and phenotypic ratios and chromosomes of the parents, the F1 heterozygote, haploid gametes, and the F2 generation. Inheritance A trait or characteristic may be under the control of one or more genes. The range of variation for a particular characteristic indicates the mode of inheritance of that characteristic. • Qualitative inheritance — simple inheritance of a characteristic under the control of single gene or a few major genes. The expression of simply inherited characteristics is discrete. That is, the phenotypic variation of the characteristic can be separated into distinct classes. Generally, the environment has little influence on the characteristic’s expression. • Quantitative inheritance — inheritance of characteristics influenced by numerous genes (multiple genes or polygenes). The involved genes have small, cumulative effects on the phenotype of the characteristic. The expression of such characteristics can be measured in quantitative units that are continuous, rather than discrete, and is often considerably influenced by the environment. Quantitative inheritance is the subject of the module on Inheritance of Quantitative Traits. The inheritance of some characteristics cannot easily be categorized as either qualitative or quantitative. These characteristics are usually under the control of one or few major genes as modified by multiple genes with small effects. Together with environmental effects, the phenotype of such characteristics may show continuous variation. Try This! – Trait Graphs Drag the correct inheritance to the appropriate trait graph Progeny Ratios To determine the mode of inheritance of a particular character, plant breeders mate plants and evaluate the performance of their offspring. The proportion of progeny exhibiting different phenotypes provides information about the proportion of progeny possessing different genotypes. • Phenotypic ratio — the proportion of progeny exhibiting different phenotypes • Genotypic ratio — the proportion of progeny possessing different genotypes These ratios are commonly determined by crossing two plants having contrasting phenotypes for a given character. The parents may or may not be homozygous. The progeny are heterozygous for the trait. Self-pollinating the F1 progeny produces the F2 generation, and so forth (Fn). In each generation, the ratio of plants displaying contrasting phenotypes for the particular trait reveals information about the genotypes of the parents, as well as gene action (e.g., dominant or recessive alleles). In the exercise concerning phenotypic and genotypic ratios, with each consecutive generation, the proportion of heterozygotes (Gg) is reduced. With continued self-pollination, the heterozygotes will segregate, decreasing the proportion of heterozygotes in the population by half each generation. Notice that the homozygotes can only produce homozygotes. Try This! – Crossing A cross is made between a plant homozygous for green seeds (GG) and a plant homozygous for white seed (gg) — a monohybrid cross. Assume: the species is diploid and normally self-pollinating, and the G allele is completely dominant. By convention, “X” means cross-pollinating, and the “” symbol indicates self-pollinating. At each generation, you will determine and fill in the missing phenotypic and genotypic ratios. You will drag a fraction from the options provided below to its respective empty box. Successive Generations Table 1 Generation Heterozygosity (%) F1 100.0 F2 50. F3 25.0 F4 12.5 F5 6.25 F6 3.12 For each successive generation of offspring resulting from one F1 individual, by the F8 generation, the population is essentially homozygous. When no further segregation for the trait occurs, all progeny derived from that F1 will “breed true” because they are homozygous for the trait. The proportion of plants that are expected to be heterozygous at any gene when starting with a heterozygous F1 and selfing can be determined by using the formula (½)n, where n = the number of segregating generations, e.g., in F2, n = 1 and in F5, n = 4. Using this we get the following proportions of heterozygous plants in F4: (½)n = (½)3 = ⅛ = 12.5%. The proportion of homozygous plants in any generation is then given by 1 − (½)n which, when algebraically converted, is equal to: $\frac{2^n - 1}{2^n}$ Applying this to F4 we get $\frac{2^3-1}{2^3} = \frac{8 - 1}{8} = {7 \over 8} = 87.5\%$ When working with actual genotypes we must remember that in any segregating generation there are two homozygous genotypes and we expect equal quantities of each. Using the example of an F1 that is Aa, in F2, we expect ¼ AA + ½ Aa + ¼ aa. In F4 we expect to be homozygous with half of those AA and half aa. Thus overall we expect the following F4 genotypic frequencies: ${7 \over 16} \textrm{AA} + {1 \over 8} \textrm{aa} + {7 \over 16} \textrm{aa}$ Scenarios under cross-pollination — with and without selection — will be discussed in more detail in the module on Population Genetics. Progeny Test There are two principal procedures that allow the plant breeder to determine the basis of phenotypes (genetic or environmental), gene action, and the genotypes of individual plants. Which procedure is used depends on the specific objectives of the breeder. Progeny Test The progeny test evaluates the genotype of an individual based on the performance of its offspring. The progeny test can be used to: 1. Distinguish heritable phenotypes from phenotypes attributable to environmental effects. 2. Determine the genotype or the allelic composition of an individual. Steps in Progeny Test 1. Hybridize (mate) two plants, A and B. 2. Grow out and self-pollinate the F1 plants. 3. Grow out and self-pollinate F2 plants. 1. Determine the phenotypic ratio of trait(s) of interest. 2. Harvest seed separately from each plant. 4. Plant a portion of the F3 seed from each phenotype separately. 1. Determine the phenotypic ratio in each group—the phenotypic ratio reveals which of the F2 plants were homozygous and which were heterozygous for the trait(s) of interest. 2. Based on the phenotype information, calculate the genotypic ratio. In this example, the phenotypic ratios of the F3 plants reveal the following genotypic information about each of the F2 parents: F2 Parent Genotype a Homozygous red b Heterozygous red c Homozygous green d Heterozygous red Both the red and green phenotypes occur in ratios consistent with those of heritable traits. Thus, there is a genetic basis for these phenotypes (i.e., these phenotypes are not just the result of environmental conditions). Testcross The testcross procedure is used to determine the genotype of an individual or linkage groups. Linkage is a condition in which genes located on the same chromosome are inherited together due to their close proximity. Linkage will be discussed in greater detail in “Linkage” module. Steps in Testcross 1. Hybridize (mate) two plants. The genotype of Parent 1 is unknown, A (?). Parent 2 is homozygous recessive for the trait of interest, aa. 1. Grow out F1 plants and evaluate the phenotypic ratio: 1. If segregating 1:1, then you know that the genotype of Parent 1 was heterozygous, Aa. 1. If all plants have the phenotype of Parent 1, than you know that Parent 1 was homozygous dominant, AA. The backcross is a special type of progeny test. It is a cross of an F1 to either of the original parents. This procedure is used extensively in basic genetic studies but not often used by plant breeders to determine genotypes of plants. Study Questions 3 For each of the following situations, identify which procedure(s) would be most appropriate. Determine Linkage To determine linkage groups, hybridize two plants: • Parent 1 is heterozygous at two (or more) loci. • Parent 2 is homozygous recessive at these loci. The interpretation of the results of this cross will be discussed in the module on Linkage. Genetic Recombination and Its Effects Develop Improved Cultivars To develop improved cultivars, plant breeders usually combine the favorable characteristics of one plant or cultivar with the desirable traits of another plant or cultivar, accumulating desirable alleles for key characters. To obtain an improved genetic combination, breeders make a series of matings, selecting the best offspring to produce the next generation. Plant breeders rely on several genetic mechanisms to obtain new genetic combinations. 1. Segregation — Homologous chromosomes derived from different parents separate and distribute randomly to cells during meiosis. 2. Recombination — Formation of new gene combinations by mating individuals having differing genotypes. Segregation Segregation is the result of the independent assortment or chance distribution of homologous chromosomes and the genes that they carry to gametes. Through meiosis, allelic pairs are separated and distributed to different cells, which subsequently undergo gametogenesis. Genes located on different chromosome pairs assort independently. That is, the chance distribution of a particular chromosome, say one of these green chromosomes, to one cell, has no effect on the distribution of a yellow chromosome. Independent assortment facilitates recombination and leads to segregation in subsequent generations. Recombination Mating two plants possessing different genotypes results in progeny with genotypes that may differ from the parental types. The progeny having genotypes that differ from the parents are referred to as “recombinants.” Try this! Recombination Exercise Mate two plants, one heterozygous and the other homozygous at the G and H loci. Determine all possible gamete types and then all possible genotypes that would result from this mating progeny. Let Parent 1 be the female and Parent 2 be the male parent in this cross. Check each step and make corrections if needed before proceeding to the next step. Table 2 Parent 1   Parent 2 Genotype GgHh X gghh Step 1: Among the following types, select the possible gamete types for the eggs, and drag the 4 appropriate types into the boxes below. Query $10$ Step 2: Among the following types, select the possible gamete types for the sperm, and drag the 4 appropriate type into the box below. Query $11$ Step 3: Fertilization: When gametes fuse, the zygote receives half of its genes from each parent. Given below are all possible combinations of the genotypes. Select the correct combinations and drag them to their respective places on the table. Query $12$ Combination of genes in sperm Combination of genes in eggs gh gh gh gh GH GgHh GgHh GgHh GgHh Gh Gghh Gghh Gghh Gghh gH ggHh ggHh ggHh ggHh gh gghh gghh gghh gghh Step 4: What is the genotypic ratio of these progeny? Query $13$ Combination of genes in sperm Combination of genes in eggs gh gh gh gh GH GgHh GgHh GgHh GgHh Gh Gghh Gghh Gghh Gghh gH ggHh ggHh ggHh ggHh gh gghh gghh gghh gghh Step 5: What is the phenotypic ratio of these progeny? Query $14$ Ratio Genotype 4/16 GgHh 4/16 Gghh 4/16 ggHh 4/16 gghh Step 6: Identify the parental types and recombinants by clicking on the correct button under each example. Query $18$ Study Questions 4 A homozygous plant that was • high yielding (Y_ = high, yy = low), • low in protein (P_ = high, pp = low), • early maturing (E_ = late, ee = early), and • with white flowers (W_ = purple, ww = white) was crossed with a homozygous plant that was low yielding, high in protein, early maturing, and with purple flowers. Option Genotype Phenotype Yield Protein Maturity Flowers A YyPpEeWw High High Late Purple B YyPPeeWw High High Early Purple C YyPpeeWw High High Early Purple D yyPpeeWW Low High Early Purple E YyppEeWw High Low Late Purple F YYPpeeww High High Early White Query $23$ Helpful Hint • 3/4 will be high yielding (Y_) • 3/4 will be high protein (P_) • all will be early maturing (ee) • 1/4 will have white flowers (ww) Let’s verify this by looking at the combinations of genes possible in the gametes. There are eight combinations. YPeW YPew YpeW Ypew yPeW yPew ypeW ypew To ascertain all the genotypes in the F2, we can create a Punnett Square with these eight combinations for the eggs and for the sperm, producing an 8 x 8 table showing 64 combinations in the F2 zygotes. Only those F2 with a Y_P_eeww genotype (indicated with an X in the table below) will have the phenotype: high yielding, high protein, early maturity, and with white flowers. Pollen Eggs YPeW YPew YpeW Ypew yPeW yPew ypeW ypew yPeW YPew   X   X   X   X YpeW Ypew   X       X yPeW yPew   X   X ypeW ypew   X Restrictions with Independent Assortment Hybrid Characteristics A breeder cannot improve a characteristic unless there is some variability for that characteristic within which to make selections. Hybridizing plants differing in their phenotypes (and genotypes) and selecting from among the recombinants provide the breeder with the opportunity to make progress towards crop improvement. However, recombination and segregation may fail to provide the expected variation for two general reasons. • Population size — A minimum of progeny from a cross must be grown out and evaluated. If the number is too small, the likelihood of the desired recombinant occurring in the population is reduced. As the number of independently assorting genes increases, the number of plants that must be evaluated increases exponentially. Thus, an adequate population is essential to make efficient progress towards the breeding goals. The minimum population size required for all genotypes to be represented in the population can be calculated as follows: 1. Determine the number of segregating gene pairs. Let that number equal “n”. 2. Calculate the minimum population size: minimum population size = 4n • Gene Interaction — Although the genes involved in epistatic and pleiotropic interactions may assort independently, their interactions often affect phenotypic and genotypic ratios. • Linkage — As stated earlier, loci in close proximity on the same chromosome tend to be transmitted together and do not assort independently. Genetic Cross-Data When analyzing data from genetic crosses, it is frequently appropriate to use some kind of statistical analysis because such data is often quantitative. One statistical procedure commonly used for testing results of segregation data is called a chi-square (χ2) test. The chi-square test is also known as a “goodness-of-fit” test. Breeders wonder if data support or fit a particular hypothesis and therefore help to explain the results. For example, does the range of phenotypes observed within the progeny of a cross-fit a particular segregation ratio, e.g., 3:1 or 9:3:3:1? The chi-square procedure helps breeders understand the significance of deviation of observed results from results predicted by the hypothesis being tested. A null hypothesis is formed that states there is no real difference between the observed and expected data. If differences are due to chance, then the hypothesis can be accepted, otherwise, the null hypothesis is rejected and the breeder can modify the hypothesis in favor of a better one. The equation used to calculate the (χ2) statistics is as follows $x^2 = \sum \frac{(\textrm{observed} - \textrm{expected})^2}{expected}$ The chi-square procedure will be covered in more detail in the Quantitative Methods course. Gene Interactions Traits When multiple genes control a particular trait or set of traits, gene interactions can occur. Generally, such interactions are detected when genetic ratios deviate from common phenotypic or genotypic proportions. • Pleiotropy — Genes that affect the expression of more than one character • Epistasisepistasis — Genes at different loci interact, affecting the same phenotypic trait. Epistasis occurs whenever two or more loci interact to create new phenotypes. Epistasis also occurs whenever an allele at one locus either masks the effects of alleles at one or more loci or if an allele at one locus modifies the effects of alleles at one or more loci. There are numerous types of epistatic interactions. Epistasis is expressed at the phenotypic level. It is important to note that genes that are involved in an epistatic interaction may still exhibit independent assortment at the genotypic level. The following slides show some examples of epistasis drawn from various types of plants. Duplicate Recessive Epistasis Duplicate recessive epistasis (also known as complementary action): 9:7 ratio observed in flower color of progeny of crosses between a pure line pea plant with purple flowers (genotype CCPP) with a pure line, homozygous recessive plant with white flowers (ccpp). The F1 plants are all purple and have a genotype of CcPp, but the F2 progeny will have a modified ratio of 9:7 because color is only produced if both genes have at least one dominant allele. These genes control flower color by controlling the expression of biochemical compounds known as anthocyanins that impart pigment to the flower. Pigmentation in this case is controlled by a two-step chemical reaction. One of these genes controls the first step and the other controls the second step. Male Gametes CP Cp cP cp Female Gametes CP CCPP Purple CCPp Purple CcPP Purple CcPp Purple Cp CCPp Purple CCpp White CcPp Purple Ccpp White cP CcPP Purple CcPp Purple ccPP White ccPp White cp CcPp Purple Ccpp White ccPp White ccpp White Dominant Epistasis Dominant epistasis (also known as masking action): 12:3:1 ratio observed in fruit color of progeny of crosses of squash. In the F2, fruits are white if the genotypes are either W_G_or W_gg because the dominant allele for the first gene (W) masks the effect of either allele for the other gene (G or g). Color is present only if the first gene is homozygous recessive (ww). Yellow squash have the genotype wwG_ and green ones have the genotype wwgg. Male Gametes Female Gametes   WG Wg wG wg WG WWGG White WWGg White WwGG White WwGg White Wg WWGg White WWgg White WwGg White WwGg Yellow wG WwGG White WwGg White wwGG Yellow wwGg Yellow wg WwGg White Wwgg White wwGg Yellow wwgg Green Duplicate Dominant Epistasis Duplicate dominant epistasis (also known as duplicate action): 15:1 ratio observed in fruit shape of progeny of crosses of the common shepherds purse. If either of the two genes involved in fruit shape (T or V) are present alone or both together (TV), then the plants will all produce triangular-shaped fruit. Only the homozygous recessive genotype (ttvv) produces a seed capsule with an ovate shape. Male Gametes Female Gametes   TV Tv tV tv TV TTVV Triangular TTVv Triangular TtVV Triangular TtVv Triangular Tv TTVv Triangular TTvv Triangular TtVv Triangular Ttvv Triangular tV TtVV Triangular TtVv Triangular ttVV Triangular ttVv Triangular tv TtVv Triangular Ttvv Triangular ttVv Triangular ttvv Ovate Epistasis Identification Identify the type of epistasis that best explains the observed effect.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.04%3A_Gene_Segregation_and_Genetic_Recombination.txt

Introduction Genes located on the same chromosome are genetically linked. Genetic linkage analysis can be used to determine the order of genes on chromosomes. Closely linked genes are not segregating independently, like genes located on different chromosomes. This has different implications, e.g., in relation to trait correlations. Moreover, linked genes can be used as genetic markers, which have become an important tool in plant breeding. Learning Objectives • Develop an understanding of the genetic basis of linkage. • Gain awareness on how to detect the occurrence of linkage. • Review the principles of genetic map construction. • Become familiar with the concept of linkage disequilibrium. Crossover and Recombination Genetic Organization Genes are physically organized on chromosomes. Each gene is located at a particular “address” (particular position on a specific chromosome, which can be identified by genetic mapping). Inheritance of genes located on different chromosomes follows the rules of independent assortment. Since plant species have multiple chromosomes, independent assortment is true for the majority of genes. In contrast, linked genes located on the same chromosome are more likely to cosegregate, i.e., being jointly transmitted to offspring more often than expected by independent assortment. The biological process that separates linked genes is the crossing-over (C.O., or crossover), which occurs during meiosis, and leads to genetic recombination. Crossing-Over Query $1$ During meiosis of diploid organisms, the chromatids of homologous chromosomes pair and form bivalents. During Meiosis I, homologous chromatids pair to physically exchange chromosome segments. The chromosomal site, where this reciprocal exchange of homologous chromosome segments takes place, is called a chiasma. Thus, crossing-over involves not completely understood mechanisms for identification of homologous sites of chromatids, breakage and rejoining of chromosomes. Genetic Distance Crossing-over events occur more or less random during meiosis. In most plant species, one to few crossing-over events occur per meiosis and chromosome. Thus, the closer the genes are physically linked on the same chromosome, the less likely they will get separated, and consequently, the less likely genetically recombinant gametes will be produced. This is the underlying principle of genetic maps: the genetic distance between genes reflects the probability of a crossing-over between linked genes. Recombination Observation of crossing-over events requires cytological methods, which can be cumbersome for large populations. In contrast, genetic recombinants can be observed at the phenotype level, or by use of DNA markers. If two linked genes with two alleles each have clear phenotypic effects, e.g., on flower color (A: red, a: white; A is dominant over a) and seed color (B: green, b: yellow; B is dominant over b), then genetic recombinants can easily be identified by determining the fraction of non-parental gametes in the offspring. Note that crossing-over also takes place in meiosis of completely homozygous individuals. However, in this case, genetic recombination cannot be observed as described above. The reason is that observation of recombinant gametes requires two (or more) different alleles at the loci, for which linkage is going to be determined. This explains why offspring saved from pure line cultivars will not segregate whereas seed harvested from F1 hybrid will segregate. The observable fraction of recombination events is also called effective recombination. Linkage Detection Linkage Phase For linkage detection, it is crucial to know the linkage phase of alleles. The linkage phase is the physical arrangement of linked genes in a chromosome. A double heterozygote with a genotype of AaBb could be in one of the two linkage phases. Conventionally, when linked dominant alleles are located on the same homologous chromosome and the linked recessive alleles are on the other homologous chromosome, for example, AB/ab, it is said the genes are linked in the coupling phase. When a dominant allele at one locus is on the same homologous chromosome as a recessive allele of the other linked gene, for example, Ab/aB, it is said that the genes are linked in repulsion phase (Figure 4). This knowledge is crucial, as linkage detection and distance estimation is based on the observed parental and non-parental gametes. Coupling and Repulsion In the case of close linkage, non-parental gametes and respective offspring are underrepresented. An example is the Australian sheep blowfly, Lucilia cuprina. Normal blowflies have a green thorax and surround themselves in a brown cocoon during their pupal stage. However, recessive genes (here marked a and b) can cause the fly to develop a purple thorax and spin a black puparium. Using Testcrosses For detection of linkage, appropriate testcrosses need to be conducted. The linkage phase is known, if two homozygous parental genotypes (AABB and aabb) are crossed to produce the respective F1 (AaBb). In this case, A and B as well as a and b are linked in coupling phase. The non-parental recombinant gametes have the genotype Ab and aB, whereas the parental gametes have the genotype AB and ab. Usually the phenotype cannot be observed in (haploid) gametes, but only in diploid plants. Thus, to determine whether two loci are linked, offspring need to be produced. This can be achieved by self pollination of the AaBb – F1, by production of doubled haploid offspring, or by a testcross. In this particular example, a backcross (BC) of the F1 to the aabb parent would be the best option. Testcross Gametes All offspring from this BC would receive an ab gamete from the aabb parent, and any of the two parental (AB, ab) or non-parental (Ab, aB) gametes from the F1. Because of dominance of A over a and B over b, all four resulting diploid genotypes in the BC1 (backcross generation 1) generation (AaBb, Aabb, aaBb, aabb) can be phenotypically discriminated, and used to count genotypes that received parental or non-parental gametes from the F1. Thus, when using this BC approach, only the crossing-over events that occurred in the F1 are monitored for linkage estimation. Chi-Square Test For detection of linkage, a Chi-Square test can be employed. The Chi-Square test compares observed with expected frequencies. In this case, the null hypothesis to determine expected frequencies is the assumption of independent assortment. Under this assumption, equal frequencies of all four gametes are expected. In the case of linkage, BC1 individuals carrying non-parental gametes are underrepresented, leading to a statistically significant Chi-Square value. This means that the null hypothesis of independent assortment would be rejected and linkage assumed. Table 1 An example of the detection of linkage in Drosophila melanogaster using a Chi-Square test. d: difference between the observed number and expected number. The significantly higher Chi-Square values reject the null hypothesis and strongly indicate the presence of linkage. Phenotypes Observed Number (o) Expected Number (e) d (o – e) d2 d2 / e Parentals: (black-bodied and normal wing plus grey-bodied, vestigial wing) 2,712 1,618 1,094 1,196,836 739.7 Recombinants: (black-bodied and vestigial wing plus grey-bodied and normal wing) 524 1,618 1,094 1,196,836 739.7 Chi-Square Results To better understand the use of Chi-Square in determining linkage, two numerical examples based on the cross schemes described in Figs. 8 and 9 are provided here. In both examples, a sample size of 2,000 BC1 individuals has been used. The Chi-Square test sums up over all squared differences between observed and expected values, divided by expected values. In example A, observed and expected values are equal, thus the Chi-Square value = 0. In example B, the squared differences between observed and expected values is in all cases 90,000, to be divided by the expected 500 = 180. As there are four genotypic classes, the Chi-Square value is 720, which is significantly larger than the tabulated value of 3.81 (p = 5%). In conclusion, example A is in agreement with independent assortment, whereas in example B, linkage has been detected. In conclusion, example A is in agreement with independent assortment, whereas in example B, linkage has been detected. Genetic Distance The same data used to determine linkage can also be used to estimate the recombination frequency between two genes (more precisely, the recombinant frequency). The recombinant frequency = (number of BC1 progeny with recombinant (non-parental) alleles / total number of BC1 progeny) x 100%. In example B of Section 2: Linkage Detection, the recombinant frequency is (200 + 200 / 2000) * 100% = 20%. The recombination frequencies between any pairs of genes provide an estimate of how close they are linked on a chromosome. The recombinant frequency in % is sometimes also called “map units” (M.U.). In this example, the genetic distance in map units between the two genes under consideration is 20 M.U. In the case of complete linkage of two genes, no recombinants would be expected. The recombinant frequency would be 0%, which represents the lower limit of recombinant frequencies. In the case of random segregation, the expected numbers of recombinant and nonrecombinant alleles are equal. Thus, the upper limit of recombinant frequencies in the case of unlinked or loosely linked genes is 50%. Even for gene pairs located at the different ends of the same chromosome, recombination frequency can reach 50%. The procedure to determine recombination frequencies between any pair of genes is called two-point analysis. Study Question 1 You have a F1 plant heterozygous at two loci that are 12 map units apart on the same chromosome. The F1 received linked recessive alleles from one parent and linked dominant alleles from the other parent. Query $4$ Study Question 2 You have a F1 plant heterozygous at two loci that are 12 map units apart on the same chromosome. The F1 received linked recessive alleles from one parent and linked dominant alleles from the other parent. Since linkage cannot be detected in the F1, you self-pollinate the F1 and evaluate the F2. What would be the F2 and testcross percentages if the F1 percentages in the case of repulsion phase of the recessive alleles? Three-Point Analysis Purpose Whereas two-point testcrosses establish linkage between pairs of genes, three-point testcrosses facilitate establishment of the order of genes on chromosomes, as a prerequisite to establishing genetic maps. If a third locus with alleles C and c (C is dominant over c) is added to the case mentioned in Genetic Distance, where A and B are linked in coupling phase and the dominant allele C is in coupling with A and B, then eight different testcross progeny would result from a backcross with the recessive parent. Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 Frequency Chart Pairwise recombination frequencies can be determined as described in the chart. • 20.8% for AC (AC recombinants are in classes 3, 4, 7, and 8; thus, the recombination rate between A and C is (52+46+4+2/500) * 100% = 20.8%) • 10% for CB (CB recombinants are in classes 5-8) • 28.4% for AB (AB recombinants are in classes 3-6). Once linkage between pairs of three (or more) genes has been established, the next question is how they are arranged in linear order on chromosomes, which could be ABC, ACB, or CAB. Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 Gene Order The most likely gene order minimizes the sum of pairwise recombination frequencies within a three-gene interval, which would be: • 38.4 for ABC (28.4% for AB + 10% for CB) • 30.8 for ACB (20.8% for AC + 10% for CB) • 49.2 for CAB (20.8% for AC + 28.4% for AB) Thus, the most likely gene order is ACB. In other words, the interval between A and B can be subdivided into the intervals between AC and CB. Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 Expressed yet another way: incorrectly ordered genes would increase the total map length because part of the recombination events would be counted twice. If ACB is the true order, then the genetic length of, e.g., ABC would be inflated, because recombinants for the segment BC would be counted two times: for the interval BC, in addition to the same interval within the segment A(C)B. Algorithms of mapping programs use this principle (minimizing the genetic distance) for three-point analyses. Double Crossovers The two-point recombination frequency between A and B (28.4%) differs from the sum of recombination frequencies for AC and CB (30.8%). The reason for this discrepancy is the occurrence of double crossover events. These are two crossovers in a single meiosis within an interval of interest and the second crossover reverses the effect of the first crossover, i.e., the second crossover returns the B allele to the original position before the first crossover. For this reason, by only taking recombinants between A and B into consideration, double crossovers cannot be observed. Because a double crossover exchanges chromosome segments within an interval of two genes, the linkage phase (coupling) of those two genes remains unchanged. Observing Double Crossovers Only by adding a gene like C in between A and B, it is possible to observe double crossovers. In the case of the interval between genes A and B, six double crossovers were observed. In consequence, recombination and crossover frequencies are not identical. The larger the genetic interval, the larger the discrepancy between recombination and crossover frequencies, because even-numbered crossover events within a pair of genes go undetected. By adding an additional gene in this interval, at least some double crossovers can be detected. This leads to detection of additional recombination events. For this reason, the recombination frequency between A and B in the example is increased, after adding C in between those two genes, because 6 double crossovers (= 12 additional recombination events) could be detected. Those 12 additional detectable recombination events explain for the 2.4% difference between recombination frequencies detected for the gene pair A and B with or without inclusion of C. Phase Analysis Recombinants resulting from double crossovers are always in the lowest frequency (class 7 and 8, respectively in this table). To determine which allele is in the middle, a convenient method is to find out which allele in the double crossover recombinants has changed its linkage phase with the other parental alleles (in classes 7 and 8, allele C/c has changed its linkage phase with the other alleles). Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 Coefficient of Coincidence and Interference Crossover events in adjacent chromosome regions might affect each other, a phenomenon called interference. Most typically, a crossover event in one region tends to suppress a crossover in the adjacent regions. The extent of interference is expressed by the coefficient of coincidence, which is equal to the observed frequency of double crossovers / expected frequency of double crossovers. Class Genotype of gamete from heterozygous parent Number Origins 1 A C B 179 } Parentals, no crossover 2 a c b 173 3 A c b 52 } Recombinants, single crossover AC 4 a C B 46 5 A C b 22 } Recombinants, single crossover CB 6 a c B 22 7 A c B 4 } Recombinants, double crossover AC, CB 8 a C b 2 The expected frequency of double crossovers is the product of two single crossovers in adjacent regions assuming there is no interference. In this example, this expected frequency is 0.21 (recombination frequency for AC) * 0.10 (recombination frequency for CB) = 0.021. The observed frequency of double crossover events is 6/500 in the example, resulting in 0.012. Thus, the coefficient of coincidence in this example is 0.012/0.021 = 0.58. Interference is defined as 1 − coefficient of coincidence, which would be 0.42 in this example. A value of zero for interference would mean that a crossover in one region does not affect crossovers in the adjacent region. Interference of 1 means, that crossovers in one region suppress crossovers in the adjacent region. Negative values are possible and have been reported in some instances, which means that crossovers in one region stimulate crossovers in the adjacent region. Map Functions Measurement Units The purpose of genetic maps is to report the length of chromosome intervals, chromosomes, and whole genomes. Since recombination frequencies converge to a value of 50% as reported above, indicating the absence of linkage, recombination frequencies are not additive and, thus, not useful to describe the distance between genes that are located far apart. When recombination frequency reaches 50%, it would be impossible to tell whether the genes are located far apart on the same chromosome or on different chromosomes. Instead, estimates of the number of crossover events are used as an additive measure of genetic map distances. The unit for measuring genetic distances is Morgan (M), or usually centiMorgan (cM). In contrast to recombination frequencies, map units expressed in cM are additive. One Morgan reflects the observation of one crossover event per single meiosis. One cM is a distance between genes that produces 1% recombinants in the offspring. Typical lengths of genetic maps in maize, for example, vary between 1,600 to 2,000 cM, which means that on average, 1.6 – 2 crossovers occur per chromosome and single meiosis in maize (maize has 10 homologous chromosome pairs). Frequency Conversion As mentioned above, direct observation of crossover events is cumbersome. For that reason, most genetic maps published to date are based on the conversion of recombination frequencies into crossover frequencies. The main obstacle to translating recombination frequencies into crossover frequencies is the variable and unknown degree of interference in different genome regions. While it has been possible in the earlier example to determine the degree of interference, and thus frequency, of double crossover events, in the genetic interval between A and B by adding C, the degree of interference between AC and CB is unknown. This could be addressed by observing segregation of further genes within these two regions (if available), but this issue could ultimately only be addressed by complete genome sequencing of all offspring in a mapping population, which at this point is still too costly. Visual Relationship Instead, map functions have been developed, that translate recombination frequencies into crossover frequencies, and thus cM (see Figure 14 below). Figure 14 clearly shows, that there is an approximately linear relationship between recombination rates (y-axis) and crossover rates (x-axis). However, with increasing map distances, recombination rates converge to 50%. In other words, gene pairs with crossover rates of 80 cM or 200 cM, respectively, would be nearly indistinguishable based on recombination rates, which would result in recombination rates between 40 and 50%. The various available map functions make different assumptions on the extent of interference. For example, the Haldane mapping function assumes absence of interference. In contrast, the Kosambi function assumes the presence of interference. Other Types of Maps Genetic maps can be generated in other ways than using testcrosses. Examples include somatic cell hybridization and tetrad analysis. In plants, interspecies addition lines such as oat-maize addition lines created by distant hybridization have been developed as tool for mapping of genes. If two genes appear on the same addition segment, they are genetically linked. Besides genetic maps, cytological and physical maps can be established. Cytological maps show gene orders along each chromosome as determined by cytological methods whereas physical maps are measured in base pairs as determined by DNA sequencing. With rapid progress in sequencing technology and an increasing number of sequenced plant genomes, physical maps gain in importance. Complex plant genomes like the maize genome are billions of base pairs long. When comparing genetic and physical maps, the order of genes is conserved. However, the relative distances between genetic and physical maps might vary substantially. The reason is that crossover events are not evenly distributed in genomes. Usually, crossover events tend to be suppressed in centromere and repetitive DNA regions, whereas they are enhanced in gene-rich regions. Factors Influencing Linkage Mapping Linkage mapping based on testcrosses can be affected by selection or incomplete penetrance, among others. Selection in the most extreme case would be due to lethality of gametes (gametic selection) or zygotes (zygotic selection). If a backcross is used for linkage detection, as described above, lethality of male gametes carrying for example the a allele would lead to only two classes of BC progeny, if AaBb is crossed as pollinator to aabb. In that case, only AaBb (parental) and Aabb (recombinant) genotypes would be obtained. Zygotic selection affects the viability of particular genotypes. If the aa genotype in the example above is lethal, then the aa offspring derived from self-pollination of an AaBb genotype would be missing. Incomplete penetrance means that a genotype that is supposed to express, for example, red flowers, has to a certain extent white flowers. In other words, there is no 100% match between genotype and phenotype, but due to environmental factors, the phenotype might differ. As for selection, incomplete penetrance alters the frequency of expected genotypes in testcrosses, which is the basis for detection of linkage. Consequences and Applications of Linkage The main application of linkage is in genetic mapping of genes using molecular markers. Once genes have been mapped and closely-linked markers identified, those markers can be used for marker-aided selection procedures. Technological progress in DNA methods has been and still is rapid, so that thousands of markers can be produced at low cost in any species of interest. Moreover, novel genomic selection strategies addressing complex inherited traits are being developed. Linkage can in some cases be confused with pleiotropy. If a favorable character (e.g. resistance) is always inherited together with an unfavorable trait (e.g., lodging), a negative pleiotropic effect might be assumed, which might alternatively be caused by two closely linked genes. Whereas close linkage can be resolved to find favorable genotypes for both traits, this is not true for pleiotropy. Linkage reduces the possible genetic variation in small populations. With increasing numbers of generations, or population sizes, genetic variation can be increased. Similarly, inbreeding reduces the opportunity for effective recombination. Linkage Disequilibrium Genotype Distribution Although allele frequencies at individual loci are expected to be stable in the case of random mating, genotype frequencies at two or more loci jointly do not achieve this equilibrium after one generation of random mating. To illustrate this point, consider two populations, one consisting of entirely AABB genotypes and the other consisting entirely of aabb genotypes. Assumed they are mixed equally and allowed to randomly mate. The first generation would consist of the three genotypes AABB, AaBb, and aabb in the proportions 1/4 : 1/2 : 1/4. However, for two loci, each with two alleles, nine genotypes are possible. (For n alleles at each locus and k loci, there are: $(\frac{n(n+1)}{2})^k$ possible genotypes). Continued random mating would produce the missing genotypes, but they would not appear at the equilibrium frequencies immediately. Equilibrium Consider the following examples based on two alleles at each of two loci: • Alleles: A a B b • Allele frequencies: PA Pa PB Pb • Gametic Types: AB Ab aB ab • Gametic Frequencies: PAB PAb PaB Pab In linkage equilibrium, the expected gamete frequencies can be calculated from the marginal allele frequencies. For example, in equilibrium, the frequency of gamete AB (PAB) would be expected to be equal to the product of the frequencies of the A allele (PA) and the B allele (PB). This is valid under the following conditions: PA + Pa = 1; PB + Pb = 1; and PAB + PAb + PaB + Pab = 1. If, for example, the allele frequencies of PA = Pa and PB = Pb are 0.5, then the frequencies of all gametes are 0.25. A measure for Disequilibrium, D = PAB – PA*PB. D = 0 in the case of equilibrium. If D differs from 0, it reflects presence of Disequilibrium. In other words, the frequency of a gamete differs from its expected frequency based on marginal probabilities of the respective individual alleles. LD and Mapping Linkage disequilibrium is the non-random association of alleles at different loci. LD is extensively used in mapping human disease genes using natural populations (Association mapping). In plants, gene mapping has been conducted mainly by using mapping families because of the ease with which mapping families are created, but LD mapping using natural populations is increasing rapidly because such populations are large in size and have much greater allelic diversity. LD Statistic D’ |D’| = $\frac{D^2_{AB}}{min (p_Ap_b,p_Ap_b)}$ for DAB < 0 |D’| = $\frac{D^2_{AB}}{min (p_Ap_b,p_Ap_B)}$ for DAB > 0 Dissipation It can be shown that after t generations of random mating, the remaining disequilibrium is given by: $D_t = D_0 (1 - c)^t$ where, D0 is the disequilibrium in generation 0 and c is the recombination fraction, with c = 0.5 for independently segregating loci, which is identical to a recombination frequency of 50% (the range of c is from 0 to 0.5, whereas the range of r is from 0% to 50%). The dissipation of disequilibrium relative to generation 0 is given in Figure 18. Recombination and LD Generally, deviations from independence at multiple loci are referred to as linkage disequilibrium, even if genetic linkage is not the cause (in other words, alleles are not physically linked). Unless two loci are known to reside on the same chromosome, the term Gametic Disequilibrium should be used to describe disequilibrium among loci. Whereas recombination and crossover frequencies, as mentioned initially, are used to describe the distance between genes from a chromosomal perspective, linkage disequilibrium is mostly used to describe a property of populations. However, both terms are closely related. Genetic Markers Overview Genetic variation results from differences in DNA sequences and, within a population, occurs when there is more than one allele present at a given locus. Such populations are referred to as populations that are polymorphic or segregating at that locus. The opposite situation is when all members of the population are homozygous for the same allele, in which case the population is said to be fixed or monomorphic for that allele. A genetic marker is a DNA sequence that exhibits polymorphism among individuals and can thus be used to identify a particular locus (although not necessarily a gene) on a particular chromosome; the marker itself may be part of a gene or may have no known function. Markers are inherited in a Mendelian fashion and facilitate the study of inheritance of a trait or sometimes a linked gene. Markers are used to identify, map, and isolate genes, select desired genotypes, and detect genetic variation or determine genetic relationships among individuals. Markers are regions of genomes that are heritable, often easy to document, and useful for detecting genetic variation. Three Types Genetic markers generally do not represent target genes of interest to a breeding program, but instead are useful as ‘signs’ or ‘tags’, particularly when they are closely linked to genes that control a trait of interest. A genetic map constructed with genetic markers is similar to a road map. Linkage groups in a genetic map represent roads whereas individual markers on each linkage group represent signs or landmarks that help plant breeders to navigate through the plant genome and find the genes of interest. There are three major categories of markers. • Morphological markers • Biochemical markers • Molecular markers Morphological Markers These types of markers (also called visible or classical markers) are phenotypic traits with only a few distinct morphs or variants (e.g., flower color or seed shape), usually due to one or perhaps two gene loci so they are not strongly affected by the environment. Inheritance patterns of visible and morphological characters have been used to map genes to particular chromosome segments and to identify linkage groups. Such markers are limited in number compared to the abundance of DNA markers, however, and may be influenced by developmental stage of the plant. Biological Markers Isozymes (sometimes called allozymes) are allelic variants of a single enzyme that share the same function, but may differ in level of activity due to differences in amino acid sequence. Isozymes are proteins for which variation can be detected by differential separation using electrophoresis, a technique for separating macromolecules (DNA, RNA, protein) on a gel by means of an electric field and specific chemical staining. Isozymes have codominant expression, meaning that both homozygotes can be distinguished from the heterozygote and neither allele is recessive. In contrast to codominant markers, dominant markers are either present or absent. In comparison to visible polymorphisms, they reveal more of the underlying genetic variation. However isozymes are gene products, so they reveal only a small subset of the actual variation in DNA sequences between individuals and do not reveal variation in the non-coding regions of the genome. In general, such markers are limited in number and have limited use in genetic mapping studies. Molecular Markers Molecular or DNA markers reveal sites of variation in DNA. Variability in DNA facilitates finer scale mapping and detection. Mapping is the process of making a representative diagram cataloging genes and other features of a chromosome and showing their relative positions. Many of these molecular markers avoid the limitations associated with visible and biochemical markers. They facilitate the evaluation of genome-wide coverage and are not affected by environmental factors or developmental stages. They allow high resolution of genetic diversity to be detected. Molecular markers have added substantial amounts of information to our genetic maps. FYI: Molecular Markers Any DNA sequence can be genetically mapped, like genes leading to plant phenotypes as long as there is a polymorphism available for the sequence to be mapped, i.e., two or more different alleles. This can basically be a single nucleotide polymorphism (SNP), a single nucleotide variant at a particular position within the target sequence, or an insertion/deletion (INDEL) polymorphism. Any target sequence can be amplified by the Polymerase chain reaction (PCR), and subsequently be visualized to generate “molecular phenotypes” comparable to visual phenotypes, that can be observed by using appropriate equipment. Various molecular methods have been developed to visualize SNPs or INDEL polymorphisms at low cost and high throughput, which will be presented in detail in the Molecular Genetics and Biotechnology course. The main use of those SNPs and INDEL polymorphisms is as molecular markers. By genetic mapping as described above, linkage between genes affecting agronomic traits or morphological characters, and DNA-based SNP or INDEL markers can be established. It can be more effective in the context of plant breeding, to select indirectly for such DNA markers, than directly for target genes. This is due to lower costs for DNA analyses, the ability to run multiple such assays (for multiple target genes) in parallel, the ability to select early and to discard undesirable genotypes or to perform selection before flowering, codominant inheritance of markers, among others.For example, both ginkgo trees (Ginkgo biloba) and asparagus (Asparagus officinalis) are dioecious species. Male plants are preferred for ginkgo tree because fruits produced from female trees have an unpleasant smell whereas male asparagus plants are preferred because of their higher yield potential. Unfortunately, sex expression will take years to occur for both species. If a DNA marker that either directly affects sex expression or is linked to genes that affect sex expression can be identified, selection of male plants can be conducted in early seedling stage rather than waiting for many years. Occurrence of environmental conditions favoring selection for disease, insect-resistant plants or drought-tolerant plants such as the prevalence of the particular disease or insect or drought is not always reliable. Selection using DNA markers can overcome these limitations as they are not affected by the environment. Polymorphism Polymorphism involves one of two or more variants of a particular DNA sequence. The most common type of polymorphism involves variation at a single base pair, also called single nucleotide polymorphism (SNP) (Figure 22). Polymorphisms can also be much larger in size and involve long stretches of DNA. Tandem repeat is a sequence of two or more DNA base pairs that is repeated in such a way that the repeats are generally associated with non-coding DNA. In contrast, SNPs can sometimes be identified that occur within coding sequences (that is within genes), as well as in non-coding DNA. Types of Biochemical/Molecular Markers There are a variety of biochemical and molecular markers available. Table 2 on the next page summarizes features of a number of the common ones: • RFLP — Restriction Fragment Length Polymorphisms • RAPD — Random Amplified Polymorphic DNA • AFLP — Amplified Fragment Length Polymorphisms • SSR — Simple Sequence Repeats (also known as microsatellites) • SNP — Single Nucleotide Polymorphisms • VNTR — Variable Number of Tandem Repeats Widely-Used Markers Table 2. Comparison among widely used molecular markers. Adapted from Nageswara-Rao and Soneji, 2008. Isozymes RFLP RAPD AFLP SSR SNP Protein- or DNA-based Protein-based DNA-based DNA-based DNA-based DNA-based DNA-based No. of loci 30-50 100s ~Unlimited ~Unlimited 10s 10s Degree of polymorphism Low-medium Meduim-high Medium-high Medium-high High High Nature of gene action Codominant Codominant Dominant Dominant Codominant Codominant Reproducibility High High Low-medium Medium-high High High Amount of DNA per sample Not applicable mg ng ng ng ng Method* Biochemical DNA-DNA hybridization PCR PCR PCR PCR Ease of array? Easy Difficult Easy Moderate Easy-moderate Easy Can be automated? Difficult Difficult Yes Yes Yes Yes Equipment cost Inexpensive Expensive Moderate Expensive Expensive Expensive Development cost Inexpensive Expensive Moderate Expensive Very Expensive Assay cost Inexpensive Expensive Moderate Expensive expensive Expensive * ‘PCR’ means Polymerase Chain Reaction amplification of genomic DNA fragments, a method that uses short, single-stranded DNA sequences, known as primers, to hybridize with the sample DNA Table 2. Comparison among widely used molecular markers. Adapted from Nageswara-Rao and Soneji, 2008. SSR and SNP Markers SSR markers remain useful to plant breeders due to their abundance and convenience with which they are assessed, but they serve most likely as linked markers. SNP markers, however can either be linked to or directly reside in a gene of interest and are hugely abundant. For these reasons, they are increasingly becoming the marker of choice. Uses of Molecular Markers Molecular markers are useful for both applied and basic genetic research. Here are some examples: Indirect selection criteria in breeding programs (marker-assisted selection) This is one of the most important and widely used molecular techniques in applied plant breeding programs today. RFLPs, SSRs, and SNPs enable breeders to indirectly select for a desired trait. Ordinarily, the DNA sequence of a molecular marker does not itself code the gene for the trait, but rather, its presence is correlated or linked with the gene for the particular trait. Thus, the breeder can indirectly select for the trait by directly selecting for the molecular marker—the DNA fragment and the gene encoding the trait are linked. The closer their physical proximity on a chromosome, the greater the probability that they will remain linked and not be separated through a recombination event in subsequent generations. As long as the gene for the trait and the marker remain linked, the marker is a useful selection criterion. Ultimately, however, potential lines still must be field-tested to verify the expression of the desired phenotype. Identify quantitative trait loci (QTLs) Molecular markers linked to genes contributing to the expression of polygenic or quantitative traits can be used to more efficiently identify and select individuals possessing the genes. It is more difficult to use conventional breeding approaches to identify plants that have accumulated the genes necessary to obtain the desired quantitative trait. Genetic mapping Molecular markers provide a means to map genes to more specific chromosome segments than is possible using visible markers. Determine genetic relationships The more molecular markers individuals have in common, the more closely related they are. Genetic Diversity and Conservation Genetic relationships within families, genera, species, or cultivars can be determined from molecular markers. Much information about the evolution of crops has been learned using molecular markers. The markers also enable breeders to monitor the genetic diversity among breeding lines to broaden the genetic base and reduce the risk of widespread genetic vulnerability to detrimental conditions. Molecular Marker ‘Fingerprints’ Individuals possessing more markers in common than could occur by random chance are closely related. Such molecular fingerprints have been used successfully in court to prove the misappropriation of proprietary breeding lines. Isolate genes Molecular markers are used to map candidate genes on a much finer scale and can eventually isolate candidate genes by positional cloning. Isolated genes can be used to study gene regulation or to directly improve agronomic performance by genetic transformation. Although molecular markers have many applications and provide useful tools to plant breeders, lines must still be evaluated under normal production conditions before their release.

textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.05%3A_Linkage.txt

"Introduction\n\nPopulation genetics is a sub-discipline of genetics that characterizes the structur(...TRUNCATED)

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"Introduction\n\nThis module focuses on inbreeding, a type of mating of individuals that is often of(...TRUNCATED)

"textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.07%3A_Inbreeding_and_H(...TRUNCATED)

"Introduction\n\nMany of the traits that plant breeders strive to improve are quantitatively inherit(...TRUNCATED)

"textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.08%3A_Inheritance_of_Q(...TRUNCATED)

"Introduction\n\nMutations are the ultimate source of all genetic variation. Mutations can occur at (...TRUNCATED)

"textbooks/bio/Agriculture_and_Horticulture/Crop_Genetics_(Suza_and_Lamkey)/1.09%3A_Mutations_and_Va(...TRUNCATED)

"Introduction\n\nNot all plant species are diploids. In fact, 75% of all angiosperms are polyploids,(...TRUNCATED)

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