1 The Science of Plants in Agriculture Plant Science 102 Study Notes for Test #3 Genes, Mendel and Meiosis What is a gene? A gene is a DNA sequence coding for a single polypeptide, t-RNA or r-RNA. The central dogma of genetics: DNA RNA Nucleic acid Nucleic acid protein Amino acid What are the components of a gene? Components of a gene include the specific gene sequence and a promoter region. Genetic variation is due to changes (mutations) in either the specific gene sequence or the promoter region. What are alleles? Different forms of a gene are called alleles. What is the difference between homozygous and heterozygous alleles? A plant can be either homozygous or heterozygous for a specific gene. It is common to use letters to symbolize genes, and upper and lower letters to distinguish different alleles (i.e. A or a). Homozygous refers to a situation where all possible copies (usually two) are the same (i.e. AA or aa). Heterozygous refers to a situation where there are different alleles of a gene (i.e. Aa). What is the difference between a recessive allele and a dominant allele? If phenotypic expression is determined by only one copy of an allele (i.e. Aa) the allele (A) is said to be dominant over the resessive allele (a). What are the two divisions of meiosis? Reduction division (Meiosis I): is the separation of homologous chromosomes. This is the division is identical to mitosis and produces diploid cells. Equational division (Meiosis II): is the separation of sister chromosomes. This results in producing four cells, having half the number of chromosomes as the original cell so results in haploid cells. What are Mendel’s two laws of genetics? 1 The law of segregation describes where a pair of different alleles (i.e. Aa) for a given gene segregates with equal frequency. So there would be an equal frequency of A gametes (haploid cells resulting from meiosis) as there would be a gametes. 2 The law of independent assortment where allelic pairs of genes will segregate independently. So if a plant is heterozygous for two trails (AaBb) will have equal frequency of the four possible gametes after meiosis. What would the gametes be? AB, Ab, aB, and ab. 2 Plant Breeding What is the difference between a self-pollinating species and an out-pollinating species: Self-pollinators are tolerant to inbreeding; have few deleterious recessive alleles; show little heterosis (hybrid advantage); and tend to have closed flowers to promote self fertilization. Out-pollinators are intolerant to inbreeding (and show a decline in performance when self pollinated); have many recessive deleterious alleles; show high heterosis; and have open flowers or other systems to promote cross fertilization. What types of cultivar can be developed? Pure line (or homozygous) cultivar; outpollinating cultivar; hybrid cultivar; hybrid cultivar. What are the major characteristics of species from which the following cultivar types are developed? And, provide an example crop where that cultivar type is developed. Cultivar type Characteristics Pure line Self-pollinating mating system, annual seed production, able to develop homozygous lines. Out-pollinating Hybrid Clone Out-pollinating species, annual, biennial or perennial, highly heterozygous and heterogeneous. Out-pollinating, annual or biennial, must show high heterosis, usually must have system of hybrid seed production. Out-pollinator, highly heterozygous and homogeneous, must allow asexual propagation. Crops wheat, barley, pea, canola alfalfa, corn, red clover corn, tomato, onion, sunflower potato, apple, bulbs, strawberry What factors would you consider when designing a breeding scheme? People, politics, economics, future trends in agriculture, diseases and other pests. How can breeders develop crops with improved profitability? By (1) increasing harvestable product over the same area; (2) increase the inherent quality of the harvested product; or (2) reduce the costs of production (i.e. improved disease resistance so avoid pesticide costs) while maintaining high yield and quality. What are the two operations in cultivar development? (1) Produce (or identify) genetic variability, and (2) select desirable recombinants (lines) from the genetically variable population. 3 How do breeders produce genetic variability? (1) Artificial hybridization between chosen parents; (2) induced mutation; (3) Interspecific (wide) crosses; (4) genetic engineering. What are most commonly used mutagens in plant breeding? Chemical, most commonly ethyl-methane-sulphonate (EMS), or radiation (X-rays or gamma rays). What types of mutation can occur? Genome mutation: which are changes in chromosome number due to addition or loss of whole chromosomes. Structural changes in chromosome: including translocations, inversions, deletions and duplications. Gene mutation (point mutation): that results in a change in a single gene. Extra nuclear mutation: which is a mutation in one of the cytoplasmic organelles. What would be the genotypic and phenotypic segregation in the F2 generation after a cross between a tall two-row barley (TTss) and a short six-row barley (ttSS), given that the tall gene (T) gene is dominant to the short gene (t) gene and the siz-row gene (S) is dominant to the two-row gene (s)? tall, 2-row (TTss) x dwarf, 6-row (ttSS) When this cross is made, the F1 shows both dominant characteristics, tall and 6-row. The F1 individuals produce equal frequencies of four kinds of gametes during meiosis (Mendel’s second law – above) (TS, Ts, tS, and ts). An easy way to illustrate the possible combination of F2 progeny is using a punnet square, where the four gamete types from one parent are listed in a row along the top, and the four kinds from the other parent are listed in a column down the left hand side. The 16 possible genotype combinations are then obtained by filling in the square, i.e.: Gametes from female parent TS Ts tS ts TS TSTS TsTS tSTS tsTS Gametes from male parent Ts tS TSTs TStS TsTs TstS tSTs tStS tsTs tstS ts TSts Tsts tSts tsts This also can be written as: Gametes from female parent TS Ts tS ts TS TTSS TTSs TtSS TtSs Gametes from male parent Ts tS TTSs TtSS TTss TtSs TtSs ttSS Ttss ttSs ts TtSs Ttss ttSs ttss 4 Collecting the like genotypes we get the following frequency of genotypes: TTSS ; 2TTSs ; TTss ; 2 TtSS; 4 TtSs; 2 Ttss; ttSS; 2 ttSs; ttss, and with the frequency of phenotypes: 9 3 3 1 T_S_ T_ss ttS_ ttss = tall and 6-row = tall and 2-row = short and 6-row = short and 2-row. If a plant breeder makes a cross between a tall homozygous barley parent (TT) and a short homozygous (dwarf) barley parent (tt). Where the tall gene (T) is completely dominant over the recessive dwarf gene (t). If the first generation (F1) is self pollinated and 800 second generation (F2) plants were grown out in the field. At harvest all the dwarf plants (short) are discarded and a single seed from each of the tall selected plants were grown out in the F3 generation. How many of the F3 plants would you expect to be short (dwarfs)? Well, only 200 (¼) of the F2 plants will be recessive short (tt) and these are discarded. The rest (600, made up from 200 TT and 400 Tt) are tall as so selected. As already stated, from the ‘selected lines 400 would be heterozygous tall (Tt) and 200 would be homozygous tall (TT). All TT would remain TT, while one quarter (¼ or 100) of the heterozygous (Tt) plants would be short in the F3 generation. So the answer would be 100 plants What are the terms in the response to selection? The response to selection is - iσh2 , where i is the intensity of selection, σ is the phenotypic standard deviation, and h2 is the heritability. What is heritability? Heritability is the proportion of total variance that is genetic. It is calculated by dividing the genetic variance by the phenotypic (or total) variance. The phenotypic variance for seed weight in an F2 population is 200 kg2 and the genetic variance in that population is 150 kg2. What is the heritability for seed weight? Heritability is genetic variance/phenotypic variance = 150/200 = 0.75. 5 Plant diseases and insects Which pests affect crop plants? Air-borne fungi, soil-borne fungi, bacteria, viruses, eelworm, insects, others include mammals. Of course weeds are also a pest to crop plants so could be included here. What are the major characteristics of the following plant pests? Pest type Air-borne fungi Soil-borne fungi Bacteria Virus Insects Description Spore forming, microscopic air-borne organisms that are usually pathotype differentiated. Spread rapidly. Spore forming, microscopic to large soil-borne organisms that have log persistence in soils. Spread slowly and usually manassisted. Microscopic prokaryotic rod-like organisms. Little pathotype differentiation. Spread slowly and man assisted. Single stranded RNA (in plants). Unable to multiply outside host (obligate). Many pathotype differentiated. Spread rate is related to vector (insects = quick; mechanical = slow). Many insects have a wide host range but some are species specific. Infestations tend to be seasonal. Can spread rapidly (winged insects) or slowly (non-flyers). Provide examples of the following pest types. Pest type Air-borne fungi Soil-borne fungi Bacteria Virus Insects Examples Potato early and late blight, wheat, barley & maize rusts, powdery mildew (many crops), cereal smuts, rice blast. Potato wart, Brassica club root, Many Vertillilium and Fusarium wilts, cereal take-all. Potato ring-rot; vascular wilts of many crops; ratoon in sugar cane; cotton blackarm. Potato virus LR, X, Y; tobacco mosaic, barley yellow dwarf, many clonal fruit viruses. Colorado potato beetle, wheat Hessian fly, many aphids, cabbage seedpod weevil. What effect do plant pests have on our crops? They reduce harvestable yield and reduce end-use quality. 6 In what sector of the ‘disease triangle’ might you expect to have disease? Susceptible Host Favorable environment Pathogen Disease Given that plants are resistant to a pathotype differentiated disease because they have at least one copies of dominant resistance gene (locks), which can only be overcome, and hence the plant become susceptible to the disease, if the disease pathotype has two copies of the appropriate recessive virulence gene (keys). Given that plant resistance genes are donated as capital letters (A, B and C) with susceptible genes as lower case letters (a, b, or c), and that disease virulent genes are donated as a’, b’ or c’ with disease avirulent genes donated as A’, B’, or C’, would the following plants be resistant or susceptible to the associated disease pathotype? Plant phenotype No resistance genes A_ bb cc A_ bb C_ A_ B_ cc Disease genotype No virulence genes A’a’ b’b’ c’c’ a’a’ B’B’ c’c’ A’A’ b’b’ c’c’ Plant response Susceptible Resistant Susceptible Resistant With respect to disease resistance what is meant by hypersensitivity? Hypersensitivity is a plant reaction in response to being infected by a disease. On infection, plant cells around the infection site die and hence the pest can’t spread through the plant. Explain the following plant resistance mechanisms: Antibiosis describes a form of plant resistance that reduces, survival, growth, development, or reproduction of pests feeding on the plant; Antixenosis describes a form of plant resistance that reduces pest preference or acceptance of the plant; Escape describes a mechanism such that a plant morphology avoid a disease. What is the difference between vertical and horizontal plant resistance? Vertical plant resistance is when the disease is controlled by a single gene, whereas horizontal disease resistance is controlled by many different genes. 7 Weed control How do weeds impact crop plants? Weeds cause yield loss as they compete for: interceptable light, water, and nutrients. Weeds ca also harbor pests, and reduce crop quality through weed seed contamination. How can weeds be controlled in crops? Mechanically by pre-plant cultivation, inter-row cultivation, or hand weeding. Culturally mainly by inter-cropping. Biologically usually by insects, and chemically using herbicides. Describe the mode of action of the following herbicide groups. Group Description Acetyl CoA Carboxytase (ACCase) inhibitors, binds to ACCase and disrupts fatty 1 acid synthesis, which leads to membrane degeneration. Acetolactate Synthase (ALS) inhibitors, binds to ALS and disrupts synthesis of 2 branched amino acids. 3 Tubulin inhibitors, interferes with cell division. Synthetic auxins, upsets plant growth regulator balance by mimicking an increase of auxins. Triazines & Ureas. Binds to a pigment in photosystem II and disrupts 5,6&7 photosynthesis. 5-enol Pyruvilshikimate-3-phosphate synthate (EPES) synthase inhibitor, binds to 9 EPES synthase and disrupts pathway, which is responsible for producing the precursors of aromatic amino acids. 4 Why is herbicide mode of action important when considering herbicides resistant weeds? Herbicide resistant weeds can arise by repeated use of herbicides within the same group and hence with the same mode of action. What is meant by the term "plant back restriction"? Several herbicides have a long residual in soils and can cause damage to certain crops if they are planted into treated soils before complete herbicide breakdown. What does IPM stand for? Integrated Pest Management. What techniques are utilized in IPM systems? Biological control where natural pest predators and parasites are encouraged on introduced. Biopesticides that are naturally occurring pesticides. Cultural control including planting resistant cultivars, planting trap crops, intercropping, having an appropriate and broad crop rotation. Mechanical & Physical control using cultivation & tillage. Reproductive & Genetic control where harmful pest genes are introduced (i.e. mass release of sterile insects), and chemical control where pesticides are used in an appropriate manner. 8 Biotechnology – molecular markers and plant transformation What are bacteriophages? Viruses that parasitizes bacteria and pass DNA into bacteria cells. What are restriction enzymes? Restriction enzymes are produced by bacteria as a defense mechanism against phages. Enzymes act like scissors by cutting phage DNA at specific sites. What are molecular markers used for? Marker assisted selection, in many cases (in plant breeding) it can be difficult to evaluate characters, and it can be easier (and quicker) to select according to a marker located closely on the same chromosome as a gene of interest. DNA finger printing can be used to identify genotypes (or cultivars), and can be used to secure proprietary ownership. This can also be used to select parents with known genetic distance. Cytological where information can be obtained regarding chromosomes (mainly in interspecific hybrids), and Saturated gene mapping. What can be achieved by plant transformation techniques that are not possible with traditional plant breeding methods? Using these techniques it is possible to transfer single genes from other species and non-plants into plant and to have these transgenes expressed and to function successfully. This effectively bypasses natural barriers which limit sexual gene transfer and allows breeders to utilize gene from completely unrelated species or to create new variability beyond that currently available in germplasm. What plant characters have been developed through plant transformation? Herbicide resistance (Roundup and Liberty Link) , insect resistance (mainly B.t. resistance), virus resistance, modifies quality (starch, amino acids, fatty acids, and fruit ripening), salt tolerance, and pharmaceuticals (edible viruses, anti-coagulants). What vectors can be used to insert genes into plants? Agrobacterium tumefacians, and particle gun (could also include viruses and electroporation). How does Agrobacterium tumefacians cause crown galls in plants? A. tumefacians cells contain Ti (tuber inducing) plasmids that insert T-DNA into the DNA of the host plant and contains genes that cause rapid cell division expressed as crown galls. Describe (briefly) the process of plant transformation. Find a gene of interest, produce a suitable construct and clone it. Develop a mechanism to insert construct into a plants DNA. Regenerate a whole plant from a single transformed cell. Check functionality of transformants.