Plant Genetics: Quiz 4 – Flashcards
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Unlock answers| Caladium |
Caladium hortulanum Grown for long-lasting and colourful leaves. Leaf traits include background colour (lemon or green), leaf shape, main vein colour, spotting, and rugosity. |
| Cao et al, 2017 |
| Crossed seven cultivars of caladium in 19 crosses to investigate inheritance of leaf traits. The first report on inheritance in Araceae plants. Looks at leaf background colour, leaf shape, main vein colour, spotting, and rugosity. |
| Centi-Morgan (cM) |
| A genetic map unit. The distance between genes for which 1 product of meiosis out of 100 is recombinant. Can be equal to 30 thousand bp to 1.6 million bp, depending on recombination frequency of the genome region. |
| Classical linkage analysis |
| Determines if individual genes assort independently or co-segregate. If there is linkage, segregation differs from this. Calculate goodness of fit for individual genes for a 3:1 ratio, and conduct a chi-square for linkage. Perform a testcross of AaBb x aabb; ratios other than 1:1:1:1 indicate linkage. Phenotypic classes are analysed, and frequency of parental and recombinant gametes are found. |
| Clone |
| To identify and isolate a DNA fragment containing a gene sequence. The sequence can be amplified by PCR or in a vetor such as a plasmid. Includes map-based cloning. |
| Codominant |
| Shows both alleles. Genotype is equal to phenotype. You can observe 1:2:1 ratio. |
| Coupling |
Cis AB/ab When two dominant alleles are linked together on a chromosome. 50% of gametes will be ab. If there were no recombination events, all progeny would be parental, with aabb up to 25%. |
| CRISPR/Cas9 |
Clustered Regulatory Interspaced Short Palindromic Repeat/CRISPR-associated protein with nuclease domains to cleave DNA May be used to determine a gene of interest. Two transgenes are used, one encoding the endonuclease, and another with the target and scaffold sequence. The sequence is designed with homology to the gene of interest, and transcribed to RNA. The RNA sequence binds to the DNA at the desired sequence and the scaffold guides Cas9 to the site where a double-stranded break (DSB) is made, leading to non-homologous end joining or homology-directed repair. |
| Deletion |
| With a mutation that is a deletion, you cannot have recombination in the area which is missing. You can only measure the distance to the edge of the deletion. |
| Determining genes |
When determining which gene is causing a trait, after map-based cloning that narrows the region of the genome down to 40 kb. There are several possible experiments: 1. Generate independent mutants by EMS. A gene with altered DNA sequence is likely the locus of interest. 2. Transform aa mutants with wild type alleles. The gene restored to A_ would be the gene of interest. 3. Indirectly knock out expression using RNAi. Can knock out multiple members or silence genes, leading to false results. 4. Search a database for t-DNA insertion knockout, and determine if it gives the same phenotype as the mutant you studied in map-based cloning. 5. Mutate and knock-out function of each candidate gene to find the original mutant phenotype. Difficult with plants. Can use CRISPR/Cas9. |
| Double crossovers (double XO) |
| Produces parental phenotype in recombinant offspring. Cause underestimation of linkage, since recombinant progeny are classified as parental. A three-point testcross is used to detect them, and a mapping function is used to account for this underestimation. |
| F2 |
Square root method Mapping can be done with F2 data, when homozygous testers are available for testcrossing. Genotype can be derived from phenotype only in the aabb class. Frequency of aabb is 6.25% with independent assortment; when values are greater than this there is linkage in coupling, and when values are lower there is linkage in repulsion. Frequency of aabb in F2 can be used to estimate frequency of alleles and work out map distance between two genes. The square root of the aabb class determines the frequency of ab gametes. Twice the amount of aabb is the number of recombinants, to count the undetectable AABB recombinants as well, which is assumed to be equal. Because all probabilities must sum to 1, you can predict other genotypes as well. This method doesn't work well for closely linked genes. |
| Fv/Fm |
| A measure of photochemical efficiency. A way to measure if the photosystem is active and can absorb light. |
| Genotyping by sequencing (GBS) |
Sequence based genotyping (SBG) A form of high-throughput genotyping. As the cost of DNA sequencing falls, it is becoming more common. Molecular marker loci are directly sequenced to identify genotypes in populations. The genotype of a SNP locus is obtained by directly sequencing DNA fragments and determining which alleles are present. Detection of polymorphisms at every locus may not be necessary. The genome is first digested with two restriction enzymes: one rare and one frequent cutter. Most restriction fragments are cut at both ends by the frequent cutter, but some are cut at one end by the rare cutter. Unique overhanging sequences of the restriction sites allows for PCR amplification of the fragments and sequencing. DNA fragments from multiple plants may be sequenced together in one sample. Adapters for rare cutter sites contain a unique ID tag for each sample. The DNA of each sample is digested and adaptors are added separately, then DNA samples are bulked for mass sequencing. One needs multiple sequence reads for each fragment to develop a consensus sequence, showing homozygosity or heterozygosity. Subsamples only potential SNP loci in a sufficient portion of the genome, yet can find tens of thousands of SNPs. Data reveals genotypes of individuals in populations, which can be used for mapping and other applications. |
| Grrain protein content (GPC) |
| A gene in wheat. Mapped to an area of 0.04 cM, with 13 - 18 candidate genes. |
| Haldane |
| A mapping function. Used in corn. |
| High-throughput genotyping |
| Allows multiple marker genotypes to be determined for many individuals simultaneously. A method initially used to map large numbers of SNPs in populations of select species. Relies on bead array, PCR with a large number of distinct primers, and colorimetric detection of genotype. Development and use is expensive; applied only to major crops. Efficiency is achieved by sequencing DNA fragments from multiple plants together in one sample. Can be used to identify mutant loci, rather than mapping: plants with mutant phenotypes are sequenced, and regions with high ratios (all plants have the same genotype) are likely to be the mutant locus. |
| Homology-directed repair (HDR) |
| A possible outcome of CRISPR/Cas9. A double-stranded break causes homologous recombination with a plasmid construct containing arms that have homology to the two ends of the break, with a sequence of interest in between. A defined sequence can be inserted into a specific location at the breakpoint. The main method of knock-in tagging, and precise pre-specified insertions or deletions using CRISPR. |
| Illumina GoldenGate |
| A form of high-throughput genotyping. A sample of DNA is immobilized on a solid support and made single stranded. Two 5' primers are added, each with a unique sequence at the 5' end for PCR amplification and a common sequence of homology to both alleles: each has a unique nucleotide that defines the SNP for both alleles. A third 3' primer contains a sequence homologous to both alleles for the 3' end of the locus, and a unique tag sequence for PCR. The three primers are hybridized to denatured DNA. Polymerase, ligase, and nucleotides are added to extend DNA from the 5' primer to join with the 3' primer. New primers, homologous to the unique 5' primer ends, containing blue or yellow fluorescent colour, are added with a 3' primer homologous to both alleles. In homozygous plants, only one 5' primer colour binds to the DNA, and in heterozygotes both colours bind. Samples from each plant are hybridized to beads containing single-stranded sequences homologous to the unique sequence tag for the locus at the 3' primer. Only PCR-amplified strands bind to the bead, and colour of each sample can be read: blue = homozygous parent 1, green (blue + yellow) = heterozygous, yellow = homozygous parent 2. Can be performed for multiple loci in a single plant, hybridizing products to a microarray of beads containing homologous sequences for each locus: you need a unique sequence for each locus. There are microarrays with sequences for hundreds of loci: you can thoroughly genotype an individual with one chip that fits into the palm of the hand. Chips are manufactured only for major crops. |
| Independent assortment |
F2 segregation should be 9 A_B_:3 A_bb:3 aaB_:1 aabb. Ratio of genotypes is 1:2:1:2:4:2:1:2:1 Genes may be on separate chromosomes. Genes on the same chromosome are 50 cM or more apart; they can be mapped using additional genes in between them. aabb = (0.25)2 = 0.0625 |
| Interference |
| When a crossover occurs, it decreases the probability of another crossover occurring nearby. Degree of interference may vary with species, genetic background, and between and within chromosomes. Makes choosing a mapping function difficult wihtout comparing the function's ability to predict segregation with three-point test data. |
| Isbell and Tipson, 1959 |
| Crossed five independently occurring, spontaneous wx mutants in all possible combinations. Each mutant tested differnt from every other mutant, and there was a characteristic, reproducible frequency of Wx pollen grains from the cross between any two wx mutants. Extra Wx pollen grains produced were caused by recombination in the waxy region. A genetic map of this regioin was made using cross data. They screened 1 million pollen grains, and mapped to 0.008 cM. Some of the recombinant pollen grains may have been contaminants, causing overestimation of map distance. Mutants were from different lines, so there is some error. |
| KASPar SNP genotyping |
| KBioscience Used when one needs to genotype a limited number of SNPs in a population. One PCR reaction produces one genotype at one locus in one individual. Similar to SSR genotyping, but does not require separation of PCR products by size. Genotypes are determined by fluorescence in a 96-well or greater plate and plate reader. Requires three components: a primer mix, master mix, and DNA template. DNA template and oligonucleotides in the master mix become single-stranded, and primers anneal to the template DNA, and elongation occurs. The double stranded DNA dissociates again, and the reverse primer anneals, and DNA elongation occurs up to and including the original forward primer. This PCR continues through many cycles, and dye-labelled oligonucleotides from the master mix are incoroprated, and they start to fluoresce since they are separated from quenchers. In a homozygote, one dyed oligonucleotide is incorporated into DNA and made visible, displaying its colour, red or blue. In a heterozygote both oligonucleotides are incorporated and both colours are displayed, red and blue together appearing green. A plate reader identifies fluorescence and genotypes are assigned to each DNA sample. It can be expensive. |
| Kosambi |
A mapping function. Used in Arabidopsis. D = 25 ln ((100 + 2r) / (100 - 2r))r |
| LEM |
| A gene in caladium that controls leaf background colour. LEM produces lemon, and lem produces green. It is independent of the gene for leaf shape, F, but linked to loci controlling spotting, main vein colour, and rugosity (S, V, and RFL). |
| Likelihood (L) |
Can be estimated for a sample. Used to calculate maximum likelihood. When you raise any number to a small exponent, it is much reduced. L (r = r hat) = (n! / a!b!c!d!)paqbrcsd a = number of A_B_ offspring b = number of A_bb offspring c = number of aaB_ offspring d = number of aabb offspring p = expected proportion of A_B_ offspring q = expected proportion of A_bb offspring r = expected proportion of aaB_ offspring s = expected proportion of aabb offspring |
| Linkage |
| F2 segregation is different from independent assortment. Parental genotypes are in high frequency. The genes are likely to be on the same chromsome and move together through meiosis. |
| Map-based cloning |
| You need two genetic lines that differ for the gene of interest as well as molecular marker loci throughout the genome. Preliminary mapping places the gene on the chromosome map between two molecular markers. The region which may contain the gene is narrowed down using other markers, until the region is as small as 0.15 cM, or 40 kb. Requires a large F2 population, 3 - 4 thousand, to generate crossover events in a small region of the genome. Relies on homozygous recessive segregates, limiting the number of mappable crossovers and precision. |
| Map distance |
| A mathematical calculation based on the number of crossover events observed. Recombination frequency is not uniform over the whole genome, so a linkage map is an abstract concept that does not put genes at specific locations on the chromosome. |
| MAPMAKER |
| A computer program that estimates linkage using maximum likelihood method. |
| Mapping |
| Determines if new mutants are allelic to described genes, establishes a genetic map, and is used in marker assisted selection in plant breeding. F1 is back-crossed to a double recessive, to look for recombinant gametes. Random sampling error can affect map distance. |
| Mapping function |
| A formula for using recombination frequencies to calculate map distances, corrected for multiple crossovers. Depends on the amount of interference that occurs, which varies with species, genotype, and between chromosomes. Always predicts more crossovers than occurs, because of interference. Includes Kosambi and Haldane. |
| Master mix |
| One of the components of KASPar SNP genotyping. Two double-stranded oligonucleotides. Identical to regions of the forward primers in the primer mix, but tagged with different fluorescent dyes: red or blue, depending on the SNP allele. For each oligonucleotide, the second strand is tagged with a quencer (Q) of fluorescence. Fluorescence can only be observed when the dye is separated from the quencher. |
| Maximum likelihood |
A method for estimating linkage used by MAPMAKER. The LOD score is estimated, the log of the ratio of the observed recombination fraction and the recombination fraction without linkage. Linkage is accepted as significant when LOD is 3 or greated, meaning that likelihood of estimated recombination fraction is at least 1000 times more probable than recombination fraction without linkage. If LOD is 7, then it is 10 million times more probable! False negatives for linkage can occur with small sample size. A computer program calculates LOD for many values of r hat, and choses the recombination fraction with the greatest LOD score as the best estimate for linkage. A normal value for maximum LOD is 3 - 4. Unlike F2 data analysis, it uses all data, not just aabb. LOD = log10 ((likelihood (r = rhat)) / (likelihood (r = 0.5))) 1. Estimate expected proportion of offspring, for all values of r from 0.01 to 0.5. 2. Estimate likelihood using these values of p, q, r, and s, with experimental observation of a, b, c, and d. 3. Estimate likelihoood using values of p, q, r, and s for recombination fraction of 0.5. p = 0.56, q = 0.19, r = 0.19, s = 0.06. 4. Estimate LOD |
| Molecular marker |
| A locus defined by a DNA sequence. Variations in sequences that can be genotyped. Genotype is phenotype. Today, mostly SNPs are used. |
| Mutant screening |
| Can screen up to 60,000 plants before a certain mutant is found. It is easier for seed traits. |
| Near isogenic line |
| Plants which are genetically identical except for at one gene. |
| Non-homologous end joining (NHEJ) |
| A possible outcome of CRISPR/Cas9. A double-stranded break causes chromosomes to be repaired through a mutation-prone process, resulting in random insertions or deletions, leading to frameshift mutations and disruption of gene function. The main method of CRISPR-mediated gene knockout. |
| Oh et al, 1997 |
| Dark screened for plants with delayed senescence. Looked at 25,000 mutants in the dark, and 9,000 in planta. Found 4 dark mutants, which had delayed senescence: oresara 1, 2, 3, and 11. Mutations were in genes that allow the plant to break down chlorophyll. In the mutants, chlorophyll remains, but the plant is not photosynthesizing. Oresara 1 is recessive. Only oresara 2 x 3 had no wild-type progeny, so they must be in the same gene. |
| Physical map |
| Actual physical location of chromosomes on the DNA. Tells the number of nucleotides that separates genes. Produced from DNA sequencing. Collinear with genetic maps, but distances placed between genes can vary. |
| Primer mix |
| One of the components of KASPar SNP genotyping. Two forward primers with unique 3' ends distinguish the two SNP alleles. There is also a reverse primer with homology to both SNP alleles. |
| Product ratio method |
A more accurate method for estimating frequency of an allele than the square root method. Takes into account data from all classes. For repulsion, z = (A_B_ x aabb) / (A_bb x aaB_) For coupling, z = (A_bb x aaB_) / (A_B_ x aabb) |
| Recombination fraction (r) |
| The fraction of recombinant genes. When it is equal to 0.01, the map distance is 1 cM. |
| Recombination frequency |
| A trait which can vary with species, individual, chromosome, and chromosome region. It affects the value of a centi-Morgan. |
| Repulsion |
Trans Ab/aB When a dominant allele is linked to a recessive allele on a chromosome. No gametes will be ab. There are no aabb at all. |
| RNAse |
| Breaks down RNA, to get N and K. |
| Russian dandelion |
| A wild species, being bred to become a crop. There is no genetic mapping data at all. If you need to do genetic analysis, you must generate a three-point testcross to correct for crossovers. You must choose a mapping function that works best, by comparing results to observations. |
| Senescence |
| Leaf senescence is controlled by an ethylene signal transduction pathway. Deteriorative events lead to cell death; there are dramatic changes in metabolism and cellular structure. Chloropphyll is broken down, and other pigments are synthesized, causing colour change in leaves. Chloroplasts are degraded. Reduced photosynthesis, and increased RNAse and peroxidase. Remobilizes nutrients from vegetative tissues to reproductive organs. Leaves turn yellow and fall off. It is a necessary trait in Arabidopsis; important for reproduction. Senescence will occur if a plant is placed in the dark. |
| Single nucleotide polymorphism (SNP) |
| A molecular marker where a single nucleotide difference can be detected between alleles at a given locus. Numerous SNPs are found when comparing genotypes of any species, allowing one to develop a detailed genetic map. One needs sequence information for two or more genotypes to find SNPs. Alleles are codominant because they can be identified in heterozygotes when assayed. |
| Three-point test cross |
| Three genes of unknown order are backcrossed, observing 8 different phenotypes. Map distance is calculated between all three points separately to map them. Double crossovers can be detected when each crossover occurs on either side of the middle gene. The number of crossovers must be multiplied by 2 before using in a mapping function, because 2 crossovers occur. |
| Waxy (wx) |
| A gene in corn. Pollen grains with mutant genotype, wx, contain only amylopectin, no starch. When stained with KI and I2 solution they stain light brown, where wild types stain black. It is easy to phenotype many gametes very quickly. The gene also governs type of starch produced in triploid cells of the endosperms. |
| Wheat |
| Hexaploid, with 19 billion bp, and 96 thousand genes. |
