LECTURE 6 TETRAD ANALYSIS Reading Ch 5 p 132 140 Problems Ch 5 solved problem III 5 20 5 24 5 27 5 30 mislabeled as the 2nd 5 29 5 31 First we went over interference see notes from last lecture TETRAD ANALYSIS IN FUNGI In the diploid organisms that we ve considered so far each individual represents only one of four potential gametes that are produced from each parent in a single meiotic event We don t know which of the other progeny in the cross are created by gametes produced by the same meiotic event So we have to analyze large numbers of progeny and use statistics to establish linkage or lack thereof and to do mapping By contrast some yeast species house all four products of a single meiosis in a sac called an ascus The haploid cells are called ascospores or haplospores and can germinate and live in the haploid state growing and dividing by mitosis Thus phenotype equals genotype in these haploid cells We will review tetrad analysis in Baker s yeast Saccharomyces cerevisiae you should review ordered tetrad analysis in Neurospora crassa on your own Life cycle of Saccharomyces cerevisiae The collection of four products of meiosis found in each ascus is called a tetrad Nomenclature conventions of Saccharomyces cerevisiae Capital letters indicate dominant alleles e g HIS4 or sometimes just Lower case letters indicate recessive alleles e g his4 Usually the wild type allele is the dominant one e g HIS4 We characterize tetrads based upon the number of parental and recombinant spores they contain Consider a cross between an a mating type haploid yeast strain of genotype HIS4 trp1 with an mating type haploid yeast strain of the opposite genotype his4 TRP1 These two genes are unlinked What are the possible kinds of tetrads that result when the resulting diploid strain undergoes meiosis Parental ditype PD A tetrad containing 4 haploid cells of the parental class Nonparental ditype NPD A tetrad containing 4 recombinant haploid cells the two parental classes have recombined to form the reciprocal nonparental combination of alleles Tetratype T A tetrad containing four kinds of haploid cells two different parental class spores and two different recombinant class spores In crosses involving 2 unlinked genes tetratypes arise when a crossover occurs between one of the two genes and its centromere If PD NPD then the two genes are unlinked either they are located on different chromosomes or they are far apart on the same chromosome Think independent assortment here 50 recombination frequency Since all Ts no matter how many are 50 parental and 50 recombinant then the only way that 50 of the total number of haploid progeny spores can be 50 recombinant is if the number of PDs 100 parental class equals the number of NPDs 100 recombinant If PD NPD then the two genes are linked When PD NPD the haploid spores of the parental class significantly outnumber the haploids of the recombinant class a sign of linkage Consider and example of two linked genes ARG3 and URA2 P arg3 ura2 x ARG3 URA2 Diploid arg3 ura2 ARG3 URA2 Meiotic products PD arg3 ura2 arg3 ura2 ARG3 URA2 ARG3 URA2 127 NPD arg3 URA2 arg3 URA2 ARG3 ura2 ARG3 ura2 3 T arg3 ura2 arg3 URA2 ARG3 ura2 ARG3 URA2 70 Recombination frequency RF NPD 1 2T total number of tetrads x 100 In our example this is 3 1 2 70 200 x 100 19 map units We will modify the equation to obtain a better estimate of map distance If you draw out the possible crossover events between two linked genes you can see the different tetrads that result see Fig 5 19 No crossovers PD Single crossover T Double crossover 2 strand PD Double crossover 3 strand T Double crossover 3 strand T Double crossover 4 strand NPD You can see how we can modify the equation to make it more accurate Remember that half 2 4 the strands recombine if there is a single crossover event and that 4 strands recombine if there is a double crossover event even if all of the strands don t participate some participate more than once Map distance total rec events total tetrads x 100 1 2 SCO DCO total tetrads x 100 Map distance 1 2 T 2 NPD 4 NPD total tetrads x 100 Map distance 1 2 T 3 NPD total tetrads x 100 For our example above map distance 1 2 70 3 3 200 x 100 22 map units This modified equation makes 2 assumptions 1 there are no more than two crossovers in the interval and 2 there is no chromosomal interference all types of DCOs occur with equal frequency We will cover the material below nest time ORDERED TETRADS AND GENE CENTROMERE DISTANCE In Neurospora crassa meiosis occurs within the tight confines of a narrow ascus resulting in the formation of ordered tetrads Because of the precise positioning of each meiotic product within the ascus one can infer the arrangement and segregation of each chromatid of homologous chromosomes during Meiosis I and II This gives information about the distance between the gene and its centromere Meiosis II is followed by mitosis each pair of genetically identical daugthers sits adjacent to one another Each ascus is thus made of up 8 haploid ascospores Consider a gene required for ascospore color ws gives black spores and ws gives white spores P ws x ws Diploid ws ws immediately undergoes meiosis If no recombination between ws gene and the centromere occurs then the resulting ascospores are arranged in a neat array with black and white spores clearly segregated from one another each type cleanly segregated to either side of the imaginary line separating the 4th and 5th ascospores This is called a first division segregation pattern Since the daughters of the mitotic division lie right next to one another we can simplify the two possible configurations to ws ws ws ws ws ws ws ws If recombination occurs between ws and the centromere then a second division segregation pattern is observed Now both types of spores are found on either side of the imaginary line between the 4th and 5th ascospores Now there are four possible configurations ws ws ws ws ws ws ws ws ws ws ws ws ws ws ws ws When an ascus shows a second division segregation pattern we know that half of the chromatids are recombinant and the other half have not participated in crossovers Thus we can calculate the distance of a gene from its centromere simply by dividing the percentage of second division octads by 2 Gene centromere distance of second division octads total octads x 100 2 To examine linkage of two genes in Neurospora we can use the same formulas as we did for Baker s yeast
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