Hartl, Jones - Genetics. Principlers and analysis - 1998 (522927), страница 52
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However, bread molds of the genus Neurospora and related organisms have the useful characteristic that themeiotic products are arranged in a definite order directly related to the planes of the meiotic divisions. We willexamine the ordered system after first looking at unordered tetrads.The Analysis of Unordered TetradsIn the tetrads, when two pairs of alleles are segregating, three patterns of segregation are possible.
For example, inthe cross AB × ab, the three types of tetrads areAB AB ab ab referred to as parental ditype, or PD.Only two genotypes are represented,and their alleles have the samecombinations found in the parents.Ab Ab aB aB referred to as nonparental ditype, orNPD. Only two genotypes arerepresented, but their alleles havenonparental combinations.AB Ab aB ab referred to as tetratype, or TT. All fourof the possible genotypes are present.It is because of the following principle that tetrad analysis is an effective way to determine whether two genes arelinked.When genes are unlinked, the parental ditype tetrads and nonparental ditype tetrads are expected in equalfrequencies (PD = NPD).The reason for the equality PD = NPD for unlinked genes is shown in Figure 4.23A for two pairs of alleles.
A, aand B, b locatedPage 151Figure 4.21Formation of an ascus containing all of the four products of a single meiosis. Each product of meiosis formsa reproductive cell called an ascospore; these cells are held together in the ascus. Segregation of onechromosome pair is shown.in different chromosomes. In the absence of crossing-over between either gene and its centromere, the twochromosomal configurations are equally likely at metaphase I, so PD = NPD. When there is crossing-over betweeneither gene and its centromere (Figure 4.23B), a tetratype tetrad results, but this does not change the fact that PD =NPD.In contrast, when genes are linked, parental ditypes are far more frequent than nonparental ditypes. To see why,assume that the genes are linked and consider the events required for the production of thePage 152Figure 4.22Life cycle of the yeast Saccharomyces cerevisiae.
Mating type is determinedby the alleles a and α. Both haploid and diploid cells normally multiply bymitosis (budding). Depletion of nutrients in the growth medium inducesmeiosis and sporulation of cells in the diploid state. Diploid nuclei are red;haploid nuclei are yellow.three types of tetrads. Figure 4.24 shows that when no crossing-over takes place between the genes, a PD tetrad isformed. Single crossing-over between the genes results in a TT tetrad. The formation of a two-strand, three-strand,or four-strand double crossover results in a PD, TT, or NPD tetrad, respectively. With linked genes, meiotic cellswith no crossovers will always outnumber those with four-strand double crossovers. Therefore,Linkage is indicated when nonparental ditype tetrads appear with a much lower frequency than parentalditype tetrads (NPD << PD).The relative frequencies of the different types of tetrads can be used to determine the map distance between twolinked genes, assuming that the genes are sufficiently close that triple crossovers and higher levels of crossing-overcan be neglected.
To determine the map distance, the number of NPD tetrads is first used to estimate the frequencyof double crossovers. The four types of double crossovers in Figure 4.24 are expected in equal frequency. Thus thenumber of tetrads resulting from double crossovers (parts C, D, E, and F of Figure 4.24) should equal four timesthe number of NPD tetrads (part F), or 4 × [NPD], in which the square brackets denote the observed number ofNPD tetrads.
At the same time, the number of TT tetrads resulting from three-strand double crossovers (parts Dand E) should equal twice the number of NPD tetrads, or 2 × [NPD] (part F). Subtracting 2 × [NPD] from the totalnumber of tetratypes yieldsPage 153Figure 4.23Types of unordered asci produced with two genes in differentchromosomes.
(A) In the absence of crossing-over, random arrangement ofchromosome pairs at metaphase I yields two different combinations ofchromatids, one yielding PD tetrads and the other NPD tetrads. (B) Whencrossing-over takes place between one gene and its centromere, the twochromosome arrangements yield TT tetrads. If both genes are closelylinked to their centromeres (so that crossing-over is rare), then few TTtetrads are produced.the number of tetratype tetrads that originate from single crossovers, or [TT] — 2[NPD].
By definition, the mapdistance between two genes equals one-half the frequency of single-crossover tetrads ([TT] — 2[NPD]) plus thefrequency of double-crossover tetrads (4[NPD]). ThusMap distanceThe 1/2 in this equation corrects for the fact that only half of the meiotic products in a TT tetrad produced by singlecrossing-over are recombinant. In the special case where the linked genes are close enough that [NPD] = 0 (nodouble crossovers), the map distance equals the percentage of TT tetrads divided by 2.
(The division by 2 isnecessary because only half the ascospores in TT tetrads are recombinant for the alleles.)A systematic format for analyzing linkage relationships for allelic pairs in unordered tetrads is presented in theform of a tree in Figure 4.25. All of the equations are based on the assumption that there is no chromatidinterference. The right-hand side summarizes the equations for linked genes that we derived on the basis of Figure4.25.
The left-hand side pertains to unlinked genes, and it shows how the frequency of tetratypes can be used tomakePage 154Figure 4.24Types of tetrads produced with two linkedgenes. In the absence of crossing-over (A), aPD tetrad is produced. With a single crossoverbetween the genes (B), a TTtetrad is produced. Among the four possibletypes of double crossovers between the genes (C,D, E, and F), only the four-strand doublecrossover in part F yields an NPD tetrad.inferences about the distance of each gene from its own centromere.As an example of the calculations for linked genes, consider a two-factor cross that yields 112 PD, 4 NPD, and 24TT tetrads.
The fact that NPD << PD (that is, 4 << 112) indicates that the two genes are linked and leads us downthe right branch of the tree. Because both TT and NPD tetrads are recovered, we take the first left fork, and becausethere are NPD tetrads, we also take the next left fork. The appropriate equation is therefore Equation (1).Substitution of the values yields a map distance ofMap distanceNote that this mapping procedure differs from that presented earlier in the chapter in that recombinationfrequencies are not calculated directly from the number of recombinant and nonrecombinant chromatids, but ratherfrom the inferred types of crossovers.As Figure 4.25 indicates, tetrad analysis yields a great deal of information about the linkage relationship betweengenetic markers and the distance of unlinked markers to their respective centromeres. However, it is not necessaryto carry out a full tetrad analysis for estimating linkage.
The alternative is to examine spores chosen at random afterallowing the tetrads to break open and disseminate their spores. This procedure is called random-spore analysis,and the linkage relationships are determined exactly as described earlier for Drosophila and Zea mays. Inparticular, the frequency of recombination equals the number of spores that are recombinant for the geneticmarkers divided by the total number of spores.The Analysis of Ordered TetradsIn Neurospora crassa, a species used extensively in genetic investigations, the products of meiosis are contained inan ordered array of ascospores (Figure 4.26). A zygote nucleus contained in a sac-like ascus undergoes meiosisalmost immediatelyPage 155Figure 4.25Tree diagram for analyzing linkage relations from unordered tetrads.after it is formed. The four nuclei produced by meiosis are in a linear, ordered sequence in the ascus, and each ofthem undergoes a mitotic division to form two genetically identical and adjacent ascospores.
Each mature ascuscontains eight ascospores arranged in four pairs, each pair derived from one of the products of meiosis. Theascospores can be removed one by one from an ascus and each germinated in a culture tube to determine itsgenotypes.Ordered asci also can be classified as PD, NPD, or TT with respect to two pairs of alleles; hence the tree diagramin Figure 4.25 can be used to analyze the linkage data. In addition, the ordered arrangement of meiotic productsmakes it possible to determine the recombination frequency between any particular gene and its centromere.
Thelogic of the mapping technique is based on the feature of meiosis shown in Figure 4.27:Homologous centromeres of parental chromosomes separate at the first meiotic division; the centromeres ofsister chromatids separate at the second meiotic division.Thus, in the absence of crossing-over between a gene and its centromere, the alleles of the gene (for example, Aand a) must separate in the first meiotic division; this separation is called first-division segregation. If, instead, acrossover is formed between the gene and its centromere, the APage 156Figure 4.26The life cycle of Neurospora crassa.
The vegetative body consists of partly segmented filaments calledhyphae. Conidia are asexual spores that function in the fertilization of organisms of the oppositemating type. A protoperithecium develops into a structure in which numerous cells undergo meiosis.and a alleles do not become separated until the second meiotic division; this separation is called second-divisionsegregation.
The distinction between first-division and second-division segregation is shown in Figure 4.27. Asshown in Figure 4.27A, only two possible arrangements of the products of meiosis can yield first-divisionsegregation: A A a a and a a A A. However, four patterns of second-division segregation are possible because of therandom arrangement of homologous chromosomes at metaphase I and of the chromatids at metaphase II.
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