Prepared by Malcolm D. Schug University of North Carolina Greensboro Powerpoint to accompany Genetics: From Genes to Genomes Third Edition Hartwell ● Hood ● Goldberg ● Reynolds ● Silver ● Veres Chapter 5 Prepared by Malcolm D. Schug University of North Carolina Greensboro Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Linkage, Recombination, and the Mapping of Genes on Chromosomes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Outline of Linkage, Recombination, and the Mapping of Genes on Chromosomes Linkage and meiotic recombination Genes linked together on the same chromosome usually assort together. Linked genes may become separated through recombination. Mapping The frequency with which genes become separated reflects the physical distance between them. Mitotic recombination Rarely, recombination occurs during meiosis. In eukaryotes mitotic recombination produces genetic mosaics. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Independent assortment Genes on different chromosomes B A B a b a b Gametes a B A b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Linkage Two genes on same chromosome segregate together. B b Gametes Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Crossing over and linkage Lead to separation of linked genes B b Gametes x Parental Recombinant Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Some genes on the same chromosome assort together more often than not. In dihybrid crosses, departures from a 1:1:1:1 ratio of F1 gametes indicate that the two genes are on the same chromosome. How we determine if two genes are on the same chromosome can be demonstrated by the white and yellow genes on the X chromosome of Drosophila. yellow (y+) – yellow body color white (w+) – white eye color Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Linkage at a sex-linked gene Deviation from 1:1:1:1 ratio of phenotypes for males Draw traits on chromosomes and work through the cross Figure 5.2 Fig. 5.2 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Designations of “parental” and “recombinant” relate to past history. Parental and recombinant classes are opposite of one another in these two crosses. Similar percentages of recombinant and parental types show that the frequency of recombination is independent of the arrangement of alleles. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Linkage in an autosomal gene Genotypes of F1 female revealed by test cross Parental class outnumbers recombinant class demonstrating linkage. Figure 5.5 Fig. 5.4 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Chi square test pinpoints the probability that ratios are evidence of linkage. Transmission of gametes is based on chance events. Deviations from 1:1:1:1 ratios can represent chance events OR linkage. Ratios alone will never allow you to determine if observed data are significantly different from predicted values. The larger your sample, the closer your observed values are expected to match the predicted values. Chi square test measures “goodness of fit” between observed and expected (predicted) results. Accounts for sample size, or the size of the experimental population Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Applying the chi square test Framing a hypothesis Null hypothesis – observed values are not different from the expected values For linkage studies – no linkage is null hypothesis Expect a 1:1:1:1 ratio of gametes. Alternative hypothesis – observed values are different from expected values For linkage studies – genes are linked. Expect significant deviation from 1:1:1:1 ratio. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Applying the chi square test to a linkage study Genotype Experiment 1 Experiment 2 A B 17 34 a b 14 28 A b 8 16 11 22 Total 50 100 Class Observed/Expected Parentals 31 25 62 50 Recombination 19 25 38 50 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Chi Square – Experiment 1 & 2 c2 = S (observed – expected)2 number expected c2 = S (31 – 25)2 + (19 – 25)2 25 25 = 2.88 Experiment 1 Experiment 2 c2 = S (62 – 50)2 + (38 – 50)2 50 50 = 5.76 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Chi square table of critical values Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Recombination results when crossing-over during meiosis separates linked genes. 1909 – Frans Janssens observed chiasmata, regions in which nonsister chromatids of homologous chromosomes cross over each other. Thomas Hunt Morgan suggested these were sites of chromosome breakage and change resulting in genetic recombination. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Reciprocal exchanges between homologous chromosomes are the physical basis of recombination. 1931 – Genetic recombination depends on the reciprocal exchange of parts between maternal and paternal chromosomes. Harriet Creighton and Barbara McClintock studied corn. Curtis Stern studied fruit flies. Physical markers to keep track of specific chromosome parts Genetic markers were points of reference to determine if particular progeny were result of recombination. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Genetic recombination between car and Bar genes on the Drosophila X chromosome Figure 5.7 Fig. 5.6 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Chiasmata mark the sites of recombination. Figure 5.8 a-c Fig. 5.7 a-c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Chiasmata mark the sites of recombination. Figure 5.8 d-f Fig. 5.5 d-f Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Recombination frequencies for pairs of genes reflect distance between them. Alfred H. Sturtevant – Percentage of recombination, or recombination frequency (RF) reflects the physical distance separating two genes. 1 RF = 1 map unit (or 1 centiMorgan) Figure 5.9 Fig. 5.8 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Summary of linkage and recombination Genes close together on the same chromosome are linked and do not segregate independently. Linked genes lead to a larger number of parental class than expected in double heterozygotes. Mechanism of recombination is crossing over. Chiasmata are the visible signs of crossing over. The farther away genes are the greater the opportunity for chiasmata to form. Recombination frequencies reflect physical distance between genes. Recombination frequencies between two genes vary from 0% to 50%. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Mapping: Locating genes along a chromosome Two-point crosses: Comparisons help establish relative gene positions. Genes are arranged in a line along a chromosome. Figure 5.11 Fig. 5.9a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Mapping: Locating genes along a chromosome Genes are arranged in a line along a chromosome. Figure 5.11 Fig. 5.9 b-d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Limitations of two point crosses Difficult to determine gene order if two genes are close together Actual distances between genes do not always add up. Pairwise crosses are time and labor consuming. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Three Point Crosses: A faster more accurate method to map genes Figure 5.12 Fig. 5.10a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

pr must be in the middle because longest distance is between vg and b Three point cross pr must be in the middle because longest distance is between vg and b Fig. 5.10b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Analyzing the results of a three point cross Look at two genes at a time and compare to parental. Figure 5.13 a,b Fig. 5.11 a,b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Analyzing the results of a three point cross Figure 5.13 c,d Fig. 5.11 c,d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Correction for Double Crossovers 252 + 241 + 131 + 118 4197 X 100 = 17.7 m.u. vg – b distance 252 + 241 + 13 + 9 X 100 = 12.3 m.u. vg – pr distance 131 + 118 + 13 + 9 X 100 = 6.4 m.u. b – pr distance Correction for Double Crossovers 131 + 118 + 13 + 9 4197 vg – b distance X 100 = 6.4 m.u. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Interference: The number of double crossovers may be less than expected. Sometimes the number of observable double crossovers is less than expected if the two exchanges are independent. Occurrence of one crossover reduces likelihood that another crossover will occur in adjacent parts of the chromosome. Chromosomal interference – crossovers do not occur independently. Interference is not uniform among chromosomes or even within a chromosome. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Measuring interference Coefficient of coincidence = ratio between actual frequency of dco and expected frequency of dco. Interference = 1 – coefficient of coincidence. If interference = 0, observed and expected frequencies are equal. If interference = 1, no double crossovers can occur. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Double recombinants indicate order of three genes. Figure 5.13 a, d Fi.g 5.11 a,d Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Summary of three point cross analysis Cross true breeding mutant with wild-type Analyze F2 individuals (males if sex linked) Parental class – most frequent Double crossovers – least frequent Determine order of genes based on parentals and recombinants Determine genetic distance between each pair of recombinants Calculate coefficient of coincidence and interference Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Do Genetic and Physical maps correspond? Order of genes is correctly predicted by physical maps. Distance between genes is not always similar on physical maps. Double, triple, and more crossovers Only 50% recombination frequency observable in a cross Variation across chromosome in rate of recombination Mapping functions compensate for inaccuracies, but are not often imprecise. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Genes chained together by linkage relationships are known as linkage groups. Figure 5.15 Fig. 5.13 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Are genetic maps and physical maps correlated? The order of genes in a genetic map is the same as the order of those same genes along the DNA molecule of a chromosome. The physical distance (amount of DNA separating genes) is not always the same as the genetic distance between genes. Factors responsible for differences in physical and genetic map distances: Double, triple, and even more crossovers 50% limit on recombination frequency observable in a cross Recombination frequency is not uniform across a chromosome. Recombination hotspots Recombination deserts Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Tetrad analysis in fungi Model organisms for understanding the mechanism of recombination because all four haploid products of meiosis are contained in ascus Ascospores within ascus germinate into haploid individuals. Saccharaomyces cerevisiae – bakers yeast Neurospora crassa – bread mold Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Saccharaomyces cerevisiae life cycle Figure 5.16 a Fig. 5.14 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Neurospora crassa life cycle Figure 5.16 b Fig. 5.14 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Neurospora crassa tetrads Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Tetrads can be characterized by the number of parental and recombinant spores they contain. Figure 5.17 a Fig. 5.15 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Figure 5.17 b,c Fig. 5.15 b,c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Figure 5.17 d,e Fig. 5.15 d,e Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Linkage is demonstrated by PDs outnumbering NPDs. Figure 5.18 Fig. 5.16 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

How crossovers between linked genes generate different tetrads Figure 5.19 Fig. 5.17 a-c Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Figure 5.19 Fig. 5.17 d-f Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Calculating recombination frequency NPD + ½ T Total tetrads RF =  100 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Confirmation that recombination occurs at the four-strand stage Figure 5.20 A mistaken Model Recombination before four-strand stage not consistent with tetrads containing recombinant spores; would be NPDs instead of Ts Fig. 5.18 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Tetrad analysis demonstrates that recombination is reciprocal. In a cross between strains with different alleles at two genes, each tetrad contains and two of each type of recombinant Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Ordered tetrads allow mapping a gene in relation to the centromere. Figure 5.22 Fig. 5.20 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Segregation patterns in ordered asci Figure 5.23 a Fig. 5.21 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Segregation patterns in ordered tetrads Figure 5.23 b Fig. 5.21 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Calculating distance to centromere SDS FDS + SDS % SDS =  100 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Example of genetic mapping by ordered tetrad analysis Figure 5.24 Fig. 5.22 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Mitotic recombination can produce genetic mosaics. Mitotic recombination is rare. Initiated by Mistakes in chromosome replication Chance exposure to radiation Curt Stern – observed “twin spots” in Drosophila – a form of genetic mosaicism. Animals contained tissues with different genotypes. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Mitotic crossing over between sn and centromere in Drosophila Figure 5.26 a Fig. 5.24 a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Essential Concepts Gene pairs that are close together on the same chromosome are linked because they are transmitted together more often than not. The recombination frequency of pairs of genes indicate how often two genes are transmitted together. Gene pairs that assort independently exhibit a recombination frequency of 50%. Statistical analysis helps determine whether or not two genes assort independently. The greater the physical distance between linked genes, the higher the recombination frequency. Genetic maps are visual representations of relative recombination frequencies. Organisms that retain all the products of meiosis within an ascus reveal the relation between genetic recombination and segregation of chromosomes during meiosis. Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Crossing over between sn and y gene Figure 5.26 b Fig. 5.24 b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Twin spots in Drosophila A form of genetic mosaicism – mistakes in chromosome replication or exposure to radiation that breaks DNA molecules lead to mitotic recombination Figure 5.25 Fig. 5.23 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Origin of twin spots and yellow spots in Drosophila Fig. 5.24a Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display

Origin of twin spots and yellow spots in Drosophila Fig. 5.24b Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display