Chapter 7. Linkage and Chromosome Mapping

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1 Chapter 7. Linkage and Chromosome Mapping

2 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 mitosis. In eukaryotes mitotic recombination produces genetic mosaics.

3 Recombination Results When Crossing- Over during Meiosis Separates Linked Genes In 1909, F. Janssens observed chiasmata, regions in which nonsister chromatids of homologous chromosomes cross over each other. T. H. Morgan suggested these were sites of chromosome breakage and change resulting in genetic recombination.

4 Chiasmata Mark the Sites of Recombination

5 Reciprocal Exchanges between Homologous Chromosomes are the Physical Basis of Recombination In 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.

6 Creighton and McClintock s Experiment in Maize Copyright 2010 Pearson Education, Inc.

7 Unlinked Genes Show a Recombination Frequency of 50% Genes on different chromosomes

8 Unlinked Genes Show a Recombination Frequency of 50% Genes far apart on the same chromosomes

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10 Copyright 2010 Pearson Education, Inc.

11 Linkage in an Autosomal Gene

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13 Copyright 2010 Pearson Education, Inc.

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17 Copyright 2010 Pearson Education, Inc.

18 Copyright 2010 Pearson Education, Inc. Single Crossover I

19 Copyright 2010 Pearson Education, Inc. Single Crossover II

20 Copyright 2010 Pearson Education, Inc.

21 Consequences of a Single Exchange between Two Nonsister Chromatids Copyright 2010 Pearson Education, Inc.

22 Consequences of a Double Exchange between Two Nonsister Chromatids Copyright 2010 Pearson Education, Inc.

23 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. 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%.

24 According to Sturtevant, In a conversation with Morgan I suddenly realized that the variations in strength of linkage, already attributed by Morgan to differences in the spatial separation of the genes, offered the possibility of determining sequences in the linear dimension of a chromosome. I went home and spent most of the night (to the neglect of my undergrad homework) in producing the first chromosomal map.

25 Recombination Frequencies for Pairs of Genes Reflect Distance between Them A. H. Sturtevant: Percentage of recombination, or recombination frequency (RF) reflects the physical distance separating two genes. 1 RF = 1 map unit (mu) or 1 centimorgan (cm)

26 According to the paper published by Sturtevant in 1931, 1) yellow-white 0.5% 2) white-miniature 34.5% 3) yellow-miniature 35.4%

27 According to the paper published by Sturtevant in 1931, 1) yellow-white: 0.5% 2) white-miniature: 34.5% 3) yellow-miniature: 35.4% Copyright 2010 Pearson Education, Inc.

28 Map of the yellow body (y), white eye (w), and miniature wing (m) Copyright 2010 Pearson Education, Inc.

29 Mapping: Locating Genes Along a Chromosome Two-point crosses: comparisons help establish relative gene positions. Genes are arranged in a line along a chromosome.

30 Mapping: Locating Genes Along a Chromosome Genes are arranged in a line along a chromosome.

31 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.

32 Question A plant of genotype is testcrossed to A a a B b b a b If the two loci are 10 m.u. apart, what proportion of progeny will be AaBb?

33 Question The A locus and D locus are so tightly linked that no recombination is ever observed between them. If AAdd is crossed to aadd, and the F 1 is intercrossed, what phenotypes will be seen in the F 2 and in what proportions?

34 Question The R locus and S loci are 40 m.u. apart. If a plant of genotype is selfed, what progeny phenotypes will be seen and in what proportions? R r S s

35 Three Point Crosses: A Faster More Accurate Method to Map Genes Copyright 2010 Pearson Education, Inc.

36 Determination of the Gene Sequence Copyright 2010 Pearson Education, Inc.

37 Copyright 2010 Pearson Education, Inc. Mapping in Maize

38 Copyright 2010 Pearson Education, Inc. Mapping in Maize

39 Copyright 2010 Pearson Education, Inc.

40 Copyright 2010 Pearson Education, Inc.

41 Parental Type Double Crossover (DCO) Copyright 2010 Pearson Education, Inc.

42 Final Map by Three Point Mapping 1. Determine the two noncrossover classes. 2. Determine the sequence of genes using DCO. 3. Determine the distance between each pair of genes. Copyright 2010 Pearson Education, Inc.

43 Another Example of Three Point Crosse

44 Analyzing the Results of a Three Point Cross Look at two genes at a time and compare to parental.

45 Analyzing the Results of a Three Point Cross

46 Double Recombinants Indicate Order of Three Genes

47 vg b distance vg pr distance b pr distance X 100 = 17.7 m.u X 100 = 12.3 m.u X 100 = 6.4 m.u Correction for Double Crossovers (DCO) vg b distance X 100 = 6.4 m.u.

48 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.

49 Interference and the Coefficient of Coincidence Coefficient of coincidence = ratio between actual frequency of double crossover and expected frequency of double crossover coefficient of coincidence (C): Observed DCO/Expected DCO Interference (I) = 1 coefficient of coincidence (C) If interference = 0: observed and expected frequencies are equal. If interference = 1: no double crossovers can occur.

50 Copyright 2010 Pearson Education, Inc. Mapping in Maize

51 Measuring Interference DCO exp = (0.223) x (0.434) = = 9.7% DCO obs = 7.8% C = Observed DCO/Expected DCO = 0.078/0.097 = I = 1 C = = % fewer double crossovers occurred than expected. Copyright 2010 Pearson Education, Inc.

52 Question The allele b gives Drosophila flies a black body and b + gives brown, the wild-type phenotype. The allele wx of a separate gene gives waxy wings and wx + gives non-waxy, the wild-type phenotype. The allele cn of a third gene gives cinnabar eyes and cn + gives red, the wild-type phenotype. A female heterozygous for these three genes is testcrossed, and 1000 progeny are classified as follows: 5 wild type; 6 black waxy cinnabar; 69 waxy cinnabar; 67 black; 382 cinnabar; 379 black waxy; 48 waxy; 44 black cinnabar Determine the order of genes on the chromosome. Determine the genetic map. Calculate coefficient of coincidence and interference.

53 Question A three-point testcross was made in corn. The results and a partial recombination analysis are shown (p = purple leaves, + = green; v = virus-resistant, + = sensitive; b = brown, + = plain). P F x pp vv bb +p +v +b x pp vv bb Determine which genes are linked. Draw a map that shows distances in map units. Calculate interference if appropriate.

54 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.

55 Genes Chained Together by Linkage Relation-ships are Known as Linkage Groups

56 Do Genetic and Physical Maps Correspond? Order of genes in correctly predicted by physical maps. Distance between genes is not always similar to 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 not often imprecise.

57 The Theoretical and Actual Percentage of Recombinant Chromatids vs. Map Distance Copyright 2010 Pearson Education, Inc.

58 Copyright 2010 Pearson Education, Inc. Undetected Double Crossover

59 Somatic Cell Hybridization The technique of somatic cell hybridization is extensively used in human genome mapping, but it can in principle be used in many different animal systems. In fact, the membranes of the two cells may fuse together and the two cells become one: a binucleate heterokaryon. The nuclei eventually fuse, creating synkaryon. Because the mouse and human chromosomes are recognizably different in number and shape, the two sets in the hybrid cells can be readily distinguished. However, in the course of subsequent cell divisions, for unknown reasons the human chromosomes are gradually eliminated from the hybrid at random.

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61 Human Chromosome Mapping If a specific human gene product is synthesized in a synkaryon containing one to three chromosomes, then the gene responsible for that product must reside on one of the human chromosomes remaining in the hybrid cell. If the human gene product is not synthesized in a synkaryon, the gene responsible is not present on those human chromosomes that remain in this hybrid cell. Synteny Testing Copyright 2010 Pearson Education, Inc.

62 Tetrad Analysis in Fungi In diploid organisms, each individual represents only one of the four potential gametes generated by each parent in a single meiotic events. Thus, our analysis has depended on inferences derived from examining the phenotypes of diploid progeny resulting from random unions of random products of meiosis. 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 : baker s yeast Neurospora crassa : bread mold

63 Saccharaomyces cerevisiae Life Cycle

64 Neurospora crassa Life Cycle

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66 Tetrads Can Be Characterized by the Number of Parental and Recombinant Spores They Contain

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68 If PD = NPD, the two genes must be unlinked, either because they reside on different chromosomes or because they lie very far apart on the same chromosome.

69 Linkage is Demonstrated by PDs Outnumbering NPDs

70 How Crossovers between Linked Genes Generate Different Tetrads

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72 Thus, if two genes are linked, the only way to generate an NPD tetrad is through a four-strand double exchange. Meioses with crossovers generating such a specific kind of double recombination must be a lot rarer than no crossingover or single crossovers, which produce PD and T tetrads, respectively. Linkage exists only when PD >> NPD, that is, when the number of PD tetrads (carrying only parental type spores) substantially exceeds the number of NPD tetrads (carrying only recombinants).

73 Calculating Recombination Frequency All of the spores in an NPD tetrad are recombinant and half of the four spores in a tetratype are recombinant. RF = NPD + ½ T 100 Total tetrads RF = 3 + ½ = 19 m.u.

74 Confirmation that Recombination Occurs at the Four-Strand Stage A Mistaken Model Recombination before four-strand stage is not consistent with tetrads containing recombinant spores would be NPDs instead of Ts.

75 Tetrad Analysis Demonstrates that Recombination is Reciprocal In a cross between strains with different alleles at two genes, every T tetrad carries one Ab and one ab spore, while every NPD tetrad contains two of each type of recombinant. We can thus concluded that meiotic recombination is almost always reciprocal, generating two homologous chromosomes that are inverted images of each other.

76 Ordered Tetrads Allow Mapping a Gene in Relation to the Centromere

77 When each of the four products of meiosis divides once by mitosis, the two genetically identical cells that result lie adjacent to each other. Because of this feature, starting from either end of the ascus, you can count the octad of ascopores as four cell pairs and analyze it as a tetrad. From the precise positioning of the four ascopore pairs within the ascus, you can infer the arrangement of the four chromatids of each homologous chromosome pair during the two meiotic divisions.

78 Segregation Patterns in Ordered Asci

79 Copyright 2010 Pearson Education, Inc. No Crossover

80 Segregation Patterns in Ordered Tetrads

81 Copyright 2010 Pearson Education, Inc. Single Crossover I

82 Copyright 2010 Pearson Education, Inc. Single Crossover II

83 Copyright 2010 Pearson Education, Inc.

84 Calculating Distance to Centromere Since second-division segregation patterns result from meioses in which there has been a crossover between a gene and its centromere, the relative number of asci with this pattern can be used to determine the gene and centromere distance. % SDS = ½ SDS Total tetradś 100

85 Example of Genetic Mapping by Ordered Tetrad Analysis

86 Question A Neurospora cross was made between one strain that carried the mating-type A and mutant allele arg-1 and another strain that carries the mating-type allele a and the wild-type allele for arg-1 (+). Four hundred linear tetrads were isolated, and these fell into the following seven classes. 1) Deduce the linkage arrangement of the mating-type locus and the arg-1 locus. 2) Include the centromere or centromeres on the map. 3) Label all intervals in map units.

87 Question A cross is made between a haploid strain of Neurospora, of genotype nic + ad and another haploid strain of genotype nic ad +. From this cross, a total of 1000 linear asci are isolated and categorized as follows: ( 총 8 점 ) 1) Map the ad and nic loci in relation to centromeres? (4 점 ) 2) Are the ad and nic loci linked? (2점) 3) Map the ad and nic loci in relation to each other (2점)

88 nic + ad nic + ad + nic + ad + nic + ad nic + ad nic + ad + nic + ad + nic + ad nic + ad + nic + ad + nic + ad nic + ad nic + ad + nic + ad + nic + ad nic + ad + nic + ad nic ad nic ad + nic ad nic ad nic + ad nic + ad + nic + ad nic ad nic ad + nic ad nic ad nic ad + nic ad nic ad + nic + ad + nic + ad nic + ad + nic + ad nic ad + nic ad nic ad + nic + ad + nic + ad nic + ad + nic + ad nic ad + nic ad nic ad nic ad + nic ad + nic ad nic ad + nic ad + nic ad nic ad nic ad + nic ad + nic ad nic ad

89 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.

90 Twin Spots in Drosophila In a y sn + /y + sn Drosophila female, most of the body is wild type, but aberrant patches showing either yellow color or singed bristles sometimes occur. In some cases, yellow and singed patches are adjacent to each other, a configuration known as twin spots.

91 Mitotic Crossingover between sn and Centromere in Drosophila

92 Crossingover between sn and y

93 Copyright 2010 Pearson Education, Inc. Did Mendel Encounter Linkage?