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Genetics 2011 Outline • Part 1 Linkage and meiotic recombination Lecture 5 • Genes linked together on the same chromosome usually assort together. together • Linked genes may become separated through recombination. Linkage, Recombination Linkage Recombination, and the Mapping of Genes on Chromosomes • Part 2 Mapping • Recombination data can be used to generate maps of relative locations of genes on chromosomes ᯘ⩶ె ⪁ᖌ • Part 3 Mitotic recombination • Rarely, recombination occurs during meiosis. • In eukaryotes mitotic recombination produces genetic mosaics. mosaics http://lms.ls.ntou.edu.tw/course/136 1 2 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 Independent assortment Linkage g Syntenic genes: two genes on same chromosome segregate together. Genes on different chromosomes A A B B A A B B a a b b a a b b Gametes A Gametes a B A b B a A B A B a b a b b 3 Lectured by Han-Jia Lin 4 Lectured by Han-Jia Lin Genetics 2011 Some genes on the same chromosome assort together more often than not. Crossing over and linkage Lead to separation of linked genes A A B B x a a b b Gametes Parental A B Recombinant a B a A Genetics 2011 b b 5 • In dihybrid crosses crosses, departures from a 1:1:1:1 ratio of F1 g gametes indicate that the two genes are on the same chromosome. • Example: the white and yellow genes on the X chromosome of Drosophila. • yellow (y+) – yellow body color • white (w+) – white eye color Lectured by Han-Jia Lin Lectured by Han-Jia Lin Genetics 2011 Detecting linkage by analyzing the progeny of dihybrid crosses: X X--linked F1 males get their only X chromosome from their mothers F1 females are dihybrids 6 Genetics 2011 Designations of “parental” and “recombinant” relate to past history history. • Compare allele configurations in F2 to P generation Note that in this cross: • 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. Deviation from 1:1:1:1 segregation ti in i F2 indicates the genes are linked Note that in this cross involving X-linked genes, only the F2 male progeny were counted 7 Lectured by Han-Jia Lin 8 Lectured by Han-Jia Lin Genetics 2011 Autosomal genes can also exhibit linkage • Detect linkage by generating a double heterozygote and crossing to homozygous recessive (testcross) • Parental class outnumbers recombinant class demonstrating linkage. Genetics 2011 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 p chance events OR linkage. • Ratios alone will never allow you to determine if observed b dd data t are significantly i ifi tl diff differentt ffrom predicted di t d values. • The larger your sample, sample the closer your observed values are expected to match the predicted values. • Chi C squa square e test es measures easu es “goodness good ess o of fit” between observed and expected (predicted) results. 9 . 5.4 Lectured by Han-Jia Lin • Accounts for sample size, or the size of the experimental population Lectured by Han-Jia Lin Genetics 2011 C Concepts t off Hypothesis H th i T Testing ti • ٿᅿଷ Genetics 2011 A l i the Applying th chi hi square test t t • Framing a hypothesis • Null hypothesis (คଷ); denoted by H0. H0 • Alternative hypothesis (ჹҥଷ); denoted by H1or Ha. • Null hypothesis – observed values are not p values different from the expected • љٯݤη • For linkage studies – no linkage is null hypothesis • Expect a 1:1:1:1 ratio of gametes. • H0Ǻࢂคޑǹᔠჸࡐ۔གྷޑ๊ܔଷ • H1ǺࢂԖޑǹᔠჸࡐ۔གྷள่݀ޑډ H1 ࢂԖ ޑᔠჸࡐ۔གྷள่݀ޑډ • ीޑᔈҔ • Alternative hypothesis yp – observed values are different from expected values • H0Ǻࣴࡐޣزགྷޑ๊ܔଷǹࣴزჹຝࢂమқޑ • H1Ǻࣴࡐޣزགྷள่݀ޑډǹࣴزჹຝࢂԖޑ 11 Lectured by Han-Jia Lin 10 • For linkage studies – genes are linked. • Expect significant deviation from 1:1:1:1 ratio. Lectured by Han-Jia Lin 12 Genetics 2011 Genetics 2011 Information needed for the chi chi-square test A l i the Applying th chichi hi-square test t t Calculate the chi-square Use data from breeding experiment • Total number of progeny • How many classes of progeny p g observed in each class • Number of offspring Consider degrees of freedom (df) in the experiment • df = N – 1 ((where N is the number of classes)) Determine a p value using chi-square value and df • Probability that the deviation from expected numbers had occurred by chance • Use table 5.1 Ca cu a e number Calculate u be o of o offspring sp g e expected pec ed in each class if there is no linkage (1:1:1:1 segregation) 13 Lectured by Han-Jia Lin 14 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 Applying pp y g the chichi-square q test to see if genes A and B are linked Critical chi chi--square values Use p value of 0 0.05 05 as cutoff Chi-square values that lie in the yellow region of this table allow rejection of the null hypothesis with >95% confidence If null hypothesis is rejected, then linkage can be postulated Fig. 5.5 Experiment p 1: Experiment 2: 15 Lectured by Han-Jia Lin 16 Lectured by Han-Jia Lin Table 5.1 Genetics 2011 Genetics 2011 Reciprocal exchanges between homologous chromosomes are the physical basis of recombination. Recombination results when crossingg over during meiosis separates linked genes genes. • 1909 – Frans Janssens observed chiasmata, regions in which nonsister chromatids h tid off h homologous l chromosomes h cross over each other. • Thomas Hunt Morgan suggested these were sites of chromosome breakage and change resulting in genetic 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. 17 Lectured by Han-Jia Lin 18 Lectured by Han-Jia Lin Genetics 2011 Genetic recombination between car and Bar genes on the Drosophila X chromosome Genetics 2011 Chiasmata mark the sites of recombination. Early prophase Leptotene and zygotene Diplotene Lectured by Han-Jia Lin Fig. 5.6 19 Fig. 5.7 a-c Lectured by Han-Jia Lin 20 Genetics 2011 Chiasmata mark the sites of recombination. Genetics 2011 Recombination frequencies for pairs of genes reflect distance between them. • Alfred H. Sturtevant – Percentage of q y recombination,, or recombination frequency (RF) reflects the physical distance separating two genes. g • 1 RF = 1 map unit (or 1 centiMorgan, cM) Terminalization – movement of chiasmata Anaphase A h – chromosome h separation occurs after chiasmata reach the telomeres Two recombinant and two parental gametes are produced Fig. 5.5 d-f 21 Lectured by Han-Jia Lin 22 Lectured by Han-Jia Lin Fig. 5.8 Genetics 2011 Genetics 2011 Properties of linked versus unlinked genes E d off Part End P t1 • You should have learned… • Genes close together on the same chromosome are linked and do not segregate independently. • Linked genes lead to a larger number of parental class l than h expected d iin d double bl h heterozygotes. • Mechanism of recombination is crossing over. • Chiasmata are the visible isible signs of crossing o over. er • The farther away genes are the greater the opportunity for chiasmata to form form. • Recombination frequencies reflect physical distance between genes. Table 5.2 23 Lectured by Han-Jia Lin 24 Lectured by Han-Jia Lin Genetics 2011 Mapping: Locating genes along a chromosome Fig. 5.9a Genetics 2011 Mapping: Locating genes along a chromosome • Two-point crosses: p help p Comparisons establish relative gene positions. positions • Genes are arranged in a line ga along chromosome. 25 Lectured by Han-Jia Lin Genes are arranged in a line along a chromosome. Fig. 5.9 b-d 26 Lectured by Han-Jia Lin Genetics 2011 Li it ti Limitations off ttwo point i t crosses Genetics 2011 Three Point Crosses: A faster more accurate method to map genes • Difficult to determine gene order if two genes are close l ttogether th • Actual distances between g genes do not always add up. • Pairwise crosses are time and labor consuming. 27 Lectured by Han-Jia Lin Fig. 5.10a Lectured by Han-Jia Lin 28 Genetics 2011 Genetics 2011 Double recombinants indicate order of three genes Analyzing the results of a three three-point cross Testcross progeny have four sets of reciprocal pairs of genotypes pr must be in the middle because longest distance is between vg and b • Most frequent pair has parental configuration of alleles • Least frequent pair results from double crossovers • Examination of double crossover class reveals which gene is in the middle Fig. g 5.10b Fig 5.10 Fig. 5 10 29 Lectured by Han-Jia Lin 30 Lectured by Han-Jia Lin Genetics 2011 Analyzing the results of a three point cross Genetics 2011 Analyzing the results of a three point cross • Look at two genes at a time and compare to parental parental. Fig. 5.11 c,d Fig. 5.11 a,b Lectured by Han-Jia Lin 31 32 Lectured by Han-Jia Lin Genetics 2011 vg – pr distance 252 + 241 + 13 + 9 4197 X 100 = 12.3 m.u. b – pr distance 131 + 118 + 13 + 9 4197 X 100 = 6.4 m.u. Genetics 2011 Interference: The number of double crossovers may be less than expected. vg – b distance 252 + 241 + 131 + 118 X 100 = 17.7 m.u. 4197 • Sometimes the number of observable d bl crossovers iis lless th double than expected t d if the two exchanges are independent. Correction for Double Crossovers 33 Lectured by Han-Jia Lin • Occurrence of one crossover reduces likelihood that another crossover will occur i adjacent in dj t parts t off th the chromosome. h • Chromosomal interference – crossovers do nott occur independently. i d d tl • Interference is not uniform among chromosomes h or even within ithi a 34 chromosome. Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 Calculation of interference in the three--point cross in Figure 5.10 three M Measuring i interference i t f • Compare observed and expected frequencies of double crossovers (DCO) • Expected probability of double crossovers is the product of the single crossover frequencies in each interval • Probability of single crossover between vg and pr is 0.123 (12.3 m.u.) • Probability off single crossover between pr and b is 0.064 (6.4 m.u.) • Interference = 1 – coefficient of coincidence. • If interference = 0, crossovers in adjacent regions occur independently of each other 35 Lectured by Han-Jia Lin • If interference = 1, no double crossovers occur Lectured by Han-Jia Lin 36 Genetics 2011 Calculation of interference in the threethreepoint cross in Figure 5.10 (cont) Genetics 2011 Do Genetic and Physical maps correspond? Expected probability of double crossovers • Order of genes is correctly predicted by physical h i l maps. • Distance between genes is not always similar on physical maps. Observed p proportion p of double crossovers • Double, triple, and more crossovers • Only 50% recombination frequency observable in a cross • Variation across chromosome in rate of recombination 37 Lectured by Han-Jia Lin • Mapping functions compensate for inaccuracies, but are not often imprecise. Lectured by Han-Jia Lin Genetics 2011 38 Genetics 2011 Are genetic maps and physical maps correlated? • Th The order d off genes iin a genetic ti map is i the th same as the order of those same genes along the DNA molecule of a chromosome. • The physical distance (amount of DNA separating gg genes)) is not always y the same as the genetic distance between genes. Genes chained together by linkage relationships are known as linkage groups. groups • 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 R bi ti ffrequency iis nott uniform if across a chromosome. • Recombination hotspots • Recombination deserts Fig. 5.13 Lectured by Han-Jia Lin 39 40 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 Saccharaomyces cerevisiae life cycle T t d analysis Tetrad l i in i fungi f i • Model organisms for understanding the mechanism of recombination because p p products of meiosis are all four haploid contained in ascus • Ascospores within ascus germinate into haploid individuals. • Saccharaomyces cerevisiae – bakers yeast p crassa – bread mold • Neurospora 41 Lectured by Han-Jia Lin Fig. 5.14 a 42 Lectured by Han-Jia Lin Genetics 2011 Genetic analysis in fungi Genetics 2011 Generation of diploid yeast cells that are heterozygous for two unlinked genes Phenotype of haploid fungi is direct representation of their genotype his4 TRP1 (a) x HIS4 trp1 () Æ his4/HIS4; trp1/TRP1 (a/) Mutations in haploids can affect appearance of cells and ability to grow under certain conditions • his4 mutant; recessive, unable to grow in absence of histidine • HIS4; dominant, grows in presence or absence of histidine • trp1 mutant; recessive, unable to grow in absence of tryptophan • TRP1; dominant dominant, grows in presence or absence of tryptophan Lectured by Han-Jia Lin 43 Fig. 5.15 a Lectured by Han-Jia Lin 44 Genetics 2011 Genetics 2011 Meiosis can generate three kinds of tetrads: Parental ditype yp (PD) ( ) Meiosis can generate three kinds of tetrads: Nonparental p ditype yp ((NPD)) his4 TRP1 (a) x HIS4 trp1 () Æ his4/HIS4; trp1/TRP1 (a/) Parental ditype (PD) - all spores with parental allele configurations (0/4 recombinants) his4 TRP1 ((a)) x HIS4 trp1 p (()) Æ his4/HIS4; trp1/TRP1 p ((a/)) Nonparental ditype (NPD) - all spores with nonparental allele configuration (4/4 recombinants) Lectured by Han-Jia Lin Fig. 5.15b 45 46 Lectured by Han-Jia Lin Fig. 5.15c Genetics 2011 Genetics 2011 Meiosis can generate three kinds of tetrads: Tetratype (T) T t d analysis Tetrad l i off unlinked li k d genes his4 TRP1 (a) x HIS4 trp1 () Æ his4/HIS4; trp1/TRP1 (a/) Tetratype (T) - four kinds of spores (2/4 recombinants) • Two have parental allele configurations • Two have recombinant allele configurations • Crossover between centromere and closest gene When genes are unlinked, number of PD = number of NPD Fig. 5.15e 47 Lectured by Han-Jia Lin Fig. 5.15d 48 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 How crossovers between linked genes generate different tetrads T t d analysis Tetrad l i off linked li k d genes When genes are lilinked, Wh k d number of PD >> number of NPD Fig 5.17 Fig. 5 17 Fig. 5.16 49 Lectured by Han-Jia Lin 50 Lectured by Han-Jia Lin Genetics 2011 How crossovers between linked genes generate different tetrads (cont) Genetics 2011 How crossovers between linked genes generate different tetrads (cont) Fig. 5.17 Fig. 5.17 51 Lectured by Han-Jia Lin 52 Lectured by Han-Jia Lin Genetics 2011 Calculating recombination frequencies in tetrad analysis Genetics 2011 Evidence that recombination takes place at the fourfour-strand stage For the cross in Figure 5.16: Fig. 5.18 53 Lectured by Han-Jia Lin 54 Lectured by Han-Jia Lin Genetics 2011 Evidence for exception to the rule that recombination is reciprocal Meiotic recombination almost always generates equal numbers of both kinds of recombinant progeny (2:2 segregation) Rare tetrads will Lectured by Han-Jia Lin t 31 13 Copyright © The McGraw-Hill Companies, Inc. Permission required to reproduce or display Hartwell et al 4th ed Genetics 2011 N Neurospora crassa life lif cycle l Fig. 5.19 55 56 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 N Neurospora f form ordered d d tetrads t t d S Segregation ti patterns tt in i ordered d d ascii • U Undergo d meiosis i i I and d II as usual, l b butt a single i l round d off mitosis after 2nd meiotic division – produces octad • Ascus is very narrow and spindle forms parallel to long axis Two genetically identical ascospores are next to each other Arrangement of chromatids can be inferred from position of ascospores FDS (first-division segregation patterns Fig. 5.20 Lectured by Han-Jia Lin Fig. 5.21 a 57 Genetics 2011 S Segregation ti patterns tt in i ordered d d tetrads t t d Number of second division tetrads is used to calculate the distance between a gene and a centromere Gene – centromere distance = divide percentage of second division tetrads by 2 58 Lectured by Han-Jia Lin Genetics 2011 Ordered tetrads help locate genes in relation to the centromere • Neurospora cross thr+ arg+ x thr arg Æ tetrads in 7 genotype classes Centromere – thr distance: Centromere – arg distance: Fig. 5.21 b Lectured by Han-Jia Lin SDS (second-division segregation patterns Fig. 5.22a 60 59 Lectured by Han-Jia Lin Genetics 2011 D t Determining i i li linkage k with ith ordered d d tetrads t t d Genetics 2011 Calculating map distance with ordered tetrads Neurospora cross thr+ arg+ x thr arg Æ tetrads in 7 genotype classes arg – thr distance: If thr and arg are linked, PD >> NPD This calculation doesn't doesn t account for double crossovers PD = 72 + 1 = 73 >> NPD = 1 + 2 = 3 Fig. 5.22b Fig. 5.22a 61 Lectured by Han-Jia Lin 62 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 Rules for tetrad analysis in ordered and unordered tetrads Table 5 5.3 3 63 Lectured by Han-Jia Lin E d off partt 2 End • 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 p y exhibit a recombination frequency q y of 50%. • Statistical analysis helps determine whether or not two genes assort independently. • The greater the physical distance between linked genes genes, the higher the recombination frequency. • Genetic maps p are visual representations p 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. 64 Lectured by Han-Jia Lin Genetics 2011 Genetics 2011 Twin spots p in Drosophila p Mitotic recombination can produce genetic mosaics. Double heterozygous Drosophila females y sn+/y+ sn yellow (y) mutant – yellow body wildtype (y+) – brown body g ((sn)) mutant – short and curled bristles singed wildtype (sn+) – long and straight bristles • Mitotic recombination is rare. • Initiated I iti t d by b • Mistakes in chromosome replication p • Chance exposure to radiation • Curt Stern – observed “twin twin spots” spots in Drosophila – a form of genetic mosaicism. • Animals contained tissues with different genotypes. 65 Lectured by Han-Jia Lin Lectured by Han-Jia Lin Genetics 2011 Mitotic crossing over between sn and centromere in Drosophila 66 Fig. 5.23 Genetics 2011 Sectored yeast colonies can arise from mitotic recombination • Diploid ADE2/ade2 • White colonies are wildtype • Red sectors are ade2/ade2 d 2/ d 2 • Size of sector indicates when recombination took place 68 67 Lectured by Han-Jia Lin Lectured by Han-Jia Lin Fig. 5.25 Genetics 2011 Genetics 2011 Mit ti Recombination Mitotic R bi ti and dC Cancer • Retinoblastoma gene (Rb) • Wild type (Rb+); mutant type (Rb-) • Heterozygote in normal somatic cells cells, homozygote in cancer cells (40%) • Mitotic Recombination takes place in where and when when~ (penetrance) E d off P End Partt 3 • You should have learned… • The cause of mitotic recombination • The twin spot in Drosophila • The relationship between mitotic recombination and human diseases 69 Lectured by Han-Jia Lin Genetics 2011 E d off the End th class l • Ask yourself, do you…. • Understand the relationship between linkage, g recombination, and crossing over. • Show how exceptions to independent assortment provide a way to map genes on chromosomes. • Explain where crossing over occurs during meiosis to give rise i tto recombinants bi t and d th the experimental i t lb basis i supporting ti that conclusion. • Derive genetic maps from two two- and three three-point point testcrosses testcrosses. • Measure positive and negative interference and understand their effect on map distances distances. 71 Lectured by Han-Jia Lin 70 Lectured by Han-Jia Lin