Chapter 6 Linkage and Chromosome Mapping in Eukaryotes

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1 Chapter 6 Linkage and Chromosome Mapping in Eukaryotes Early Observations By 1903 Sutton pointed out likelihood that there were many more unit factors than chromosomes in most species Shortly, observations made that some genes or unit factors seemed to segregate as if they were somehow linked together (linkage ) Eventually shown to be part of same chromosome and transmitted as a single unit (again sort of because of recombination ) Crossing Over Birds of a Feather Flock Together Segregation not always random, but then linkage isn t generally complete Crossing over or recombination of alleles between homologs occurs Degree of linkage is inversely related to the interlocus distance (closer = stronger linkage) Linkage maps were the first chromosome maps Or genes of a chromosome segregate together Genes on separate chromosomes do follow independent assortment Genes on the same chromosome exhibit linkage Complete if no crossing over Linkage decreases with increased interlocus distance recombination Linkage and Recombination Recombination occurs during meiosis Produces recombinant or crossover gametes (as opposed to parental or noncrossover gametes) Examples of parental and crossover gametes are shown in Figure 6-1 1

2 Linkage Ratio If two loci are located on the same chromosome, how similar is the F 2 ratio to that expected for independent assortment or to that expected as if they were inherited as a single unit factor? Genes on the same chromosome Linkage group Example: Complete Linkage Drosophila, bw (brown eyes) and hv (heavy wing vein) are closely linked and both wild types (red, thin) are dominant Cross homozygous bw hv + with bw + hv homozygote F 1 is bw hv + /bw + hv (red with thin veins) F 2 has only 3 phenotypes (not four) at a 1:2:1 ratio (brown, thin) : (red, thin) : (red, heavy) See Figure 6-2, also include testcross results repulsion phase coupling phase 2

3 Crossover Frequency Reflects Distance of Locus Separation Thomas Morgan and his undergraduate student Alfred Sturtevant, 1911 Morgan discovered X-linkage Crosses with 2 X-linked loci gave puzzling results Cross of yellow-bodies (y) and white-eyed (w) females with wt males (gray, red) F 1 females wt, males double recessive phenotype F 2 showed 99.5% parental phenotypes (yw or wtwt), but 0.5 percent were ywt or wtw (y and w had become separated ), Figure 6-3 cross A Thomas Hunt Morgan, the first nativeborn American to win the Nobel Prize (1933), founder of modern genetics Thomas Hunt Morgan ( ) A.H. Sturtevant 3

4 Fruit fly Experiments Continue More X-linked locus crosses more confusion Second cross of w locus and m locus (miniature) gave only 65.5% parental phenotypes (Figure 6-3 cross B) Questions Why did the loci separate? Why did the frequency vary with the pair of loci studied? Morgan s Proposals Loss of linkage due to crossovers at chiasmata observed by Janssens (now points of genetic exchange), coined term crossing over Distance of separation affects probability of a chiasma form between them Morgan s Student: Sturtevant Realized that Morgan s ideas, if correct would allow the construction of genetic maps based upon recombination frequencies (linkage map) Neglected [undergraduate] homework that night and constructed first genetic map showing order and separation of different genes on a chromosome Crossover frequencies Yellow, white 0.5 percent White, miniature 34.5 percent Yellow, miniature 35.4 percent Map shown Figure 6.4 4

5 Simple Linkage Map Autosomal Linkage Sturtevant and colleague, Calvin Bridges, show linkage not limited to X-chromosome by 1923 (genes on autosomes behave similarly) Drosophila studies aided by the fact that crossing over doesn t happen in males in this species Not all species behave the same Single Crossovers Not all single crossovers occurring between homologous chromosomes will separate any given two loci during meiosis Figure 6-5 Probability increases as the % of the chromosome separating the two loci increases Note that a single crossover, even if it occurs 100% of the time in the region between the two loci, only separates the two alleles in half of the gametes produced does not effect the other two chromatids of the tetrad 20% recombinant gametes involves 40% of tetrads (Fig 6-6) Gametes from Single Crossover Multiple Crossovers Single crossover frequencies give distance between two loci but order can be more difficult to determine based only upon this type of data Example of a double crossover using 3 marker loci are shown in Figure Frequency of a double crossover is expected to the the product of the two individual frequencies Individual probabilities of 0.20 and 0.30 are going to both occur about 0.06 For 0.03 and 0.02 you get (lots of flies to look over ) 5

6 Double-Crossover Three-Point Mapping in Drosophila Criteria for a 3-point mapping cross Organism producing crossover games must be heterozygous at all loci to be studied All genotypes of gametes must be able to be determined from offspring phenotypes Sufficient numbers of offspring must be produced to have a representative sample of all crossover classes Three-point mapping experiment Offspring are a combination of parental (most common), single-crossover and double-crossover (least common) phenotypes 3-Point Mapping Map constructed at bottom based upon frequencies of different phenotypes Determining Gene Order Assumptions There are only 3 possible orders for 3 genes (w-y-ec, y-ec-w, and y-w-ec depending upon which is in the center), each producing different double-crossover phenotypes/genotypes in gametes Parental phenotype likely most common Single-crossover phenotypes less common Double-crossover phenotype most rare, also the center allele is always matched to From data determine which order most fits data best Mapping Genes in Maize Autosomal loci Gene order unknown Allele arrangements in heterozygous female F 1 not known Symbol + used for wt alleles (not pr + ) Cross and possibilities shown Fig 5-10a Results shown Fig 5-10b 6

7 Interference mu = single + double frequencies If the single crossover frequency between v and pr is and for pr and bm, then the double crossover frequency would be expected to be X = But is is Difference is result of interference (I) Coefficient of coincidence = observed/expected (0.804) The value for I here is = (positive) Interference is commonly positive but can be negative Inaccuracy in Distance Measurements Not all recombination events yield detectable recombinant chromosomes Double recombination between two loci Accuracy of distance estimates decrease with actual distance of separation of the loci under study 7

8 Genetic Maps Drosophila, maize and mouse genes extensively mapped (and genomes sequenced) Drosophila Four chromosomes (or linkage groups) Mutant variants of nearly all genes found Crossovers Involve Physical Exchange Between Chromatids Are chiasmata of meiotic prophase I related to crossing over? 1930s Zea mays, Creighton and McClintock Drosophila, Stern Zea mays Chromosome 9, c locus for endosperm color, wx locus for waxy carbohydrate in endosperm Each P generation also has a unique visible chromosome anomaly (dark staining blob, translocation) Mitotic Recombination Discovered by Stern in Drosophila Synapsis observed during mitosis Also occurs in some fungi Exchanges Between Sister Chromatids Sister Chromatid Exchanges (SCEs) Harlequin chromosomes BUdr in chromosomes, after replication only one chromatid contains Budr Stimulated by DNA damage Bloom syndrome Gene encodes DNA helicase 8

9 Linkage Analyses in Haploid Organisms Form diploid cells (zygotes) by the fusion of reproductive cells (gametes or isogametes when look identical) Common organisms Chlamydomonas and Neurospora species Neurospora Ordered asci (singular ascus) Sacks containing 8 spores in a string Each spore has genetic information from one single strand of a DNA duplex from each tetrad in the original zygotes Somatic Cell Hybridization 1960s Fuse mouse and human cell to form heterokaryon (cell has two diploid genomes) Nuclei fuse Human chromosomes lost until stable karyotype is achieved (number of and specific ones retained is quite variable) Karyotyping reveals which human chromosomes retained by each stable cell line Study of panel of different cell lines determines which specific human chromosome required for expression/presence of gene or gene product Synteny testing Tranlocations of portions of chromosomes Molecular Biology DNA markers RFLP, minisatellites PCR, microsatellites SNPs Chromosome walking or jumping Physical maps III Gp/gp 21 0 Fa/fa 78 A/a I/i 204 Le/le V/v R/r 60 A/a / I/i / Fa/fa / Le/le / V/v / Gp/gp / R/r ( ) / The garden pea, Pisum sativum, the model system used by Gregor Mendel; published Lamprecht H Agri Hort Gen 6:

10 Why Didn t Mendel Find Linkage? 10