Crossing Over of John Edward’s Chromosomes Linkage & Mapping Linkage Eukaryotic chromosomes are long ds DNA molecules Typical chromosome contains thousands of genes (loci) Linkage loci located on the same chromosome linked loci tend to be transmitted as a unit Linkage Because they are a group of genes linked together, chromosomes are functionally linkage groups # of linkage groups = the # of types of chromosomes Crossing over causes loci that are far apart on the same chromosome to sometimes independently assort known as incomplete linkage Crossing Over Produce Recombinant Phenotypes Crossing over (meiotic recombination) Occurs during prophase I of meiosis at the bivalent stage zygotene - pachytene - diplotene Non-sister chromatids of homologous chromosomes exchange DNA segments Linkage Prevents Independent Assortment Figure 5.1 Crossing Over May Produce Recombinant Phenotypes These are termed parental gametes Figure 5.1 gametes with a combination of alleles NOT found in the original chromosomes as a result of meiotic recombination These are called nonparental or recombinant gametes Example of Linkage Bateson and Punnett conducted a cross in sweet pea involving two traits Flower color and pollen shape Dihybrid cross expected to give 9:3:3:1 phenotypic ratio of F2 phenotypes Observed linkage Called it coupling Example of Linkage x Purple flowers, long pollen (PPLL) Red flowers, round pollen (ppll) F1 offspring Purple flowers, long pollen (PpLl) Self-fertilization F2 offspring phenotypes Observed Observed Expected Expected Ratio Ratio number number (9:3:3:1) P NP NP P Purple flowers, long pollen Purple flowers, round pollen Red flowers, long pollen Red flowers, round pollen 296 19 27 85 15.6 1.0 1.4 4.5 240 80 80 27 9 3 3 1 Morgan Provided Evidence for the Linkage of Several X-linked Genes The first direct evidence of linkage came from studies of Thomas Hunt Morgan Morgan investigated several traits that followed an X-linked pattern of inheritance Body color Eye color Wing length Linkage to a Particular Chromosome x / y+ w+ m+ Y y w m/y w m F1 generation Sex linkage of all traits places them all on X chromosome x y+w+ m+/ y w m F2 generation P P F1 generation contains wild-type females and yellow-bodied, white-eyed, miniature-winged males. ywm/Y Females Tan body, red eyes, normal wings Tan body, red eyes, miniature wings Tan body, white eyes, normal wings Tan body, white eyes, miniature wings Yellow body, red eyes, normal wings Yellow body, red eyes, miniature wings Yellow body, white eyes, normal wings Yellow body, white eyes, miniature wings 439 208 1 5 7 0 178 365 Males Total 319 193 0 11 5 0 139 335 758 401 1 16 12 0 317 700 P Males P Females Morgan observed a much higher proportion of the combinations of traits found in the parental generation Morgan’s explanation: All three genes are located on the X chromosome Therefore, they tend to be transmitted together as a unit Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-13 Morgan Provided Evidence for the Linkage of Several X-linked Genes However, Morgan still had to interpret two key observations Why did the F2 generation have a significant number of nonparental combinations? Why was there a quantitative difference between the various nonparental combinations? Reorganize Morgan’s data considering pairs of genes separately Gray body, red eyes 1,159 Yellow body, white eyes 1,017 Gray body, white eyes Yellow body, red eyes Total 17 12 2,205 Red eyes, normal wings 770 White eyes, miniature wings 716 Red eyes, miniature wings White eyes, normal wings Total 401 318 2,205 But this nonparental combination was rare It was fairly common to get this nonparental combination Figure 5.4 These parental phenotypes are the most common offspring These recombinant offspring are not uncommon because the genes are far apart Figure 5.4 These recombinant offspring are fairly uncommon because the genes are very close together These recombinant offspring are very unlikely 1 out of 2,205 it is product of single cross over probabilities Incomplete Linkage Linked loci that are sometimes separated by recombination are inherited in a pattern between linked and independently assorting not always together not present in normal dihybrid ratios The percentage of offspring with the loci linked vs those with the loci separated is a measure of the physical distance separating the loci on the chromosome Alfred Sturtevant’s Analysis An undergraduate in the laboratory of T. H. Morgan Constructed first genetic map in 1911 Sturtevant wrote: “In conversation with Morgan … I suddenly realized that the variations in the length of linkage, already attributed by Morgan to differences in the spatial orientation of the genes, offered the possibility of determining sequences [of different genes] in the linear dimension of the chromosome. I went home and spent most of the night (to the neglect of my undergraduate homework) in producing the first chromosome map, which included the sex-linked genes, y, w, v, m, and r, in the order and approximately the relative spacing that they still appear on the standard maps.” Linkage and Genetic Maps Estimating the relative distances between linked genes, based on the amount of recombination occuring between them allows us to generate genetic maps If the genes are far apart many recombinant offspring If the genes are close very few recombinant offspring Number of recombinant offspring X 100 Total number of offspring The units of distance are called map units (mu) They are also referred to as centiMorgans (cM) One map unit is equivalent to 1% recombination frequency Map distance = Linkage Analysis and Mapping Genetic mapping experiments are typically accomplished by carrying out a testcross Example of a two-point mapping cross Cross of two linked genes affecting bristle length and body color in fruit flies s = short bristles + = normal bristles e = ebony body color + = gray body color One parent double recessive (homozygous recessive at both loci) – s/s ; e/e Other parent is heterozygous at both loci (+/s ; +/e) Chromosomes are the product of a crossover during meiosis in the heterozygous parent Recombinant offspring are fewer in number than nonrecombinant offspring Figure 5.9 5-47 Linkage Analysis and Mapping The phenotype data are used to estimate the distance between the two loci Number of recombinant offspring X 100 Map distance = Total number of offspring = 76 + 75 542 + 537 + 76 + 75 X 100 = 12.3 map units Therefore, the s and e genes are 12.3 map units apart from each other along the same chromosome Trihybrid Crosses Data from trihybrid crosses can also yield information about map distance and gene order The following experiment outlines a common strategy for using trihybrid crosses to map genes In this example, we will consider fruit flies that differ in body color, eye color and wing shape b = black body color b+ = grey body color pr = purple eye color pr+ = red eye color vg = vestigial wings vg+ = normal wings Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-59 Step 1: Cross two true-breeding strains that differ at three loci. Female is mutant for all three traits Male is homozygous wildtype for all three traits The goal in this step is to obtain F1 individuals that are heterozygous for all three genes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-60 Step 2: Perform a testcross by mating F1 female heterozygotes to homozygous recessive, male flies During gametogenesis in the heterozygous female F1 flies, crossovers may produce new combinations of the 3 alleles Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-61 Step 3: Collect data for the F2 generation Number of Observed Offspring (males and females) Phenotype Gray body, red eyes, normal wings 411 Gray body, red eyes, vestigial wings 61 Gray body, purple eyes, normal wings 2 Gray body, purple eyes, vestigial wings 30 Black body, red eyes, normal wings 28 Black body, red eyes, vestigial wings 1 Black body, purple eyes, normal wings 60 Black body, purple eyes, vestigial wings 412 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-62 Analysis of the F2 generation flies will allow us to map the three genes The three genes exist as two alleles each Therefore, there are 23 = 8 possible combinations of offspring If the genes assorted independently, all eight combinations would occur in equal proportions It is obvious that they are far from equal In the offspring of crosses involving linked genes, Parental phenotypes occur most frequently Double crossover phenotypes occur least frequently Single crossover phenotypes occur with “intermediate” frequency Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-63 The combination of traits in the double crossover tells us which gene is in the middle A double crossover separates the gene in the middle from the other two genes at either end In the double crossover categories, the recessive purple eye color is separated from the other two recessive alleles Thus, the gene for eye color lies between the genes for body color and wing shape Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-64 Step 4: Calculate the map distance between pairs of genes To do this, one strategy is to group the data according to pairs of phenotypes resulting from non-crossovers, single crossovers, & double crossovers Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-65 Phenotype Number of Observed Offspring Gray body, purple eyes, vestigial wings 30 Black body, red eyes, normal wings 28 Gray body, red eyes, vestigial wings 61 Black body, purple eyes, normal wings 60 Gray body, purple eyes, normal wings 2 Black body, red eyes, vestigial wings 1 Single crossover between b and pr 30 + 28 = 0.058 1,005 Single crossover between pr and vg 61 + 60 Double crossover, between b and pr, and between pr and vg 1+2 = 0.120 1,005 = 0.003 1,005 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-71 To determine the map distance between the genes, we need to consider both single and double crossovers To calculate the distance between b and pr Map distance = (0.058 + 0.003) X 100 = 6.1 mu To calculate the distance between pr and vg Map distance = (0.120 + 0.003) X 100 = 12.3 mu To calculate the distance between b and vg The double crossover frequency needs to be multiplied by two Because both crossovers are occurring between b and vg Map distance = (0.058 + 0.120 + 2[0.003]) X 100 = 18.4 mu Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-72 Step 5: Construct the map Based on the map unit calculation the body color and wing shape genes are farthest apart The eye color gene is in the middle The data is also consistent with the map being drawn as vg – pr – b (from left to right) In detailed genetic maps, the locations of genes are mapped relative to the centromere Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-69 Alternatively, the distance between b and vg can be obtained by simply adding the map distances between b and pr, and between pr and vg Map distance = 6.1 + 12.3 = 18.4 mu Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-73 Interference The product rule allows us to predict the likelihood of a double crossover from the individual probabilities of each single crossover P (double crossover) = P (single crossover X P (single crossover between b and pr) between pr and vg) = 0.061 X 0.123 = 0.0075 Based on a total of 1,005 offspring the expected number of double crossover offspring is = 1,005 X 0.0075 = 7.5 Interference Therefore, we would expect seven or eight offspring to be produced as a result of a double crossover However, the observed number was only three! Two with gray bodies, purple eyes, and normal sings One with black body, red eyes, and vestigial wings This lower-than-expected value is due to a common genetic phenomenon, termed positive interference The first crossover decreases the probability that a second crossover will occur nearby Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-75 Interference (I) is expressed as I = 1 – C where C is the coefficient of coincidence Observed number of double crossovers C= Expected number of double crossovers C= 3 7.5 = 0.40 I = 1 – C = 1 – 0.4 = 0.6 or 60% This means that 60% of the expected number of crossovers did not occur Since I is positive, this interference is positive interference Rarely, the outcome of a testcross yields a negative value for interference This suggests that a first crossover enhances the rate of a second crossover The molecular mechanisms that cause interference are not completely understood However, most organisms regulate the number of crossovers so that very few occur per chromosome Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-77 Somatic Cell Hybridization Fusion of human cell and rodent cell Eventually, most human sequences lost Lines carrying sub-sets of human chromosomes are compared to determine which express the given gene product entire chromosomes, portions (arms) of chromosomes have to have a detectible DNA sequence or protein Process of elimination determines on which chromosome (region of chromosome) a gene is located Hybrid Panel Other Mapping Techniques Cytogenetic Mapping Have a mutant phenotype Perform karyotype Observe altered chromosomes aneuploidies translocations deletions normal Aberrant chromosome probably location of gene translocation t(14;17) Patau Syndrome - Trisomy 13 Karyotype: 47, 13+ Partial Chromosomal Deletions cri-du-chat - 46, 5pFragile X mental retardation – 46, Xp- Monosomy loss of an entire chromosome lethal in all cases for autosomes XO – Turner’s syndrome is only viable monosomy Mapping Strategies Deletion Mapping Cross recessive heterozygote to deletion heterozygote lines appearance of recessive phenotype localizes recessive gene within deletion interval Complementation Determines if two phenotypes are caused by same mutant gene Complementation Analysis Complementation Analysis Complementation Analysis of Eye Color Mutations Complementation groups white, cherry, coral, apricot, buff W1, wch , wc , wa , wb Alleles of white gene garnet ruby vermillion carnation Complementation Analysis Mapping with Unknown Phase of Linkage When you have heterozygotes, but do not know what the phenotypes of the parents were. 12 possible diploid arrangements for 3 linked loci bm + pr + + + bm pr bm + bm brown midrib, virescent seedling, purple aleurone pr Use Results of Cross to Determine Phase of Linkage NCOs = parental chromosomes = phase DCOs = fewest Do not directly detect middle locus NCO Gives 3 Possibilities for Phase 12 possible diploid arrangements for 3 linked loci bm + pr + + + bm pr bm + bm pr DCO Gives Loci Order DCO Defines 1 of these 3 Possibilities for Phase Because a DCO was required to group all mutant loci on same homologue 12 possible diploid arrangements for 3 linked loci bm + pr + + + bm pr bm + bm pr