1. PowerPoint Presentation Materials to accompany Genetics: Analysis and Principles Robert J. Brooker Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display CHAPTER 5 LINKAGE AND GENETIC MAPPING IN EUKARYOTES

3. INTRODUCTION Each chromosome is likely to carry many hundred or even thousands of different genes The transmission of such genes will violate Mendel’s law of independent assortment 5-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

4. The term linkage has two related meanings 1. Two or more genes can be located on the same chromosome 2. Genes that are close together tend to be transmitted as a unit Linkage influences inheritance patterns 5-3 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5.1 LINKAGE AND CROSSING OVER

5. Chromosomes are called linkage groups They contain a group of genes that are linked together The number of linkage groups is the number of types of chromosomes of the species For example, in humans 22 autosomal linkage groups An X chromosome linkage group A Y chromosome linkage group Genes that are far apart on the same chromosome may independently assort from each other This is due to crossing-over 5-4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

6. Crossing Over May Produce Recombinant Phenotypes In diploid eukaryotic species, linkage can be altered during meiosis as a result of crossing over Crossing over Occurs during prophase I of meiosis replicated sister chromatid homologues associate as bivalents Non-sister chromatids of homologous chromosomes exchange DNA segments Figure 5.1 illustrates the consequences of crossing over during meiosis 5-5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

7. 5-6 Figure 5.1 The haploid cells contain the same combination of alleles as the original chromosomes The arrangement of linked alleles has not been altered

8. 5-7 Figure 5.1 These haploid cells contain a combination of alleles NOT found in the original chromosomes These are termed parental or non- recombinant cells This new combination of alleles is a result of genetic recombination These are termed nonparental or recombinant cells

9. Bateson and Punnett Discovered Two Traits That Did Not Assort Independently In 1905, William Bateson and Reginald Punnett conducted a cross in sweet pea involving two different traits Flower color and pollen shape This is a dihybrid cross that is expected to yield a 9:3:3:1 phenotypic ratio in the F 2 generation However, Bateson and Punnett obtained surprising results Refer to Figure 5.2 5-8 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

10. Figure 5.2 5-9 A much greater proportion of the two types found in the parental generation

11. Bateson and Punnett Discovered Two Traits That Did Not Assort Independently They suggested that the transmission of the two traits from the parents was somehow coupled The two traits are not easily assorted in an independent manner However, they did not realize that the coupling is due to the linkage of the two genes on the same chromosome 5-10 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

12. 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 Figure 5.3 illustrates an experiment involving three traits Body color Eye color Wing length 5-11 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

13. 5-12 Figure 5.3

14. 5-13 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Morgan observed a much higher proportion of the combinations of traits found in the parental generation P Males P Females Morgan’s explanation: All three genes are located on the X chromosome Therefore, they tend to be transmitted together as a unit

15. However, Morgan still had to interpret two key observations 1. Why did the F 2 generation have a significant number of nonparental combinations? 2. Why was there a quantitative difference between the various nonparental combinations? 5-14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Morgan Provided Evidence for the Linkage of Several X-linked Genes

16. 5-15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Gray body, red eyes1,159 Yellow body, white eyes1,017 Gray body, white eyes 17 Yellow body, red eyes 12 Total2,205 Let’s reorganize Morgan’s data by considering the pairs of genes separately Red eyes, normal wings 770 White eyes, miniature wings 716 Red eyes, miniature wings 401 White eyes, normal wings 318 Total2,205 It was fairly common to get this nonparental combination But this nonparental combination was rare

17. To explain these data, Morgan considered the previous studies of the cytologist F.A. Janssens Janssens had observed chiasmata microscopically And proposed that crossing over involves a physical exchange between homologous chromosomes Morgan shrewdly realized that crossing over between homologous X chromosomes was consistent with his data He assumed crossing over did not occur between the X and Y chromosome The three genes were not found on the Y chromosome 5-16 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

18. Morgan made three important hypotheses to explain his results 1. The genes for body color, eye color and wing length are all located on the X-chromosome They tend to be inherited together 2. Due to crossing over, the homologous X chromosomes (in the female) can exchange pieces of chromosomes This created new combination of alleles 3. The likelihood of crossing over depends on the distance between the two genes Crossing over is more likely to occur between two genes that are far apart from each other Figure 5.4 illustrates how crossing over provides an explanation for Morgan’s trihybrid cross 5-17 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

19. 5-18 Figure 5.4 These parental phenotypes are the most common offspring because the genes are far apart These recombinant offspring are not uncommon

20. These recombinant offspring are fairly uncommon

21. These double recombinant offspring are very unlikely 1 out of 2,205

22. This method is frequently used to determine if the outcome of a dihybrid cross is consistent with linkage or independent assortment 5-20 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Chi Square Analysis

23. Genetic mapping is also known as gene mapping or chromosome mapping Its purpose is to determine the linear order of linked genes along the same chromosome Figure 5.8 illustrates a simplified genetic linkage map of Drosophila melanogaster 5-42 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5.2 GENETIC MAPPING IN PLANTS AND ANIMALS

25. The recombination frequencies between linked genes are additive, and the frequency of exchange is an estimate of the relative distance between two genes along the chromosome.

26. 5-43 Figure 5.8 Each gene has its own unique locus at a particular site within a chromosome

27. 5-44 Genetic maps are useful in many ways 1. They allow us to understand the overall complexity and genetic organization of a particular species 2. They can help molecular geneticists to clone genes 3. They improve our understanding of the relationships among different species 4. They can be used to diagnose, and perhaps, someday to treat inherited human diseases 5. They can help in predicting the likelihood that a couple will produce children with certain inherited diseases 6. They provide helpful information for improving agriculturally important strains through selective breeding programs Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

28. 5-45 Genetic maps allow us to estimate the relative distances between linked genes, based on the likelihood that a crossover will occur between them Experimentally, the percentage of recombinant offspring is correlated with the distance between the two genes If the genes are far apart  many recombinant offspring If the genes are close  very few recombinant offspring Map distance = Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Number of recombinant offspring Total number of offspring X 100 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

29. 5-46 Genetic mapping experiments are typically accomplished by carrying out a testcross A mating between an individual that is heterozygous for two or more genes and one that is homozygous recessive for the same genes Figure 5.9 provides an example of a testcross This cross concerns two linked genes affecting bristle length and body color in fruit flies Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display e = ebony body color e + = gray body color s = short bristles s + = normal bristles One parent displays both recessive traits It is homozygous recessive for the two genes (ss ee) The other parent is heterozygous for the two genes The s and e alleles are linked on one chromosome The s+ and e+ alleles are linked on the homologous chromosome

30. 5-47 Figure 5.9 Chromosomes are the product of a crossover during meiosis in the heterozygous parent Recombinant offspring are fewer in number than nonrecombinant offspring

31. 5-48 The data at the bottom of Figure 5.9 can be used to estimate the distance between the two genes Map distance = Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Number of recombinant offspring Total number of offspring X 100 Therefore, the s and e genes are 12.3 map units apart from each other along the same chromosome 76 + 75 542 + 537 + 76 + 75 X 100= = 12.3 map units

32. The first genetic map was constructed in 1911 by Alfred Sturtevant He was an undergraduate who spent time in the laboratory of Thomas Hunt Morgan 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.” 5-49 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Alfred Sturtevant’s Experiment

33. Sturtevant considered the outcome of crosses involving six different mutant alleles All of which are known to be recessive and X-linked 5-50 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Mutant allele (Phenotype) Wild-type allele (Phenotype) y (yellow body color)y + (gray body color) w (white eye color)w + (red eye color) w-e (eosin eye color)w-e + (red eye color) v (vermillion eye color)v + (red eye color) m (miniature wings)m + (normal wings) r (rudimentary wings)r + (normal wings) Therefore, his genetic map contained only 5 genes y, w, v, m and r Alleles of the same gene Alleles of different genes that affect eye color Alleles of different genes that affect wing length

34. The Hypothesis The distance between genes on a chromosome can be estimated from the proportion of recombinant offspring This provides a way to map the order of genes along a chromosome 5-51 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis Refer to Figure 5.10

35. 5-52 Figure 5.10

36. The Data 5-53 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Alleles Concerned Number Recombinant/ Total Number Percent Recombinant Offspring y and w/w-e214/21,736 1.0 y and v1,464/4,55132.2 y and r115/32435.5 y and m260/69337.5 w/w-e and v471/1,58429.7 w/w-e and r2,062/6,11633.7 w/w-e and m406/89845.2 v and r17/573 3.0 v and m109/40526.9

37. Interpreting the Data 5-54 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display In some dihybrid crosses, the percentage of nonparental (recombinant) offspring was rather low For example, there’s only 1% recombinant offspring in the crosses involving the y and w or w-e alleles This suggests that these two genes are very close together Other dihybrid crosses showed a higher percentage of nonparental offspring For example, crosses between the v and m alleles produced 26.9% recombinant offspring This suggests that these two genes are farther apart

38. 5-55 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display To construct his map, Sturtevant assumed that the map distances would be more accurate among genes that are closely linked Therefore, his map is based on the following distances y – w (1.0), w – v (29.7), v – r (3.0) and v – m (26.9) Sturtevant also considered other features of the data to deduce the order of genes For example, Percentage of crossovers between w and r was 33.7 Percentage of crossovers between w and v was 29.7 Percentage of crossovers between v and r was 3.0 Therefore, the gene order is w – v – r Where v is closer to r than it is to w

39. 5-56 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Sturtevant collectively considered all these data and proposed the following genetic map Sturtevant began at the y gene and mapped the genes from left to right yw vmr 129.7323.9 130.733.757.60

40. 5-57 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A close look at Sturtevant’s data reveals two points that do not agree very well with his genetic map The y and m dihybrid cross yielded 37.5% recombinants But the map distance is 57.6 The w and m dihybrid cross yielded 45.2% recombinants But the map distance is 56.6 So what’s up? As the percentage of recombinant offspring approaches a value of 50%- The likelihood of multiple crossovers increases Even numbers of crossover won’t be seen as recombination So the observed recombinations tend to underestimate the actual measure of map distance Refer to Figure 5.11

41. 5-58 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Multiple crossovers set a quantitative limit on measurable recombination frequencies as the physical distance increases A testcross is expected to yield a maximum of only 50% recombinant offspring Figure 5.11

42. 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 5-59 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Trihybrid Crosses b = black body color b + = gray body color pr = purple eye color pr + = red eye color vg = vestigial wings vg + = normal wings

43. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-60 Step 1: Cross two true-breeding strains that differ with regard to three alleles. 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 Do not have a JPG for this

44. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-61 Step 2: Perform a testcross by mating F 1 female heterozygotes to male flies that are homozygous recessive for all three alleles During gametogenesis in the heterozygous female F 1 flies, crossovers may produce new combinations of the 3 alleles Do not have a JPG for this

45. 5-62 Step 3: Collect data for the F 2 generation

46. 5-63 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Analysis of the F 2 generation flies will allow us to map the three genes The three genes exist as two alleles each Therefore, there are 2 3 = 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

47. 5-64 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 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 Do not have a JPG for this

48. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-65 Step 4: Calculate the map distance between pairs of genes To do this, one strategy is to regroup the data according to pairs of genes From the parental generation, we know that the dominant alleles are linked, as are the recessive alleles This allows us to group pairs of genes into parental and nonparental combinations Parentals have a pair of dominant or a pair of recessive alleles Nonparentals have one dominant and one recessive allele The regrouped data will allow us to calculate the map distance between the two genes

50. 5-66 Parental offspringTotalNonparental OffspringTotal Gray body, red eyes (411 + 61) 472 Gray body, purple eyes (30 + 2) 32 Black body, purple eyes (412 + 60) 472 Black body, red eyes (28 + 1) 29 944 61 The map distance between body color and eye color is Map distance = 61 944 + 61 X 100= 6.1 map units

51. 5-67 Parental offspringTotalNonparental OffspringTotal Gray body, normal wings (411 + 2) 413 Gray body, vestigial wings (30 + 61) 91 Black body, vestigial wings (412 + 1) 413 Black body, normal wings (28 + 60) 88 826 179 The map distance between body color and wing shape is Map distance = 179 826 + 179 X 100 = 17.8 map units

52. 5-68 Parental offspringTotalNonparental OffspringTotal Red eyes, normal wings (411 + 28) 439 Red eyes, vestigial wings (61 + 1) 62 Purple eyes, vestigial wings (412 + 30) 442 Purple eyes, normal wings (60 + 2) 62 881 124 The map distance between eye color and wing shape is Map distance = 124 881 + 124 X 100 = 12.3 map units

53. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 5-69 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 b prvg 6.112.3

54. A physical map of a chromosome or a genome that shows the physical locations of genes and other DNA sequences of interest. Physical maps are used to help scientists identify and isolate genes by positional cloning.