Quiz Section 4 Molecular analysis of inheritance: An amphibian puzzle

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1 Genome 371, Autumn 2018 Quiz Section 4 Molecular analysis of inheritance: An amphibian puzzle Goals: To illustrate how molecular tools can be used to track inheritance. In this particular example, we will see how one such tool can give us a glimpse into the peculiar mode of inheritance in Bufo baturae, a species of toad found in the Karakoram mountain range in Pakistan. Be warned, this is not a fully solved puzzle, there are lots of unanswered questions! Outline of exercise Part 1. Defining the puzzle with Bufo baturae reproduction. Part 2. Brainstorm: come up with possible solutions and how to see if that s what happens Part 3. Playing detective: Using the available clues to deduce how these toads reproduce. Part 4. Take-home material. Part 1. Reproduction in a Bufo species. Shown below is a species of toad found in the mountains of Pakistan, along with a typical karyotype from a mitotic cell in this species. 1. What is the chromosome number N in this toad (i.e., how many chromosomes per genome)? As an example: N for humans is What is the ploidy in this toad? How many double-stranded DNA molecules (i.e., double helices) are represented in the karyotype shown? 3. What do you think is the puzzle/problem regarding reproduction of this toad? Genome 371 QS4 Worksheet 1

2 Part 2. How might reproduction work in a triploid species? Work in your groups to sketch one or more idea(s). Genome 371 QS4 Worksheet 2

3 Part 3. (a) DNA content analysis: For a diploid organism, what do you expect to see if you compare the amount of DNA per cell in somatic cells vs. gametes? Look first at the data from a diploid toad species (top). Do the data match your expectations? Then examine the data for blood cells and gametes from our Bufo baturae triploid toad. What do you conclude about the ploidy of the gametes from this species? Do the data help you eliminate one or more models from the previous page? Genome 371 QS4 Worksheet 3

4 (b) Gametocytes (i.e., germline cells that can give rise to gametes) from male (left) and female (right) Bufo baturae in early prophase of Meiosis I with synapsed chromosomes. Describe what you see. How do you interpret the figure? Are these data consistent with what you concluded on the previous page? Genome 371 QS4 Worksheet 4

5 (c) PCR analysis of two microsatellite marker loci that are known to be on the same chromosome. Results for a male and female toad and 13 of their offspring are shown. The thin horizontal dotted lines are to help you see the alignment of bands across the gel. Based on the genotypes shown, deduce the parental allele arrangements. Genome 371 QS4 Worksheet 5

6 Genome 371, Autumn 2018 QS 4 Part 4 Homework. 1. Diagram metaphase of Meiosis I that generated the eggs responsible for offspring #s 1 and 5 on page 5 of the worksheet. Mark the alleles on all chromatids. 2. Diagram the outcome you would have seen on the gel on page 5 of the worksheet if loci A and B had been on separate chromosomes. What would be different about the results? 3. Consider the following two scenarios. One is that it is always the same chromosome set that undergoes copy number increase or decrease during gametogenesis (e.g., if the chromosome with allele A1 undergoes copy number increase in one female during oogenesis, is it the same chromosome that undergoes copy number increase during oogenesis in her female offspring, and her female offspring, and so on). Alternatively, it could be that a randomly chosen chromosome set is targeted for copy number change in each generation (e.g., the chromosome with allele A1 in one female, and the chromosome with allele A3 in one of her female offspring, and the chromosome with allele A2 in another female offspring, and so on). How could you experimentally determine which of these two scenarios is correct? What results would you expect under each scenario? Genome 371 QS4 Worksheet 6

7 4. The maps below depict the cut sites ( A ) for the restriction enzyme AseI in a region of the human genome. This sequence exists as two alleles: allele I and an inversion of the portion indicated by the curly bracket, giving rise to allele II (grey arrow = gene in the region). If you cut a sample of human DNA with AseI and did Southern blot analysis to identify the genotype at this locus, which probe would you find more informative, the small probe or the large probe? Illustrate your answer by predicting the Southern blot results with each probe for a person who is homozygous for allele I, a person who is homozygous for allele II, and a heterozygous individual (use the gel image). 1 kb = 1000 base pairs. Genome 371 QS4 Worksheet 7

8 5. This question is a continuation of the triploid toad story you saw in quiz section. We are now tracking alleles of a gene G: a wild type allele (G) and two deletion mutations (g 1 and g 2). DNA from a male, a female and eight of their offspring was obtained, cut with a restriction enzyme and analyzed by Southern hybridization to a probe that can distinguish G, g 1 and g 2 alleles. The results are shown on the Southern blot image, where thick bands have twice as much hybridizing DNA as thin bands. What you deduced in quiz section: Adult Bufo baturae toads are triploid. Leading up to female meiosis, one set of chromosomes is duplicated and the duplicated chromosomes pair up with each other in meiosis. Thus, oocytes are tetraploid and eggs are diploid. Leading up to male meiosis, one set of chromosomes is destroyed such that spermatocytes are diploid and sperm are haploid. (a) Write down the genotypes of the female and male toads shown on the Southern blot. Male: (b) In the grid below the blot, write the egg and sperm genotypes that made up each offspring. (c) Complete the following statements based on the Southern blot results The chromosome destroyed in the male during gametogenesis had allele The chromosome that was duplicated in the female had allele (d) Sketch the pairing arrangement that must have occurred during metaphase I of meiosis in the female, marking the alleles clearly: Genome 371 QS4 Worksheet 8

9 5. (cont d from previous page) (e) Toads with two or more wild type G alleles are green, while toads with fewer than 2 wild type G alleles are brown. If the same male and female toads from the previous page are mated again, what progeny ratio of brown and green toads do you expect? Show your reasoning. Assume that the same chromosomes undergo duplication/destruction as before and that both g 1 and g 2 are complete loss of function alleles.!"!"!" Genome 371 QS4 Worksheet 9