Meiosis, Recombination and Mapping

1. Meiosis, Recombination and Mapping • Meiosis • Why bother to map your gene? • Mapping a mutant defining a single gene • Mapping a sequence • Material for you: • This presentation • PDFs of important papers • Review of basic Genetics • Problem sets with answers • Definitions of IBM map words • Contact me anytime!

2. Dempsy. Maize Handbook

3. Meiosis takes about 5 days, then there is about 8-10 more days to shed

4. Maize meiosis

5. Three major processes in meiotic prophase

6. Leptotene 1. Chromosomes become visable as threads 2. The Axial Element is installed

7. Leptotene-Zygotene Transition Zygotene The bouquet is formed Dramtic transient change in chromatin structure It all begins… Pairing Synapsis Recombination

8. Pachytene 1. Chromosomes are completely synapsed 2. Recombination is completed 3. Interference probably works here

9. METAPHASE I ANAPHASE I METAPHASE ANAPHASE METAPHASE II ANAPHASE II MITOSIS MEIOSIS Sister Chromatid cohesion and chiasmata holds bivalents together until the Metaphase to Anaphase to transition

10. Diplotene- Diakinesis 1. SC falls apart 2. Chromosomes further condense 3. Homologs held together by chiasmata

11. Homologous Chromosome Pairing

12. Synapsis

13. Axial element Lateral Element

15. Synapsis starts at the chromosome ends Anderson and Stack, Chr. Res 2002

16. Complete Synapsis Anderson et al, Genetics 2003

17. Recombination

18. 5’ 3’ Lichten

19. 600 500 400 300 200 100 0 Zygotene Leptotene Early pachytene Late pachytene Number of RAD51 foci Distribution of RAD51 recombination protein in normal maize meiosis Diplotene Pachytene Leptotene Mid zygotene Late zygotene Early zygotene Wojtek Pawlowski

21. Early Recombination Nodules in TEM Anderson, et al Genetics 2001

22. Allers and Lichten Cell 2001

23. Patterns of distribution of RAD51 recombination protein in normal maize meiosis.

24. Organisms rely on crossovers to segregate chromosomes… … but most organisms make very few crossovers per chromosome pair Anne Villeneuve

26. Inna Golubovskaya

27. Break

28. The Genetic Maps of Maize There 1176 genetic maps of maize. We will only use the old fashion “genetic” map and the IBM2 map

29. Where is your gene? • Relative to other genes? • One the chromosome? • Why do you care? • How do you find out?

30. Your gene relative to other Genes • The genetic map is based on an abstract principle: the percent recombination between two genes (or markers). • The amount of recombination is not uniform along the length of the chromosome- but that does not matter for mapping. f1 an1 rhl d8 150 170 184 197

31. Your gene on the chromosome

32. Why do you care? • It makes to whole map better. • Maybe there is already a sequence mapped to your gene! • Maybe there will be soon • Maybe you think that the mutant of your gene is just like a mutant in yeast. Using the yeast sequence, get some maize GSSs, map, do they map to your gene? • Map-based cloning may (will) be possible

33. How do you map a gene? • You need a DIFFERENCE to map- two alleles • First localize in a general region • Next fine structure mapping

35. How do you map two genes relative to each other?

36. y10 y10 y10 Y10 lg2 lg2 lg2 Lg2 Gametes: { y10 lg2 Parental Y10 Lg2 { y10Lg2 Recombinant Y10lg2 y10 lg2 Test Cross: Heterozygote cross multiply recessive (or co-dominant) tester line. The genotype of the gametes of the “tested” parent is also the phenotype of the progeny.

37. y10 y10 y10 Y10 lg2 lg2 lg2 Lg2 y10 lg2 y10 lg2 Genotype Phenotype Number of Progeny Yellow, liguleless 48 Y10 Lg2 y10 lg2 42 Wildtype y10Lg2 y10 lg2 6 Yellow Y10lg2 y10 lg2 liguleless 4 { Parentals { Recombinants

38. y10 y10 y10 Y10 lg2 lg2 lg2 Lg2 Number of Progeny Recombinants Total Progeny X 100 = cM 48 P: 42 6 6+4 48+42+6+4 X 100 = 10% = 10cM R: 10 cM between y10 and lg2 4

39. How can you determine gene order?? Three (or more) factor cross

40. True breeding miniature, white-eyed, yellow bodied fly True Breeding wild type X X mwy mwy MWY MWY Test cross X F1 mwy mwy MWY mwy Phenotype (Genotype) Number Min, white, yellow m w y 3501 Yellow y + + 25 White eyes w + + 3 Min m + + 1720 Yellow, white eyes y w + 1734 White eyes, min w m + 35 Min, yellow m y + 6 Wild type + + + 3471

41. The number of progeny per class is a clue. mwy +++ Parental: Parental P Single recombinants SR SR Single recombinants Double Recombinants DR Put the 8 progeny types into 4 reciprocal classes. m w y 3501 + + + 3471 m + + 1720 y w + 1734 w m + 35 y + + 25 w + + 3 m y + 6

42. M w y + + + Parental: m w y 3501 + + + 3471 m + + 1720 + w y 1734 w m + 35 + + y 25 + w + 3 m + y 6 P Recombinants Total Progeny SR X 100 = cM SR DR

43. M w y + + + Parental: m w y 3501 + + + 3471 m + + 1720 + w y 1734 w m + 35 + + y 25 + w + 3 m + y 6 P Recombinants Total Progeny SR X 100 = cM SR DR Recombination between m and w: (1720 + 1734 + 3 + 6) / 10495 = 33cM

44. M w y + + + Parental: m w y 3501 + + + 3471 m + + 1720 + w y 1734 w m + 35 + + y 25 + w + 3 m + y 6 P Recombinants Total Progeny SR X 100 = cM SR DR Recombination between m and w: (1720 + 1734 + 3 + 6) / 10495 = 33cM Recombination between w and y: (35 + 25 + 3 + 6) / 10495 = 0.7cM

45. M w y + + + Parental: m w y 3501 + + + 3471 m + + 1720 + w y 1734 w m + 35 + + y 25 + w + 3 m + y 6 P Recombinants Total Progeny SR X 100 = cM SR DR Recombination between m and w: (1720 + 1734 + 3 + 6) / 10495 = 33cM Recombination between w and y: (35 + 25 + 3 + 6) / 10495 = 0.7cM Recombination between m and y: (1720 + 1734 + 35 + 625) / 10495 = 33.5cM

46. M w y + + + Parental: m w y 3501 + + + 3471 m + + 1720 + w y 1734 w m + 35 + + y 25 + w + 3 m + y 6 P Recombinants Total Progeny SR X 100 = cM SR W is in the middle, because it took two crossovers to separate it from the other allele DR Recombination between m and w: (1720 + 1734 + 3 + 6) / 10495 = 33cM Recombination between w and y: (35 + 25 + 3 + 6) / 10495 = 0.7cM Recombination between m and y: (1720 + 1734 + 35 + 625) / 10495 = 33.5cM

47. M w y + + + Parental: m w y 3501 + + + 3471 m + + 1720 + w y 1734 w m + 35 + + y 25 + w + 3 m + y 6 P Recombinants Total Progeny SR X 100 = cM SR DR Recombination between m and w: (1720 + 1734 + 3 + 6) / 10495 = 33cM Recombination between w and y: (35 + 25 + 3 + 6) / 10495 = 0.7cM Recombination between m and y: (1720 + 1734 + 35 + 625) / 10495 = 33.5cM 33 cM 0.7cM m w y

48. Crossover Interference: Crossover in one region of a chromosome reduces the probability of a second crossover nearby. If no interference: Freq. of d.c.o. involving interval x and y = (RF x) (RF y) x y Exp dco=2.3% 33 cM 0.7cM Obs dco =0.08 m w y “coefficient of coincidence” C = obs d.c.o. / exp d.c.o. C = 1 intervals are independent C = 0 complete interference C=0.08/2.3

49. How can you map your MUTANT? In most cases, you must do crosses First localize it to a chromosome arm by: -Crossing to many TB or T-waxy translocation lines and scoring. This takes two (TB) to three (T-waxy) generations. Then, make crosses to close markers, and map. A possible discussion topic. And/Or -Make a mapping population, and use molecular markers to localize, and to fine map

50. Molecular Markers for mapping: RFLPs SSRs AFLPs Indels SNPs