1. Training session on Drosophila mating schemes Andreas Prokop

2. STEP 1: Remind yourself of the key differences between mitosis and meiosis: crossing-over / interchromosomal recombination during prophase I ( ➊ ) separation of homologous chromosomes during telophase I ( ➋ ) an additional division in meiosis ( ➌ ) This document is one part of a Drosophila genetics training package, the entire strategy of which is described in detail elsewhere (see link).link It is important that you view this PowerPoint file in presentation mode.

3. synapsis - interchromosomal recombination separating homologous chromosomes haploid Mitosis and meiosis separating sister chromatids generating sister chromatids for each of the homologous chromosomes diploid ➊ ➋ ➌

4. STEP2: Remind yourself of the basic rules of Drosophila genetics: law of segregation independent assortment of chromosomes linkage groups and recombination (recombination rule) balancer chromosomes and marker mutations

5. Law of segregation / linkage groups Homologous chromosmes are separated during meiosis

6. Law of segregation / linkage groups each offspring receives one parental and one maternal chromosome loci on the same chromsome are passed on jointly (linkage) 1 2 1

7. intra-chromosomal recombination takes place randomly during oogenesis Recombination rule: there is no recombination in males (nor of the 4 th chromosome) Complication: recombination in females

8. 7 instead of 3 different genotypes wildtype heterozygous homozygous mutant Complication: recombination in females

9. If the mutant alleles (blue and orange) were both lethal in homozygosis, which of these genotypes would fail to survive?” Balancers and stock keeping lethal mutations are difficult to keep as a stock and will eventually segregate out (i.e. be replaced by wt alleles)

10. remedy in Drosophila: balancer chromosomes Balancers and stock keeping

11. balancers carry easily identifiable dominant and recessive markers

12. Balancers and stock keeping balancers carry easily identifiable dominant and recessive markers balancers are homozygous lethal or sterile

13. balancers carry easily identifiable dominant and recessive markers balancers are homozygous lethal or sterile the products of recombination involving balancers are lethal Balancers and stock keeping only hetero- zygous flies survive and maintain the stock With balancers lethal mutations can be stably kept as stocks. In mating schemes, balancers can be used to prevent unwanted recombination. Balancers and their dominant markers can be used strategically to follow marker- less chromosomes through mating schemes.

14. 'X' indicates the crossing step; female is shown on the left, male on the right sister chromosomes are separated by a horizontal line, different chromosomes are separated by a semicolon, the 4 th chromosome will be neglected maternal chromosomes (inherited from mother) are shown above, paternal chromosomes (blue) below separating line the first chromosome represents the sex chromosome, which is either X or Y - females are X/X, males are X/Y generations are indicated as P (parental), F1, 2, 3.. (1 st, 2 nd, 3 rd.. filial generation) to keep it simple: dominant markers start with capital, recessive markers with lower case letters Rules to be used here:

15. Now apply your knowledge: follow a step-by-step explanation of a typical crossing task experienced during routine fly work you will be prompted to make your choices at each step of the mating scheme; take this opportunity before forwarding to see a solution

16. Do not yet start with the cross. You will first be asked a couple of questions! Task: To study the potential effect of a 2 nd chromosomal recessive lethal mutation m (stock 1) on brain development, you want to analyse certain neurons in the brain of m/m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2 nd chromosomal P- element insertion P(lacZ,w + ) (stock 2). To perform the experiment, you need to recombine m and P(lacZ,w + ) onto the same chromosome. Design a suitable mating scheme. Tip: w + on the P-element gives orange eyes when in white mutant background (w on 1 st )., Hu

17. Task: To study the potential effect of a 2 nd chromosomal recessive lethal mutation m (stock 1) on brain development, you want to analyse certain neurons in the brain of m/m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2 nd chromosomal P- element insertion P(lacZ,w + ) (stock 2). To perform the experiment, you need to recombine m and P(lacZ,w + ) onto the same chromosome. Design a suitable mating scheme. Tip: w + on the P-element gives orange eyes when in white mutant background (w on 1 st )., Hu What is the genotype of the recombinant fly stock you want to generate?, m

18. Task: To study the potential effect of a 2 nd chromosomal recessive lethal mutation m (stock 1) on brain development, you want to analyse certain neurons in the brain of m/m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2 nd chromosomal P- element insertion P(lacZ,w + ) (stock 2). To perform the experiment, you need to recombine m and P(lacZ,w + ) onto the same chromosome. Design a suitable mating scheme. Tip: w + on the P-element gives orange eyes when in white mutant background (w on 1 st )., Hu What is the genotype of the embryos you will analyse?, m For further information about concepts of lethality and stock keeping, see file "02-ConceptsDrosophila" on this LINK.LINK

19. Identify the eye colours of these flies Task: To study the potential effect of a 2 nd chromosomal recessive lethal mutation m (stock 1) on brain development, you want to analyse certain neurons in the brain of m/m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2 nd chromosomal P- element insertion P(lacZ,w + ) (stock 2). To perform the experiment, you need to recombine m and P(lacZ,w + ) onto the same chromosome. Design a suitable mating scheme. Tip: w + on the P-element gives orange eyes when in white mutant background (w on 1 st )., Hu

20. Identify all other markers of these flies, Hu Task: To study the potential effect of a 2 nd chromosomal recessive lethal mutation m (stock 1) on brain development, you want to analyse certain neurons in the brain of m/m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2 nd chromosomal P- element insertion P(lacZ,w + ) (stock 2). To perform the experiment, you need to recombine m and P(lacZ,w + ) onto the same chromosome. Design a suitable mating scheme. Tip: w + on the P-element gives orange eyes when in white mutant background (w on 1 st ). Cy If Hu Sb

21. , Hu Cy If Hu Sb Identify the balancer chromosomes Task: To study the potential effect of a 2 nd chromosomal recessive lethal mutation m (stock 1) on brain development, you want to analyse certain neurons in the brain of m/m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2 nd chromosomal P- element insertion P(lacZ,w + ) (stock 2). To perform the experiment, you need to recombine m and P(lacZ,w + ) onto the same chromosome. Design a suitable mating scheme. Tip: w + on the P-element gives orange eyes when in white mutant background (w on 1 st ).

22. , Hu Define the first cross! cross Does it matter which stocks you choose ♀♀ & ♂♂ from? Task: To study the potential effect of a 2 nd chromosomal recessive lethal mutation m (stock 1) on brain development, you want to analyse certain neurons in the brain of m/m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2 nd chromosomal P- element insertion P(lacZ,w + ) (stock 2). To perform the experiment, you need to recombine m and P(lacZ,w + ) onto the same chromosome. Design a suitable mating scheme. Tip: w + on the P-element gives orange eyes when in white mutant background (w on 1 st ).

23. Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. Since you will select females in F1, it does not matter whether you choose females from stock 1 or 2. The outcome it identical for females. Test it out! Selecting F1 stock 1stock 2

24. Now select gender and genotype! Selecting F1 Y + ; second? ; third? m first? stock 1stock 2 Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange.

25. m Selecting F1 Y + first? ; second? ; third? take females (to allow for recombination) select against curly wings (to have P- element & mutation) stock 1stock 2 Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange.

26. Challenge: how to select for the F2 flies carrying correctly recombined chromosomes? Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. each layed egg has its individual recombination history In the germline of the selected females, recombination takes place P(lacZ,w + ) m m no recombination recombination * Remember: recombination occurs at random! If the chromosomal positions of m and P(lacZ,w + ) are known, the recombination frequency can be calculated (see genetic manual); typically between 20-100 single crosses are required. * gonad haploid gametes * * * * * * * * * * Designing the F1 cross ++ m

27. , Hu Males from which stock below? Designing the F1 cross 1 st step: stabilise recombinant chromosomes with a balancer ++ m Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange., Hu

28. F2 selection F2 ; + TM6b + Sb ; not important here; ignored hereafter third?second?first? Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. ++ m, Hu P(lacZ,w + ),[m]* CyO P(lacZ,w + ),[m]* If [m]* CyO [m]* If [m]* = potentially present w Y + Y w w + w

29. F2 P(lacZ,w + ),[m]* CyO P(lacZ,w + ),[m]* If [m]* CyO [m]* If select for orange eyes, for If, against Cy ; w Y + Y w w + w + TM6b + Sb ; select for white back-ground, to see orange eyes Define your selection criteria for 2 nd and 1 st chromosomes choosing males is preferable for reasons explained later Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. third?second?first?

30. Selecting recombinants ; w Y P(lacZ,w + ),[m]* If F2, Hu Choose female from available stocks Key strategy: backcross to "m" stock Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. Before explaining this choice, let's do the next crossing step. Task: determine whether the recessive mutation m has indeed recombined with P(lacZ,w + ) in these males

31. ; + + m CyO X stock 1 ; w Y P(lacZ,w + ),[m]* If F2 Now it becomes clear why If marked males were chosen: stock 1 is w +, therefore F3 animals will not display white mutant background. In those animals, the P(lacZ,w + ) chromosome cannot be identified by eye colour. It cannot be distinguished from the m chromosome, and can therefore not be selected to establish a stable recombinant stock. Having an "asymmetric" marker distribution (If vs. Cy) solves this problem. performing the back cross Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. Males need to be chosen to prevent recombination of the If chromosome (not a balancer!) with the potentially recombinant chromosome. Furthermore, remember that each F2 individual derived from the recombination cross, reflects a unique potential crossing-over event, and these flies need to be crossed individually. For single crosses, males are preferable because they can mate several females. Even if they die early, fertilised females store enough sperm to lay eggs for a while. Hence, the likelihood that a single male successfully establishes a stable daughter generation is considerably higher than a single female.

32. How can you identify the recombinants? Define your criteria! performing the back cross F3 m P(lacZ,w + ),[m]* CyO P(lacZ,w + ),[m]* CyO If m If Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. ; + + m CyO X stock 1 ; w Y P(lacZ,w + ),[m]* If F2 2 nd ? ; first? + Y + w single males!

33. How can you identify the recombinants? Define your criteria! performing the back cross F3 m P(lacZ,w + ),[...]* CyO P(lacZ,w + ),[m]* CyO If m If Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. ; + + m CyO X stock 1 ; w Y P(lacZ,w + ),[m]* If F2 2 nd ? ; first? + Y + w Alternatively... lethality as a selection criterion: if there are flies which are neither If nor Cy, then m is in heterozygous condition and [m]* is not present.

34. performing the back cross F3 m P(lacZ,w + ),[m]* CyO P(lacZ,w + ),[m]* CyO If m If Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. ; + + m CyO X stock 1 ; w Y P(lacZ,w + ),[m]* If F2 2 nd ? ; first? + Y + w... if all flies are either If or Cy, then [m]* is present, and these homozygous flies die. What flies do you select? Define your criteria! for Cy, against If

35. performing the back cross F3 m P(lacZ,w + ),[m]* CyO P(lacZ,w + ),[m]* CyO If m If Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. ; + + m CyO X stock 1 ; w Y P(lacZ,w + ),[m]* If F2 2 nd ? ; first? + Y + w Cross ♂♂ and ♀♀ to establish a stock. In the next generation you could start selecting for orange eyes for future purposes. TASK DONE!

36. Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. F2 P(lacZ,w + ),[m]* CyO P(lacZ,w + ),[m]* If [m]* CyO [m]* If ; w Y + Y w w + w + TM6b + Sb ; third?second?first? What would have happened if you had chosen the CyO males?

37. performing the back cross F3 m P(lacZ,w + ),[m]* CyO P(lacZ,w + ),[m]* CyO m CyO Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. ; + + m CyO X stock 1 ; w Y P(lacZ,w + ),[m]* CyO F2 2 nd ? ; first? + Y + w This is a "symmetric" constellation and therefore a DEAD END! Recombinants cannot be identified: because of w + background (red box), "orange eyes" cannot be used for selection. Cy alone does not distinguish between flies carrying m versus P(lacZ,w + ),[m]* single males!

38. Lethality can also be used as criterion in a different strategy where potential recombinants are first bred into stocks. Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. ; w Y P(lacZ,w + ),[m]* CyO F2 single males! Selecting recombinants, Hu Choose female from available stocks

39. F3 If P(lacZ,w + ),[m]* CyO P(lacZ,w + ),[m]* CyO If CyO Task: Recombine P(lacZ,w + ) with the lethal mutation m! Note that P(lacZ,w + ) is orange. 2 nd ? ; first? w Y w w ; w Y P(lacZ,w + ),[m]* CyO F2 single males! Establishing stable stocks, Hu What flies do you select? Define your criteria! for Cy, for orange eyes against If Now cross ♂♂ and ♀♀ to establish a stock. If there are non-Cy flies in F4, then [m]* is not present, if all F4 flies are Cy you have a recombinant! TASK DONE!

40. Now continue with independent crossing tasks (Suppl. Mat. 4 under this link)link for further info on flies and fly genetics see: http://www.flyfacility.ls.manchester.ac.uk/forthepublic