Training session on Drosophila mating schemes

1. Training session on Drosophila mating schemes

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)

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

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 1 • each offspring receives one parental and one maternal chromosome • loci on the same chromsome are passed on jointly (linkage) 2 1

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

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

9. Balancers and stock keeping • remedy for work on mice: genotyping of every new generation through PCR analysis of tail tips - impossible in flies! • lethal mutations are difficult to keep as a stock and will eventually segregate out

10. Balancers and stock keeping • lethal mutations are difficult to keep as a stock and will eventually segregate out • remedy in Drosophila:balancer chromosomes

11. Balancers and stock keeping • 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 and stock keeping only heterozygous flies survive and maintain the stock • balancers carry easily identifiable dominant and recessive markers • balancers are homozygous lethal or sterile • the products of recombination involving balancers are lethal • During mating schemes, balancers can be used to prevent unwanted recombination - providing an additional means to the recombination rule. • You can use balancers and their dominant markers strategically to follow marker-less chromosomes through mating schemes.

14. Rules to be used here: • '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 4th 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.. (1st, 2nd, 3rd.. filial generation) • to keep it simple: dominant markers start with capital, recessive markers with lower case letters

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. Task:To study the potential effect of a 2nd chromsomal recessive lethal mutation m(stock 1) on brain development, you want to analyse certain neurons in the brain of m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2nd 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. , Hu Identify the eye colours of these flies

17. CyO If TM6b, Hu Sb CyO * * * * Task:To study the potential effect of a 2nd chromsomal recessive lethal mutation m(stock 1) on brain development, you want to analyse certain neurons in the brain of m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2nd 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. , Hu Identify all other markers of these flies

18. Task:To study the potential effect of a 2nd chromsomal recessive lethal mutation m(stock 1) on brain development, you want to analyse certain neurons in the brain of m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2nd 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. CyO If TM6b, Hu , Hu Sb CyO Identify the balancer chromosomes

19. cross Task:To study the potential effect of a 2nd chromsomal recessive lethal mutation m(stock 1) on brain development, you want to analyse certain neurons in the brain of m mutant embryos. These neurons can be specifically labelled with ß-Gal using a 2nd 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. , Hu Define the first cross! Assign ♀ & ♂ to these stocks

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

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

22. m + + gonad haploid gametes * Remember: recombination occurs at random * * * In the germline of the selected females, recombination takes place P(lacZ,w+) * * no recombination m * each layed egg has its individual recombination history P(lacZ,w+) m recombination * Task: Recombine P(lacZ,w+) with lethal mutation m Designing the F1 cross Challenge: how to select for the F2 flies carrying correctly recombined chromosomes?

23. choose the stock for the males m + + Stocks available: 1st step: stabilise recombinant chromosomes with a balancer Task: Recombine P(lacZ,w+) with lethal mutation m Designing the F1 cross

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

25. w P(lacZ,w+),[m]* Y CyO + + P(lacZ,w+),[m]* TM6b Y If w + [m]* w Sb CyO + [m]* w If [m]*= potentially present Task: Recombine P(lacZ,w+) with lethal mutation m Identify the eye colours! first second third F2 ; ; not important here; ignored hereafter

26. w P(lacZ,w+),[m]* Y CyO + + P(lacZ,w+),[m]* TM6b Y If w + [m]* w Sb CyO + [m]* w If select for white back-ground, to see orange eyes select for orange eyes, for Cy, against If [m]*= potentially present Task: Recombine P(lacZ,w+) with lethal mutation m Define your selection criteria for 2nd and 1st chromosomes first second third F2 ; ; not important here; ignored hereafter

27. F2 w P(lacZ,w+),[m]* ; Y CyO Choose female from available stocks Key strategy: backcross to "m" stock Task: Recombine P(lacZ,w+) with lethal mutation m Selecting recombinants Challenge: determine whether recessive "m" is present

28. F2 w P(lacZ,w+),[m]* ; Y CyO Problem: Each individual represents a unique recombination event. Solution: perform many(1) parallel single crosses, in each using ONE potentially recombinant male(2) and 3 to 5 females of stock1. (1) If the chromosomal positions of m and P(lacZ,w+) are known, the recombination frequency can be calculated; typically between 20-100 single crosses are required. (2) Males can mate several females. Even if they die early, females store enough sperm to lay eggs for a while. Hence, the likelihood that a single male successfully establishes a large enough daughter generation is considerably higher than a single female. Task: Recombine P(lacZ,w+) with lethal mutation m performing the back cross + m X ; stock 1 + CyO

29. F2 w P(lacZ,w+),[m]* ; Y CyO F3 2nd? m P(lacZ,w+),[m]* All flies Cy? If yes, the lethal mutation "m" is present on the putatively recombinant chromosome. m CyO To establish the recombinant stock, you need to distinguish these two genotypes, i.e. select for orange eyes. CyO P(lacZ,w+),[m]* CyO CyO Task: Recombine P(lacZ,w+) with lethal mutation m performing the back cross + m X ; stock 1 + CyO Can you spot the recombinants? Define your criteria!

30. F2 w P(lacZ,w+),[m]* ; Y CyO F3 1st? + w To establish the recombinant stock, you need to distinguish these two genotypes, i.e. select for orange eyes. ; + Y Task: Recombine P(lacZ,w+) with lethal mutation m performing the back cross + m X ; stock 1 + CyO Since you have w+ background, this strategy does not work m P(lacZ,w+),[m]* m CyO CyO P(lacZ,w+),[m]* CyO CyO

31. F2 w P(lacZ,w+),[m]* ; Y CyO Task: Recombine P(lacZ,w+) with lethal mutation m X Rethink your strategy: • given the complexity of genetic crosses, trial and error is often unavoidable • careful planning is pivotal!

32. single males! F2 aid 1 stock w P(lacZ,w+),[m]* ; Y CyO to establish stable fly stocks cross single potentially recombinant males to the balancer stock Task: Recombine P(lacZ,w+) with lethal mutation m X Choose female from available stocks • 2 possibilities: • You could use strategy 1, but add a parallel F1 cross between stock 1 and aid 1 to bring m into w mutant background (thus preparing it for the backcross in F2). Try whether it works for you! • Here we will use an alternative strategy, establishing stable stocks and test for lethality.

33. F2 aid 1 stock w P(lacZ,w+),[m]* ; Y CyO 2nd? If P(lacZ,w+),[m]* • for Cy • for orange eyes • against If • against white eyes w w CyO P(lacZ,w+),[m]* ; If CyO w Y CyO CyO take males & females Task: Recombine P(lacZ,w+) with lethal mutation m establishing stable stocks X F3 1st? select!

34. w w P(lacZ,w+),[m]* P(lacZ,w+),[m]* ; ; Y Y CyO CyO 2nd? All flies Cy? If yes, the marker "m" is present on the putatively recombinant chromosome. P(lacZ,w+),[m]* P(lacZ,w+),[m]* 1 w w P(lacZ,w+),[m]* CyO 2 ; CyO P(lacZ,w+),[m]* w Y maintain as stock CyO CyO 1 Task: Recombine P(lacZ,w+) with lethal mutation m select stable stocks X F3 F4 1st?

35. Now continue with independent crossing tasks