Training session on Drosophila mating schemes Andreas Prokop

Training session on Drosophila mating schemes Andreas Prokop

• This document is one part of a Drosophila genetics training package, the entire strategy of which is described in detail elsewhere (see link). • It is important that you view this Power. Point file in presentation mode. 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 )

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

STEP 2: 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

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

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

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

Balancers and stock keeping • lethal difficult keep as a stock; they in will gradually If each ofmutations the mutantare alleles (bluetoand orange) were lethal homozygosis, which of these genotypes fail to survive? ” be lost (i. e. be replaced would by wt alleles in subsequent generations)

Balancers and stock keeping • lethal mutations are difficult to keep as a stock; they will gradually be lost (i. e. be replaced by wt alleles in subsequent generations) • remedy in Drosophila: balancer chromosomes

Balancers and stock keeping • balancers carry easily identifiable dominant and recessive markers

Balancers and stock keeping • balancers carry easily identifiable dominant and recessive markers • balancers are homozygous lethal or sterile (red cross)

Balancers and stock keeping only parental genotypes survive and maintain the stock • balancers carry easily identifiable dominant and recessive markers • balancers are homozygous lethal or sterile (red cross) • recombination of balancers is either suppressed or causes lethality (black cross) • Through using 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 markerless chromosomes through mating schemes.

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 4 th chromosome will be neglected (crossed out) • 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 (animals may be indicated as "X / Y or X" if both genders are being used or can be used) • generations are indicated as P (parental), F 1, 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 (but note that Fly. Base nomenclature is more complex)

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

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 Pelement insertion P(lac. Z, w+) (stock 2). To perform the experiment, you need to recombine m and P(lac. Z, 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). Do not yet start with the cross. You will first be asked a couple of questions! stock 3: , 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 Pelement insertion P(lac. Z, w+) (stock 2). To perform the experiment, you need to recombine m and P(lac. Z, 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). For further information about concepts of lethality and stock keeping, see file "02 -Concepts. Drosophila" under this LINK. stock 3: What is the genotype of the recombinant fly stock you want to generate? , Hu , m

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 Pelement insertion P(lac. Z, w+) (stock 2). To perform the experiment, you need to recombine m and P(lac. Z, 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 stock 3: What is the genotype of the embryos you will analyse? , m or: , m m (if you use a backcross of stock 1 with the recombinant stock for your experiment)

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 Pelement insertion P(lac. Z, w+) (stock 2). To perform the experiment, you need to recombine m and P(lac. Z, 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). stock 3: , Hu 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 Pelement insertion P(lac. Z, w+) (stock 2). To perform the experiment, you need to recombine m and P(lac. Z, 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 stock 3: If Cy Identify all other markers of these flies , Hu Sb

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 Pelement insertion P(lac. Z, w+) (stock 2). To perform the experiment, you need to recombine m and P(lac. Z, 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 stock 3: If Cy , 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 Pelement insertion P(lac. Z, w+) (stock 2). To perform the experiment, you need to recombine m and P(lac. Z, 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). cross , Hu stock 3: Define the first cross! Does it matter which stocks you choose ♀♀ & ♂♂ from?

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

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

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. Selecting F 1 stock 2 first? second? third? m + ; Y stock 1 ; • take females (to allow for recombination) • select against curly wings (to have Pelement & mutation) Remember that: P stands for P + m

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. Designing the F 1 cross + m + Remember: recombination occurs at random! If the chromosomal positions of m and P(lac. Z, w+) are known, the recombination frequency can be calculated (see genetic manual); typically between 20 -100 single crosses are required. In the+germline of the P(lac. Z, w ) selected females, recombination takes no recombination place gonad haploid gametes * * * m * * P(lac. Z, w+) m recombination each layed egg has its individual recombination history Challenge: how to select for the F 2 flies carrying correctly recombined chromosomes?

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. Designing the F 1 cross + m stock 3: + Males from which stock below? , Hu 1 st step: stabilise recombinant chromosomes with a Cy. O balancer (either stock 1 or 3; here we use stock 3) , Hu

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. F 2 selection , Hu m + F 2 + first? second? w Y P(lac. Z, w+), [m]* Cy. O + Y P(lac. Z, w+), [m]* If w w + w ; [m]* Cy. O third? + TM 6 b, Hu ; + Sb [m]* If [m]* = potentially present not important here; ignored hereafter

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. Define your selection criteria for 2 nd and 1 st chromosomes choosing males is preferable for reasons explained later F 2 first? second? w Y P(lac. Z, w+), [m]* Cy. O + Y P(lac. Z, w+), [m]* If w w ; + w select for white back-ground, to see orange eyes [m]* Cy. O [m]* If select for orange eyes, for If, against Cy third? + TM 6 b, Hu ; + Sb

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. Selecting recombinants F 2 w P(lac. Z, w+), [m]* ; Y If Task: determine whether the recessive Before explaining this choice, mutation m has indeed recombined with let's do the next crossing step. P(lac. Z, w+) in these males Choose female from available stocks stock 3: Key strategy: backcross to "m" stock , Hu

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. performing the back cross F 2 w P(lac. Z, w+), [m]* ; Y If X + m ; + Cy. O stock 1 Now it becomes clear why If marked males were chosen: stock 1 has the wildtype white allele, therefore F 3 animals will not display white eyes. In those animals, the P(lac. Z, 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. Males need to be chosen to prevent recombination of the If chromosome (which is is not a balancer!) with the potentially recombinant chromosome. Furthermore, remember that each F 2 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 males 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.

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. performing the back cross single males! F 2 F 3 w P(lac. Z, w+), [m]* ; Y If first? X 2 nd? m P(lac. Z, w+), [m]* + Y + w ; m If Cy. O P(lac. Z, w+), [m]* Cy. O If + m ; + Cy. O stock 1 How can you identify the recombinants? Define your criteria! lethality as selection criterion

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. performing the back cross F 2 F 3 w P(lac. Z, w+), [m]* ; Y If first? X 2 nd? m P(lac. Z, w+), [. . . ]* + Y + w ; m If Cy. O P(lac. Z, w+), [m]* Cy. O If + m ; + Cy. O stock 1 How can you identify the recombinants? Define your criteria! lethality as selection criterion Possibility 1: if there are surviving flies carrying neither If nor Cy, then [m]* is not present (i. e. m is in heterozygous, viable condition).

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. performing the back cross F 2 F 3 w P(lac. Z, w+), [m]* ; Y If first? X 2 nd? m P(lac. Z, w+), [m]* + Y + w ; m If Cy. O P(lac. Z, w+), [m]* Cy. O If + m ; + Cy. O stock 1 How can you identify the recombinants? Define your criteria! lethality as selection criterion Possibility 2: if all flies are either If or Cy, then [m]* is present, because the homozygous animals (which carry neither If nor Cy) die.

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. performing the back cross F 2 F 3 w P(lac. Z, w+), [m]* ; Y If first? X + m ; + Cy. O stock 1 2 nd? m P(lac. Z, w+), [m]* + Y + w ; m If Cy. O P(lac. Z, w+), [m]* Cy. O If What flies do you select? Define your criteria!

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. performing the back cross F 2 F 3 w P(lac. Z, w+), [m]* ; Y If first? X + m ; + Cy. O stock 1 2 nd? m P(lac. Z, w+), [m]* + Y + w ; m If Cy. O P(lac. Z, w+), [m]* Cy. O If for Cy, against If Cross ♂♂ and ♀♀ to establish a stock. In the next generation you could start selecting for orange eyes (i. e. w background) for future purposes. TASK DONE!

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. What would have happened if you had chosen the Cy. O males in F 2? F 2 first? second? w Y P(lac. Z, w+), [m]* Cy. O + Y P(lac. Z, w+), [m]* If w w + w ; [m]* Cy. O [m]* If third? + TM 6 b ; + Sb

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. performing the back cross single males! F 2 F 3 w P(lac. Z, w+), [m]* ; Y Cy. O first? X 2 nd? m P(lac. Z, w+), [m]* + Y + w ; m Cy. O P(lac. Z, w+), [m]* Cy. O + m ; + Cy. O stock 1 You know that you have a recombinant, but cannot select the recombinant chromosome to establish a stable line. Thus, the blue and green box cannot be distinguished because you have a "symmetric" constellation: (1) due to the w+ background (red box), "orange eyes" cannot be seen. (2) both are balanced with Cy. O. You can solve this by first establishing a stock and then performing the selection step.

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. single males! F 2 w P(lac. Z, w+), [m]* ; Y Cy. O , Hu w Which stock do you choose to establish a stable line? Choose female from available stocks stock 3: , Hu

Task: Recombine P(lac. Z, w+) with the lethal mutation m! Note that P(lac. Z, w+) is orange. Establishing stable stocks single males! F 2 F 3 w P(lac. Z, w+), [m]* ; Y Cy. O first? w 2 nd? If P(lac. Z, w+), [m]* w Y w w ; , Hu If Cy. O P(lac. Z, w+), [m]* Cy. O 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 F 4, then [m]* is not present, if all F 4 flies are Cy you have a recombinant! TASK DONE!

Now continue with independent crossing tasks (Suppl. Mat. 4 under this link) for further info on flies and fly genetics see: • https: //droso 4 schools. wordpress. com/why-fly • http: //www. flyfacility. ls. manchester. ac. uk/forthepublic