Training session on Drosophila mating schemes I would have pages 1-12 as one separate presentation to act as an accompaniment to the Genetics document. Then have the problem as a separate task

This document is one part of a Drosophila genetics training package, the entire strategy of which is described in detail elsewhere (see link). 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)

generating sister chromatids for each of the homologous chromosomes 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

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

Homologous chromosmes are separated during meiosis Law of segregation / linkage groups Homologous chromosmes are separated during meiosis Take mitosis out of the title

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 Take mitosis out of the title 6

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

7 instead of 3 different genotypes Complication: recombination in females wildtype heterozygous homozygous mutant Maybe leave the 1:2:1 ratio in the diagram by the strict gene linkage section 7 instead of 3 different genotypes 8

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) 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) 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

only heterozygous flies survive and maintain the stock 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 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.

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

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 2nd chromosomal 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 when in white mutant background (w on 1st). , Hu Do not yet start with the cross. You will first be asked a couple of questions 16

Identify the eye colours of these flies Task: To study the potential effect of a 2nd chromosomal 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 when in white mutant background (w on 1st). , Hu Identify the eye colours of these flies 17

Identify all other markers of these flies Task: To study the potential effect of a 2nd chromosomal 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 when in white mutant background (w on 1st). CyO If * TM6b, Hu Sb , Hu Identify all other markers of these flies

Identify the balancer chromosomes Task: To study the potential effect of a 2nd chromosomal 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 when in white mutant background (w on 1st). CyO If TM6b, Hu , Hu CyO Sb Identify the balancer chromosomes 19

Does it matter which stocks you choose ♀♀ & ♂♂ from? Task: To study the potential effect of a 2nd chromosomal 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 when in white mutant background (w on 1st). cross , Hu Define the first cross! Does it matter which stocks you choose ♀♀ & ♂♂ from? 20

Task: Recombine P(lacZ,w+) with the lethal mutation m Selecting F1 stock 2 stock 1 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!

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

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

* * m P(lacZ,w+) m Designing the F1 cross + Task: Recombine P(lacZ,w+) with the lethal mutation m Designing the F1 cross + m each layed egg has its individual recombination history gonad haploid gametes * In the germline of the selected females, recombination takes place P(lacZ,w+) m no recombination recombination * Remember: recombination occurs at random I think if you rearrange the ‘no crossover’ chromosomes side by side to the ‘crossover’ chromosomes it will look more clearer and then perhaps the haploid gametes diagram underneath Challenge: how to select for the F2 flies carrying correctly recombined chromosomes?

m Designing the F1 cross + Stocks available: Task: Recombine P(lacZ,w+) with the lethal mutation m Designing the F1 cross + m Males from which stock below? Stocks available: 1st step: stabilise recombinant chromosomes with a balancer

F2 m w Y + P(lacZ,w+),[m]* CyO If [m]* + TM6b Sb ; ; F2 selection + Task: Recombine P(lacZ,w+) with the lethal mutation m F2 selection + m first? second? third? F2 w Y + P(lacZ,w+),[m]* CyO If [m]* [m]* = potentially present + TM6b Sb ; ; Mention here that * means that we are not sure of it’s presence not important here; ignored hereafter

F2 w Y + P(lacZ,w+),[m]* CyO If [m]* + TM6b Sb ; ; Task: Recombine P(lacZ,w+) with the lethal mutation m Identify the eye colours! first second third F2 w Y + P(lacZ,w+),[m]* CyO If [m]* [m]* = potentially present + TM6b Sb ; ; Mention here that * means that we are not sure of it’s presence not important here; ignored hereafter 27

F2 w Y + P(lacZ,w+),[m]* CyO If [m]* + TM6b Sb ; ; Task: Recombine P(lacZ,w+) with the lethal mutation m Define your selection criteria for 2nd and 1st chromosomes choosing males is preferable for reasons explained later first second third F2 w Y + select for orange eyes, for Cy, against If P(lacZ,w+),[m]* CyO If [m]* [m]* = potentially present select for white back-ground, to see orange eyes + TM6b Sb ; ; Mention here that * means that we are not sure of it’s presence not important here; ignored hereafter 28

Selecting recombinants Task: Recombine P(lacZ,w+) with the lethal mutation m Selecting recombinants ; w Y P(lacZ,w+),[m]* CyO F2 Next task: determine whether the recessive mutation m has indeed recombined with P(lacZ,w+) in the chosen males Choose female from available stocks Key strategy: backcross to "m" stock

performing the back cross Task: Recombine P(lacZ,w+) with the lethal mutation m performing the back cross ; w Y P(lacZ,w+),[m]* CyO F2 + m X ; stock 1 + CyO Problem: Each male individual represents a potentially 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.

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

performing the back cross Task: Recombine P(lacZ,w+) with the lethal mutation m performing the back cross ; w Y P(lacZ,w+),[m]* CyO F2 + m X ; stock 1 + CyO F3 2nd? If the lethal mutation "m" was recombined onto the P(lacZ,w+) chromosome, all surviving animals carry the CyO balancer. m P(lacZ,w+),[m]* CyO If so, establish the recombinant stock by selecting for P(lacZ,w+), i.e. for orange eyes.

performing the back cross Task: Recombine P(lacZ,w+) with the lethal mutation m performing the back cross ; w Y P(lacZ,w+),[m]* CyO F2 + m X ; stock 1 + CyO ; F3 1st? Since you have w+ background, this strategy does not work m P(lacZ,w+),[m]* + w Y m CyO If so, establish the recombinant stock by selecting for P(lacZ,w+), i.e. for orange eyes. CyO P(lacZ,w+),[m]* CyO

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

Task: Recombine P(lacZ,w+) with the lethal mutation m single males! ; w Y P(lacZ,w+),[m]* CyO F2 aid 1 stock 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, first establishing stable stocks then testing for lethality. to establish stable fly stocks cross single potentially recombinant males to the balancer stock

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

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

Now continue with independent crossing tasks (Suppl. Mat Now continue with independent crossing tasks (Suppl. Mat. 4 under this link)