Training session on Drosophila mating schemes Andreas Prokop.

Training session on Drosophila mating schemes

Andreas Prokop

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).

• It is important that you view this PowerPoint file in presentation mode.

http://flybase.org/static_pages/news/articles/2013_03/Roote_Prokop_Mating_scheme_package.html

synapsis -interchromosomal

recombination

separatinghomologous

chromosomes

hap

loid

hap

loid

Mitosis and meiosis

separatingsister

chromatids

generating sister chromatids for each of the homologous chromosomes

dip

loid

➊ ➋➌

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

Law of segregation / linkage groups

Homologous chromosmes are separated during meiosis

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

intra-chromosomal recombination takes

place randomly during oogenesis

Recombination rule:

there is no recombination in males (nor of the 4th chromosome)

Complication: recombination in females

7 instead of 3 different genotypes

wildtypeheterozygoushomozygous

mutant

Complication: recombination in females

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)

• 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 carry easily identifiable dominant and recessive markers



• balancers are homozygous lethal or sterile


• balancers are homozygous lethal or sterile

• the products of recombination involving balancers are lethal


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.

• '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

Rules to be used here:

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

Do not yet start with the cross. You will first be

asked a couple of questions!

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/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



, Hu

What is the genotype of the recombinant fly stock

you want to generate?

, m



, Hu

What is the genotype of the embryos you

will analyse?

, m, m

For further information about concepts of lethality and stock keeping, see file "02-ConceptsDrosophila"

on this LINK.

Identify the eye colours of these flies



, Hu

, Hu

Identify all other markers of these

flies

, Hu



Cy

Cy

If HuSb

, Hu, Hu

Cy

Cy

If HuSb

Identify the balancer chromosomes



, Hu

Define the first cross!

cross

Does it matter which stocks you choose ♀♀ & ♂♂ from?



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

Now select gender and genotype!

Selecting F1

Y

+

;

second?

;

third?

m

first?

stock 1stock 2


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


Challenge: how to select for the F2 flies carrying correctly

recombined chromosomes?


each layed egg has its individual recombination history

In the germline of the selected females,

recombination takes place

P(lacZ,w+)

m

P(lacZ,w+) 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 haploidgametes

****

** ****

Designing the F1 cross

+ +m

, Hu

Males from which stock below?

Designing the F1 cross

1st step: stabilise recombinant chromosomes with a balancer

+ +m


, Hu

F2 selection

F2

;

+

TM6b

+

Sb

;

not important here; ignored hereafter

third?second?first?


+ +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

F2P(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 2nd and 1st

chromosomes

choosing males is preferable for reasons

explained later


third?second?first?

Selecting recombinants

;w

Y

P(lacZ,w+),[m]*

IfF2

, Hu

Choose female from available stocks

Key strategy: backcross to "m" stock


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

;+

+

m

CyOX stock 1;

w

Y

P(lacZ,w+),[m]*

IfF2

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


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.

How can you identify the recombinants?

Define your criteria!


F3m

P(lacZ,w+),[m]*

CyOP(lacZ,w+),[m]*

CyOIf

mIf


;+

+

m

CyOX stock 1;

w

Y

P(lacZ,w+),[m]*

IfF2

2nd?

;

first?

+

Y

+

w

single males!

How can you identify the recombinants?



F3m

P(lacZ,w+),[...]*

CyOP(lacZ,w+),[m]*

CyOIf

mIf


;+

+

m

CyOX stock 1;

w

Y

P(lacZ,w+),[m]*

IfF2

2nd?

;

first?

+

Y

+

wAlternatively...

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.


F3m

P(lacZ,w+),[m]*

CyOP(lacZ,w+),[m]*

CyOIf

mIf


;+

+

m

CyOX stock 1;

w

Y

P(lacZ,w+),[m]*

IfF2

2nd?

;

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?


for Cy, against If


F3m

P(lacZ,w+),[m]*

CyOP(lacZ,w+),[m]*

CyOIf

mIf


;+

+

m

CyOX stock 1;

w

Y

P(lacZ,w+),[m]*

IfF2

2nd?

;

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!


F2P(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?


F3m

P(lacZ,w+),[m]*

CyOP(lacZ,w+),[m]*

CyOCyO

mCyO


;+

+

m

CyOX stock 1;

w

Y

P(lacZ,w+),[m]*

CyOF2

2nd?

;

first?

+

Y

+

wThis 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!

Lethality can also be used as criterion in a different strategy where potential recombinants are first bred into stocks.


;w

Y

P(lacZ,w+),[m]*

CyOF2

single males!

Selecting recombinants

, Hu

, Hu

Choose female from available stocks

F3If

P(lacZ,w+),[m]*

CyOP(lacZ,w+),[m]*

CyOCyO

IfCyO


2nd?

;

first?

w

Y

w

w

;w

Y

P(lacZ,w+),[m]*

CyOF2

single males!

Establishing stable stocks

, Hu

What flies do you select?


• 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!

Now continue with independent crossing tasks (Suppl. Mat. 4

under this link)

for further info on flies and fly genetics see:http://www.flyfacility.ls.manchester.ac.uk/forthepublic

Training session on Drosophila mating schemes Andreas Prokop.

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Transcript of Training session on Drosophila mating schemes Andreas Prokop.