Transcript m - Figshare

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 PowerPoint 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
➊
separating
sister
chromatids
➋
➌
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
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 4th chromosome)
Complication: recombination in females
wildtype
heterozygous
homozygous
mutant
7 instead of 3 different genotypes
Balancers and stock keeping
•If lethal
mutations
difficult
to keep
as both
a stock
and
eventually
the mutant
alleles are
(blue
and orange)
were
lethal
in will
homozygosis,
which
of these
genotypes
would fail
segregate
out
(i.e. be replaced
bytowtsurvive?”
alleles)
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
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 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 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/m mutant
embryos. These neurons can be specifically labelled with ß-Gal using a 2nd chromosomal Pelement 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).
Do not yet start with the
cross. You will first be
asked a couple of
questions!
, Hu
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 Pelement 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
What is the genotype of
the recombinant fly stock
you want to generate?
,m
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 Pelement 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).
For further information
about concepts of lethality
and stock keeping, see file
"02-ConceptsDrosophila"
on this LINK.
, Hu
What is the genotype
of the embryos you
will analyse?
,m
,m
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 Pelement 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
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 Pelement 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).
Cy
If
Cy
Identify all other
markers of these
flies
, Hu
Sb
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 Pelement 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).
Cy
If
Cy
, Hu
Sb
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/m mutant
embryos. These neurons can be specifically labelled with ß-Gal using a 2nd chromosomal Pelement 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?
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
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! Note that P(lacZ,w+) is orange.
Selecting F1
stock 2
first?
second?
third?
m
+
;
Y
stock 1
;
Now select
gender and
genotype!
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
Selecting F1
stock 2
first?
second?
third?
m
+
;
Y
stock 1
;
• take females (to allow
for recombination)
• select against curly
wings (to have Pelement & mutation)
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
Designing the F1 cross
+
m
+
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.
In the+germline of the
P(lacZ,w
)
selected females,
recombination takes
no recombination
place
gonad
haploid
gametes
*
*
*
*
*
m
*
*
P(lacZ,w+)
m
recombination
each layed egg has its individual
recombination history
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.
Designing the F1 cross
+
m
+
Males from which
stock below?
, Hu
1st step: stabilise recombinant
chromosomes with a balancer
, Hu
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
F2 selection
, Hu
m
+
F2
+
first?
second?
w
Y
P(lacZ,w+),[m]*
CyO
+
Y
P(lacZ,w+),[m]*
If
;
third?
+
TM6b
;
w
w
[m]*
CyO
+
w
[m]*
If
[m]* = potentially
present
+
Sb
not important here;
ignored hereafter
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
Define your selection
criteria for 2nd and 1st
chromosomes
choosing males is
preferable for reasons
explained later
F2
first?
second?
w
Y
P(lacZ,w+),[m]*
CyO
+
Y
P(lacZ,w+),[m]*
If
;
+
TM6b
;
w
w
[m]*
CyO
+
w
[m]*
If
select for white back-ground, to
see orange eyes
third?
select for orange eyes, for
If, against Cy
+
Sb
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
Selecting recombinants
F2
w
P(lacZ,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(lacZ,w+) in these males
Choose female from available stocks
, Hu
Key strategy: backcross to "m" stock
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
performing the back cross
F2
w
P(lacZ,w+),[m]*
;
Y
If
X
+
m
;
+
CyO
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.
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.
stock 1
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
performing the back cross
single males!
F2
F3
w
P(lacZ,w+),[m]*
;
Y
If
X
stock 1
2nd?
first?
m
P(lacZ,w+),[m]*
+
Y
+
w
+
m
;
+
CyO
m
If
;
CyO
P(lacZ,w+),[m]*
CyO
If
How can you identify
the recombinants?
Define your criteria!
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
performing the back cross
F2
F3
w
P(lacZ,w+),[m]*
;
Y
If
X
stock 1
2nd?
first?
m
P(lacZ,w+),[...]*
+
Y
+
w
+
m
;
+
CyO
m
If
;
CyO
P(lacZ,w+),[m]*
How can you identify
the recombinants?
Define your criteria!
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.
Alternatively...
CyO
If
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
performing the back cross
F2
F3
w
P(lacZ,w+),[m]*
;
Y
If
X
2nd?
first?
m
P(lacZ,w+),[m]*
+
Y
+
w
m
If
;
+
m
;
+
CyO
stock 1
... 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!
CyO
P(lacZ,w+),[m]*
CyO
If
for Cy, against If
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
performing the back cross
F2
F3
w
P(lacZ,w+),[m]*
;
Y
If
X
stock 1
2nd?
first?
m
P(lacZ,w+),[m]*
+
Y
+
w
+
m
;
+
CyO
m
If
;
Cross ♂♂ and ♀♀ to
establish a stock. In
the next generation you
could start selecting for
orange eyes for future
purposes.
TASK DONE!
CyO
P(lacZ,w+),[m]*
CyO
If
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
What would have
happened if you had
chosen the CyO males?
F2
first?
second?
w
Y
P(lacZ,w+),[m]*
CyO
+
Y
P(lacZ,w+),[m]*
If
;
third?
+
TM6b
;
w
w
[m]*
CyO
+
w
[m]*
If
+
Sb
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
performing the back cross
single males!
F2
F3
w
P(lacZ,w+),[m]*
;
Y
CyO
X
stock 1
2nd?
first?
m
P(lacZ,w+),[m]*
+
Y
+
w
+
m
;
+
CyO
m
CyO
;
CyO
P(lacZ,w+),[m]*
CyO
CyO
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]*
This is a "symmetric"
constellation and therefore
a DEAD END!
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
Selecting recombinants
single males!
F2
w
P(lacZ,w+),[m]*
;
Y
CyO
, Hu
Lethality can also be used as criterion in a different strategy
where potential recombinants are first bred into stocks.
Choose female from available stocks
, Hu
Task: Recombine P(lacZ,w+) with the lethal mutation m! Note that P(lacZ,w+) is orange.
Establishing stable stocks
single males!
F2
F3
w
P(lacZ,w+),[m]*
;
Y
CyO
, Hu
2nd?
first?
If
P(lacZ,w+),[m]*
Define your criteria!
• for Cy, for orange eyes
w
Y
w
w
What flies do you
select?
If
CyO
;
CyO
P(lacZ,w+),[m]*
CyO
CyO
• 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