RAD projects, Chris Jiggins Lab, Cambridge
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Genome sequence assembly
Mapping of wing pattern QTL
Population Genetics
Identifying genes of major effect:
Insecticide resistance in pest moths
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Heliconius melpomene genome project
-11 X genome coverage (454)
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-Contigs only 2 kb (6 kb max)
-Hope to make a dense RAD
linkage map to make a genome
scaffold
Patricio Salazar, PhD candidate
♂ 11440
♀ 11445
♂ 11437
♀ 11470
P
Grandparents
♂ 11458
F1
Parents
F2
n=131
♀ 12032
PC 1
Measure appearance
The Isles of Scilly. Re-investigate meadow brown populations.
Dave Hosken, U. of Exeter in Cornwall
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Variable spot pattern in female Meadow Brown
Two Spots (ventral)
No spots (ventral)
EB Ford. Spot pattern diversity between islands
Large Islands
Small Islands
Replace AFLP methods with RAD for population genetics
Diamondback moth, Plutella xylostella (L.)
a
Q
a
T
Q
21 days at 18°C
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Global Pest of
Broccoli,
cabbage,
canola,
cauliflower
Identify mutations causing resistance to;
Bt : Bacillus thuringiensis
Spinosad
a
Q
S
ICP
Synthetic
pyrethroids
a
T
Q
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Spinosad resistance is a single
recessive gene
SS
RR
a
Q
Perform RAD on backcross
progeny and parents.
a
T
Q
RS
There is no crossing over during
oogenesis.
31 chromosome patterns should
be identified by RAD tags in the
progeny. Genes on the same
chromosome inherited from the
RS mother are linked
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Crosses between susceptible and resistant insects
SS
RR
a
Q
RR
RS
a
T
Q
Which RAD markers segregate
Controls
1S:1R
control
RS
RS RR
RR
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Bioassay
0S:1R
Create RAD library using F1 mother, backcross father and 22 progeny
-took a while, many hurdles
Create two further libraries using same parents and another 22 progeny
a
Q
SbfI Digest then biorupt
a
T
Q
Modified adapters
24 clones Sanger sequenced
Unmodified adapters
24 clones Sanger sequenced
Library sequenced (illumina)
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gDNA for digest
a
Q
a
T
Q
Sheer P1 ligated gDNA
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Final PCR
amplification of
RAD library
a
Q
a
T
Q
Average fragment size = 339 bp
24 clones contain ~ 2 genes and >5 repetitive elements
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Thanks to Genepool for sequencing
a
Q
a
T
Q
Eric Johnson and Paul Etter for helpful information and adapters
John Davey for driving all the bioinformatics and organisation
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