Expert meeting: Walter Salzburger
Location: RBINS (Brussels)
June 8
Authored by: Sofie Derycke
Expert meeting: Walter Salzburger
08/06/2015
Present : Loic Kéver, Koen Herten, Jos Snoeks, Erik Verheyen, Sofie Derycke, Maarten Van Steenberge, Nicolas Lichilin
Excused : Eric Parmentier, Pascal Poncin, Jeroen Van Houdt
10.00h – 11.15h: Seminar by Walter Salzburger: Adaptive radiation in Darwin’s
dreamponds (open to all interested scientists)
This talk gave a thorough overview of the adaptive radiation in African cichlids and of the
current genomic work (with specific focus on the evolution of egg spots) in Walter
Salzburgers lab.
11.15h-12.40h: Presentation GENBAS project + discussion (GENBAS partners only)
Overview of the aims of the project and critical points to decide upon:
1/ Behavioural part: at which point in the mating process will we kill the females?
2/ Transcriptome part: which brain parts will we focus on?
3/ Population genomics part: which restriction enzyme to use?
12.40h-14h: Lunch at l’ Oriento
14h-16h: Continuation discussion critical points
Expert meeting: Walter Salzburger | June 8
Summary of ideas and suggestions offered by Walter Salzburger:
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1/ Behavioural experiments:
- Comment on control treatment: we want to have the gene expression at the
moment the female decides to mate with the male. An ideal control for that is
putting females in the same tank that is divided with an invisible barrier to a mix
of males and females. So the only difference is the presence/absence of males for
these controls. Our controls consist of females that have never been in contact
with males, but they were maintained in a separate tank, this choice was based
on the fact that we want to detect all cues (chemical, auditive and visual)
occurring during the mating process and because we cannot control chemical
and auditive cues when females would have been placed in the same tank.
- Comment on decision process: how long is the decision signal detectable in the
hormone level of the brains? This is important, if the female decides eg three
days before the actual egg laying we can miss the signal. Check papers by Hans
Hoffman, and contact him if this information is not present in his pubications.
- Comment on experimental setup: the timing of the killing should be as
standardized as possible to reduce variability as much as possible and to be able
to detect the signal in the RNAseq data. This will also reduce the number of
biological replicates needed to detect the signal. We should very carefully think
about the set-up of these mating experiments. A good setup would eg be that
gravid females are separated from the males by a barrier in the same tank, and
when you think the female is ready to spawn (belly is bigger), then the barrier is
removed and the female is exposed for a standardized amount of time (eg 30
min) to the male. If she does not respond, she is separated and exposed later de
novo. Ideally, she should also get the choice between males. Walter suggested to
keep two males on either side of the tank, and place the female in the middle. He
also believes that the female will start laying eggs just by seeing the male; so if
the male is separated by a net from the female and the female cannot go into the
bower, seeing a male will induce egg laying. It would be good to see what
happens if you destroy or add some materials in the middle of the bower during
the night, when the fish are sleeping. Loic will try some settings with gravid O.
ventralis, after the ethograms are finished.
3/ Population genomics part:
- Mapping reads against reference genome: it is completely normal to loose ca 25% of
the reads, these are linked to repetitive units and transposable elements, so even if
we would map the reads against Ophthalmotilapia we would loose reads. They
always map the reads against the fully annotated genome of tilapia; if you do it
against your own reference genome, you eventually end up with looking to those
data that can be linked to the annotated genome anyway because the rest of the data
is not interpretable. The unmappeble reads of all species can then be stored in a
separate folder, and these reads can be used to generate contigs that can be used to
create an artificial chromosome.
- If we want to know the genomic regions that are differentiated between species, we
need as much as the genome as possible, and 40 000 loci (the number we now get
with PstI or ApeKI) will for sure not be enough. He would not use reduced genome
libraries anymore, but instead sequence the genomes with a lower coverage (10 to
15x at population level) and then in a lower number of specimens/populations. This
has the added advantage that no PCR is needed, so no PCR bias is generated.
- The reduced genome approach (RAD or GBS) is perfect to look at phylogeographic
patterns and differences between populations. Here, it is better to use a rare
frequent cutter (PstI) and aim for a high coverage, so that the SNP’s can be called
with high accuracy. Based on these results we can selectively pick a much smaller
number of specimens from sympatric and allopatric populations and sequence the
whole genome (they run 48 samples at once).
Expert meeting: Walter Salzburger | June 8
2/ Transcriptomics part:
- store extra tissue in RNA later (gill and gonads) as potential controls for later
qPCR assays. Gills are not under hormone control and can thus be used as
negative controls.
- Dissecting the brains and analysing them separately is definetely the best choice
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