sRAD with grasshoppers
outline:
1.
sRAD protocol for grasshoppers
2.
complexity reduction
3.
the issue of repetitive DNA
4.
applications of sRAD
Claudius Kerth, Roger Butlin – Sheffield University
sRAD – the promise
 ~1000 markers per grasshopper genome
 codominant SNPs and dominant presence/absence of RAD tags
 ~5 pence per scored marker
→ cost-effective alternative to genetic markers like AFLP, microsatellites or cDNA
sequences
sRAD protocol for grasshoppers
36 DNA
samples
pooled
DNA shearing to below
1000 bp fragment size
DNA digestion with SbfI
sheared
library
restricted
DNA samples
select 100 bp size range on
gel at around 567 bp
fragment size
P1 adapter ligation
pool DNA samples
barcoded
DNA samples
size selected
library
blunt-ending and A-tailing of
sheared fragments
sRAD protocol for grasshoppers
enriched
library
A-tailed library
P2Y adapter ligation
gel purification of library
P2Y adapter
ligated library
restriction with 3
4-base cutters
complexity
reduced library
purified library
selective PCR of P1insert-P2Y constructs
validation of library by
cloning and sequencing
validated library
determination of molar
concentration
sRAD protocol for grasshoppers
quantified library
paired end Solexa
sequencing
sRAD tags
sorting by barcode, alignment
of short reads, SNP calling
Why complexity reduction ?
•
grasshopper genome is 10-14Gbp large
•
an 8-base cutter is expected to yield 152,588 – 213,623 fragments per grasshopper genome
•
DNA shearing step doubles that number
•
with 20x minimum coverage and 64 individuals more than 1 billion reads would be necessary
• we want to afford only one lane of an illumina flow cell for a start,
i. e. about 5 million reads
•
we want to score presence/absence of RAD sequences
→ number of fragments in the library needs to be reduced in a systematic way !
complexity reduction with additional frequent
cutting restriction enzymes and selective PCR
P2Y adapter
ligated library
restriction with 3
4-base cutters
complexity
reduced library
selective PCR of P1insert-P2Y constructs
enriched
library
~1200 uncut fragments expected,
~58x coverage per allele
complexity reduction
4-base cutter restriction enzymes that don’t cut P1 adapters with the first 36 barcodes nor the P2Y adapter:
(checked with NEBcutter)
the issue of repetitive DNA
1. Overrepresentation of repetitive “junk” DNA in the sRAD library:
• methylation sensitivity of the restriction enzyme is probably not the
solution for insects:
- no overrepresentation of heterochromatin sequences in m5C-antibody
purified DNA in Drosophila (Salzberg, 2004, Biochem Biophys Res Commun)
- methylation patterns vary extremely between species, developmental
stages, cell types and castes in social insects (Kucharski et al., Science, 2009)
the issue of repetitive DNA
2. paralogous RAD sequences interfering with SNP calling:
• paired-end reads might help to distinguish some paralogous RAD sequences
• exceptionally high coverage across individuals at a putative locus is an indication of two or more
underlying loci (threshold?)
• low nucleotide identity within a contig is an indication of two or more underlying loci (threshold?)
• trinucleotide positions in a single individual will clearly indicate duplicated loci
Bailey et al., Science, 2002
Looking for new candidate genes for
hybrid sterility in a grasshopper hybrid
zone
Two subspecies of Chorthippus parallelus form a hybrid zone in the Pyrenees
Hybrid male sterility

F1 hybrid males with parents from
outside the hybrid zone are almost
completely sterile

analysis of backcross hybrids suggests
that few genes of major effect are
causing hybrid sterility
Llewellyn, A. (2008) The Genetics of Male Sterility in a Grasshopper Hybrid
Zone. PhD thesis, Univerity of Sheffield, Department of Animal and Plant
Sciences
Hybrid male sterility
Shuker, D. M.; Underwood, K.; King, T. M. & Butlin, R. K.
(2005), Proc R Soc B, 272, 2491–2497
steep clines for sterility but shallow clines for stridulatory peg number
→ differential introgression across the hybrid zone
Genome scan for loci under selection
against introgression
 sample of 96 individuals from a transect
across the hybrid zone
 genotype them with sRAD markers
 test for selection using neutral distribution
of FST's AND cline widths
Beaumont, M. A. & Nichols, R. A. (1996) Evaluating loci for use
in the genetic analysis of population structure. Proc R Soc Lond
B, 263, 1619-1626
follow-up studies
 Which of the outlier loci are linked with hybrid male sterility?
→ analyis of association with hybrid male sterility in laboratory hybrids
 Are the outlier loci disproportionately located on the X-chromosome?
→ X- vs. autosomal location can be inferred from the marker phenotypes in this study
(male grasshoppers have genotype X0)
 What genes are in the vicinity of the outlier fragments?
→ the genetic surroundings of the outlier fragments can be analysed with the library of
restriction fragments and the RAD sequences
Looking for new candidate genes for
mating behaviour and speciation in
grasshoppers

great diversity in song within the subfamily
Gomphocerinae

many species pairs have little or no
intrinsic post-zygotic isolation

hybrid males are behaviourally sterile due
to a disrupted calling song
→ song and associated female preference
are likely to be speciation phenotypes
in these grasshoppers
after Heller, K.-G., Korsunovskaya, O., Ragge, D.R., Vedenina,
V., Willemse, F., Zhantiev, R.D. & Frantsevich, L. (1998)
Check-List of European Orthoptera. Articulata, Beiheft 7, 1-61
The species pair C. parallelus and C.
montanus
 sibling species
 morphologically and genetically very
similar
 parapatric in large parts of Europe
 ecologically divergent
 F1 hybrid males are probably sterile
von Helversen, O. & von Helversen, D. (1994) The neural basis of
behavioural adaptations. chapter Forces driving coevolution of
song and song recognition in grasshoppers, 253-284
 C. parallelus females do not discriminate against C. montanus males
→ hybridisation is probably still going on in nature
simple difference in song
 simple and purely quantitative
difference in song,
involved in a strong asymmetric
behavioural isolation between the
species
 F1 hybrid males produce songs with
intermediate syllable duration
von Helversen, O. & von Helversen, D. (1987) Aims and methods in
neurology. chapter Innate receiver mechanisms in the acoustic
communication of orthopteran insects. Manchester Univ. Press, 104-150
Genome scan for loci under selection
between C. parallelus and C. montanus
 sample 15 individuals from each of 3
genetically independent sympatric
populations of Chorthippus montanus and
C. parallelus across Europe
 genotype them with sRAD markers
 perform FST -outlier test for selection
We expect outlier loci to be linked with genes for ecological adaptation, intrinsic hybrid sterility
and differences in mating behaviour
Genetic map of parallelus/montanus
genome
 parental individuals from geographically
distant source populations
 raise F1 generation at around February
2010
 raise F2 generation at around July 2010
 genotype backcross family with sRAD
markers
QTL study of song differences between
C. parallelus and C. montanus

record the song of ~600 backcross
males

test for association between marker
alleles and phenotype

look whether outlier loci from the
genome scan coincide with QTL
regions

conduct sign test for selection on
QTL
Doerge, R. W. (2002) Mapping and analysis of quantitative trait loci in experimental
populations. Nat Rev Genet, 3, 43-52
What can we expect to find out?
 Are song differences between sister species caused by many genes of small effect or few
genes of major effect?
 Are song genes primarily sex-linked?
 Did the syllable duration of the song diverge due to selection or genetic drift?
 Do candidate genes for mating behaviour in Drosophila fall into QTL regions for song?
 The cross can be used to map other traits that are involved in reproductive isolation, like
sterility or cuticular hydrocarbons
Thank you for your
attention, sRADers !