sRAD with grasshoppers

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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
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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

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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
size selected library
barcoded DNA samples
blunt-ending and A-tailing of sheared fragments
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sRAD protocol for grasshoppers
A-tailed library
enriched library
P2Y adapter ligation
gel purification of library
P2Y adapter ligated library
purified library
restriction with 3 4-base cutters
validation of library by cloning and sequencing
selective PCR of P1-insert-P2Y constructs
complexity reduced library
validated library
determination of molar concentration
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sRAD protocol for grasshoppers
quantified library
paired end Solexa sequencing
sRAD tags
sorting by barcode, alignment of short reads, SNP
calling
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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

? number of fragments in the library needs to be
reduced in a systematic way !
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complexity reduction with additional frequent
cutting restriction enzymes and selective PCR
1200 uncut fragments expected, 58x coverage
per allele
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complexity reduction
4-base cutter restriction enzymes that dont cut
P1 adapters with the first 36 barcodes nor the
P2Y adapter
(checked with NEBcutter)
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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)
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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
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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

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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
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Hybrid male sterility
Shuker, D. M. Underwood, K. King, T. M.
Butlin, R. K. (2005), Proc R Soc B, 272,
24912497

• steep clines for sterility but shallow clines for
  stridulatory peg number
• ? differential introgression across the hybrid
  zone

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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
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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

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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
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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

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simple difference in song

• simple and purely quantitative difference in
  song,
• involved in a strong asymmetric behavioural
  isolation between the species

• the syllables in the song of C. montanus are at
  all temperatures 2 3 times longer than those
  in the song of C. parallelus

• 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
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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
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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

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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
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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

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Thank you for your attention, sRADers !