Developing RAD markers as a resource for
plant breeding using the perennial ryegrass
Lolium perenne.
Matt Hegarty, Danny Thorogood, Justin Pachebat, Wayne Powell
Institute of Biological, Environmental & Rural Sciences (IBERS)
Aberystwyth University
• IBERS have identified a need for high-throughput SNP discovery in all of
our primary plant breeding and trait mapping programmes.
• Several avenues are being pursued to develop SNPs for genotyping:
- Next-generation sequencing of transcriptomes (mRNA-seq) to
develop EST-based SNPs.
- Resequencing of polymorphic loci in different genotypes.
• These are good for finding SNPs but a downstream assay system must still
be developed (i.e. Illumina GoldenGate chips).
RAD mapping
• BBSRC Tools & Resources Development grant (£120K) to utilise RADseq
to map self-incompatibility loci in Lolium perenne and investigate usefulness
of technique for mapping in crop species.
• Lolium represents a good model for testing RAD in crops:
- Moderately large genome (4Gbp)
- Diploid
- Highly heterozygous outbreeder
• If RAD proves useful for SNP discovery and trait mapping in Lolium, will
apply to other IBERS forage/bioenergy crops (clover, Miscanthus).
Self-incompatibility
• Many plants have the ability to recognise and reject their own pollen or that
of close relatives.
• This serves to prevent inbreeding.
• Works on a ‘lock and key’ basis – incompatible pollen either triggers a
receptor on the stigma surface which results in a cascade designed to stop
pollen tube growth OR is susceptible to stigma proteins entering the tube.
• Has evolved many times and involves different molecular interactions in each
major plant family.
• Two main forms: gametophytic (based on pollen haploid genotype) or
sporophytic (based on diploid genotype of pollen donor).
reciprocal cross S‐mapping population P235/59 Z‐mapping population P235/58 S Z x S Z S Z x S Z parents 11 12 12 11 12 11 11 12 progeny S Z S Z S Z S Z S associated tags/SNPs + + ‐ + Z associated tags/SNPs ‐ + + + 2
2
12 11
12 12
11 12
12 12
Due to unequal male transmission of alleles, F1 progeny inherit a particular set of
markers associated with SI loci and can thus be used as a linkage mapping population.
Research plan
• Initial deep-sequencing (50x each allele) of parental genotypes to be carried
out using a variety of restriction enzymes to determine optimal tag density
and identify SNPs.
• A “bulk-segregant” approach taken to 15 F1 progeny for each of 4 distinct SI
genotypes (can be determined biochemically) at 10x coverage per allele.
• Initial genotyping accuracy validation via custom GoldenGate assay.
• RAD genotypes used to create a linkage map (via JOINMAP software).
• SI-associated loci tested in a wider number of individuals using small-scale assays.
• Attempt to position SI loci on linkage map and also tie RAD map to existing maps.
Acknowledgements
Mark Blaxter, John Davey (Edinburgh GenePool)
Wayne Powell, Justin Pachebat, Danny Thorogood (IBERS)
Eric Johnson (Floragenex)