Developing SNP assays for Lolium perenne: a pilot study for LpCCR1

Perennial ryegrass (Lolium perenne) is used extensively as proprietary forage in dairy farming, because of its high yield and high protein content. In order to increase the energy released from perennial ryegrass during digestion, we aim to improve the digestibility of the cell wall carbohydrate fraction. This can be achieved by reducing the lignin content. As we follow a candidate gene association mapping approach, we are currently studying the genetic diversity of genes involved in the lignin biosynthesis pathway. Tu et al. (2010) showed that downregulation of LpCCR1, one of the genes coding for the enzyme cinnamoyl-coA reductase, leads to a significant reduction in lignin content. Therefore, LpCCR1 was selected as a pilot candidate gene for developing SNP assays.
Through phylogenetic analysis, five bona fide CCR genes in L. perenne were identified in the available RNA-Seq database containing transcript sequences of 14 genotypes (Ruttink et al., 2012). For SNP identification, LpCCR1 was Sanger sequenced for an additional 6 genotypes with diverging cell wall digestibility. This way, 103 trustworthy SNPs were identified in the LpCCR1 transcript, resulting in an overall SNP density of 7.5 SNPs per 100 nucleotides.
KASP assays were used for genotyping a selection of 12 SNPs in the AM population. 92% of these assays were validated, indicating that they are efficient in genotyping a SNP dense species. As unknown SNPs within the primer binding sites might decrease efficiency, this also shows that the genetic diversity within the 20 genotypes is sufficient for capturing most SNPs of LpCCR1.
As common SNPs are required for a powerful association mapping, a minor allele frequency (MAF) threshold of 10% was set. Over a genome distance of 5000 bp, these common SNPs were found to be in strong linkage disequilibrium within a non-stratified subpopulation. We conclude that the best approach for genotyping other candidate genes is to select common SNPs at the ends of the transcript and check for LD decay. If LD is strong, a limited set of common SNPs is sufficient for AM purposes.