Haplotype Variation for Genes Involved in Biomass and Lignin Biosynthesis in Switchgrass

Switchgrass (Panicum virgatum), a tetraploid and outbreeding grass native to US prairies, has agronomic characteristics that make it an exceptionally promising feedstock for bioenergy production. Switchgrass has high genotypic and phenotypic variation,  and we aimed to study haplotype variation in switchgrass germplasm for genes involved in biomass production and biomass quality. Fifty four primer pairs, each spanning ~1 kb regions, were designed against 14 biomass genes and 7 lignin biosynthesis genes. The genomic regions were amplified from a set of 372 genotypes representing the biodiversity present in lowland and upland switchgrass. For each genotype, the 54 amplicons were pooled and sequenced. The Illumina reads were aligned with the switchgrass genomic sequences corresponding to the 54 amplicons using Bowtie 2. The assembled reads were mined for single nucleotide polymorphisms (SNPs) using Samtools. An average of 5,000,000 reads with a read length of ~90bp were obtained for each genotype. About 280M reads mapped to the reference sequences with an average coverage of 50X. A total of 5248 SNPs were identified which corresponds to a SNP density of, on average, 1 SNP every 17 bp. The SNP polymorphism level varied depending on the gene analyzed. A population structure analysis was performed on the SNP data. The haplotype information was further used to establish the extent of linkage disequilibrium (LD) in those 21 genes. Knowledge on LD is key in determining strategies to establish marker-trait associations in switchgrass.