SNP Discovery by Amplicon Sequencing to Study Genetic Diversity in Brassica A Genome

The aim of the study was to analyze SNP marker diversity in a collection of  Brassica rapa accessions from different countries, mostly oilseed types, which was gathered from genebanks. Amplicon sequencing was used for SNP discovery, applying the recently released B.rapa genome sequence for primer design. One hundred annotated genes involved in fatty acid or glucosinolate metabolism, or defence were selected, as well as genes situating evenly in the genome to end up with 384 amplicons. Over 80 % (=315) of the designed primer pairs worked well and amplified only one product, and were used for amplification from 8 different genotypes. All the amplicons were sequenced in one 454 sequencing run on GS Junior System. SNP or indel differences between genotypes were visually scored. To supplement the collection of SNPs from the resequencing effort we also used public databases. For some amplicons, more than one SNP/indel was selected, so 262 different genes were included in the total amount of 384 SNPs. SNP genotyping was carried out using Illumina BeadXpress. The final amount of functioning and polymorphic SNPs was 209. A total of 901 plants from 62 accessions was analyzed. Thirty-four percent of the genetic variation was due to differences between accessions. Genetic diversity within accessions was very variable: the percentage of polymorphic loci ranged from 1 to 91%, expected heterozygosity (H_e) from 0.003 to 0.318, and observed heterozygosity (H_o) from 0.002 to 0.315. Principal coordinates analysis separated e.g. B.rapa ssp. oleifera accessions from B.rapa  ssp. trilocularis and ssp. dichotoma accessions.