ddRAD sequencing-based identification of inter-genepool SNPs and association analysis in Brassica juncea

Narrow genetic base, complex allo-tetraploid genome and presence of repetitive elements have led the discovery of single nucleotide polymorphisms (SNPs) in Brassica juncea (AABB; 2n = 4x = 36) at a slower pace. Double digest RAD (ddRAD) - a genome complexity reduction technique followed by NGS was used to generate a total of 23 million paired-end reads from three genotypes each of Indian (Pusa Tarak, RSPR-01 and Urvashi) and Exotic (Donskaja IV, Zem 1 and EC287711) genepools.

Sequence data analysis led to the identification of 10,399 SNPs in six genotypes at a read depth of 10x coverage among the genotypes of two genepools. A total of 44 hyper-variable regions (nucleotide variation hotspots) were also found in the genome, of which 93% were found to be a part of coding genes/regions. The functionality of the identified SNPs was estimated by genotyping a subset of SNPs on MassARRAY® platform among a diverse set of B. juncea genotypes. SNP genotyping-based genetic diversity and population studies placed the genotypes into two distinct clusters based mostly on the place of origin. The genotypes were also characterized for six morphological traits, analysis of which revealed a significant difference in the mean values between Indian and Exotic genepools for six traits. The association analysis for six traits identified a total of 45 significant marker-trait associations on 11 chromosomes of A- and B- group of progenitor genomes.

Despite narrow diversity, the ddRAD sequencing was able to identify large number of nucleotide polymorphisms between the two genepools. Association analysis led to the identification of common SNPs/genomic regions associated between flowering and maturity traits, thereby underscoring the possible role of common chromosomal regions-harboring genes controlling flowering and maturity in Brassica juncea.