High-throughput polymorphism detection and genotyping in Brassica napus using next-generation RAD sequencing

Abstract

Background: The complex genome of rapeseed (Brassica napus) is not well understood despite the economic importance of the species. Good knowledge of sequence variation is needed for genetics approaches and breeding purposes. We used a diversity set of B. napus representing eight different germplasm types to sequence genome-wide distributed restriction-site associated DNA (RAD) fragments for polymorphism detection and genotyping.

Results: More than 113,000 RAD clusters with more than 20,000 single nucleotide polymorphisms (SNPs) and 125 insertions/deletions were detected and characterized. About one third of the RAD clusters and polymorphisms mapped to the Brassica rapa reference sequence. An even distribution of RAD clusters and polymorphisms was observed across the B. rapa chromosomes, which suggests that there might be an equal distribution over the Brassica oleracea chromosomes, too. The representation of Gene Ontology (GO) terms for unigenes with RAD clusters and polymorphisms revealed no signature of selection with respect to the distribution of polymorphisms within genes belonging to a specific GO category.

Conclusions: Considering the decreasing costs for next-generation sequencing, the results of our study suggest that RAD sequencing is not only a simple and cost-effective method for high-density polymorphism detection but also an alternative to SNP genotyping from transcriptome sequencing or SNP arrays, even for species with complex genomes such as B. napus.

Figure 1

Restriction-site associated DNA (RAD) clusters with different numbers of RAD tags across eight B. napus inbreds.

Figure 2

Distribution of restriction-site associated DNA polymorphisms (single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels)) along the read length.

Figure 3

Number of polymorphisms and number of reads per restriction-site associated DNA (RAD) clusters across eight B. napus inbreds. Violin plot denotes 9,552 RAD clusters with single nucleotide polymorphisms (SNPs), and triangles denote 59 RAD clusters with insertions/deletions (InDels). Black dots denote the median number of reads per RAD cluster.

Figure 4

Transitions and transversions within 20,795 biallelic single nucleotide polymorphisms (SNPs) detected among eight B. napus inbreds..

Figure 5

Correlation of Modified Roger’s distances (MRD) between pairs of eight B. napus inbreds. MRD estimates were determined with simple sequence repeat (SSR) markers and polymorphisms from restriction-site associated DNA (RAD) sequencing.

Figure 6

Representation of B. napus restriction-site associated DNA (RAD) information and unigenes (UG). (a) RAD clusters, single-nucleotide polymorphisms (SNPs), and insertions/deletions (InDels) in the B. rapa sequence, B. rapa chromosomes, and B. rapa coding sequence (CDS) after BLAST searches and (b) UG with RAD clusters and polymorphisms, and UG with RAD clusters and polymorphisms that were assigned a function after BLAST searches against the UniProtKB/Swiss-Prot dataset.

Figure 7

Distribution of restriction-site associated DNA (RAD) information in B. rapa . Dispersal of RAD clusters, RAD clusters with single nucleotide polymorphisms (SNPs), and RAD clusters with insertions/deletions (InDels) across the ten chromosomes of B. rapa.

Figure 8

Gene Ontology (GO) term representation. GO term representation (%) of all B. napus unigenes (UG), UG with restriction-site associated DNA (RAD) clusters, UG with single nucleotide polymorphisms (SNPs), and UG with insertions/deletions (InDels) according to GO slim categories.