Evaluation of Linkage Disequilibrium Pattern and Association Study on Seed Oil Content in Brassica napus Using ddRAD Sequencing

Abstract

High-density genetic markers are the prerequisite for understanding linkage disequilibrium (LD) and genome-wide association studies (GWASs) of complex traits in crops. To evaluate the LD pattern in oilseed rape, we sequenced a previous association panel containing 189 B. napus inbred lines using double-digested restriction-site associated DNA (ddRAD) and genotyped 19,327 RAD tags. A total of 15,921 RAD tags were assigned to a published genetic linkage map and the majority (71.1%) of these tags was uniquely mapped to the draft reference genome “Darmor-bzh.” The distance of LD decay was 1,214 kb across the genome at the background level (r2 = 0.26), with the distances of LD decay being 405 kb and 2,111 kb in the A and C subgenomes, respectively. A total of 361 haplotype blocks with length > 100 kb were identified in the entire genome. The association panel could be classified into two groups, P1 and P2, which are essentially consistent with the geographical origins of varieties. A large number of group-specific haplotypes were identified, reflecting that varieties in the P1 and P2 groups experienced distinct selection in breeding programs to adapt their different growth habitats. GWAS repeatedly detected two loci significantly associated with oil content of seeds based on the developed SNPs, suggesting that the high-density SNPs were useful for understanding the genetic determinants of complex traits in GWAS.

Dissection of the genetic architecture of three seed‐quality traits and consequences for breeding in Brassica napus

“…With the rapid development and low cost of next‐generation sequencing, GWAS based on millions of markers has become a powerful and popular approach in dissecting the genetic architecture of complex agronomic traits for crops such as rice (Huang et al ., ; Yano et al ., ) and maize (Li et al ., ; Xiao et al ., ). In our previous study, we evaluated the pattern of linkage disequilibrium in the 189 association panels using 10 343 markers generated by double‐digested restriction‐site associated DNA (ddRAD) and carried out an initial dissection of the genetic determinants of oil content as test of GWAS in rapeseed (Wu et al ., ). In this study, direct resequencing of 238 rapeseed lines provided a high‐resolution genomic variation map consisting of 4 542 011 SNPs and 628 666 INDELs (Figure and Table S2).…”

“…In this study, direct resequencing of 238 rapeseed lines provided a high‐resolution genomic variation map consisting of 4 542 011 SNPs and 628 666 INDELs (Figure and Table S2). This enabled us to perform a more exhaustive GWAS for three important seed‐quality traits including EAC, GSC and SOC, using 3 824 552 SNPs and INDELs in the same rapeseed association panel (Wu et al ., ). The high marker density helps identifying numerous variations within genes, which facilitated identification of a more complete set of candidate genes responsible for each trait.…”

“…A total of 238 rapeseed cultivars or inbred lines were collected from the major production areas of the world to represent the genetic diversity of B. napus cultivars (Table S1); 189 of the 238 cultivars or inbred lines prefixed with letter ‘g’ were employed for phenotyping and genome‐wide association mapping in this study (Table S1). The 189 cultivars or inbred lines had been well described in previous study and were classified into two main groups based on both population structure and principal component analysis: P1 group with 136 lines mainly originated from China and P2 group with 53 lines mainly originated from Europe, Canada and Australia (Wu et al ., ). Field trials were performed using a random block design with three replications in each environment from 2009 to 2012 at Huazhong Agricultural University, Wuhan, China, in the winter–spring growing seasons.…”

“…The nonsingleton variants, which were present in two or more lines, were retained as ‘raw variants’ for further analysis. To obtain high‐quality variants, raw variants were further filtered using the following criteria: (i) relative heterozygosity ( H R ) less than 0.2 (Wu et al ., ), which was used to discriminate simple SNPs/INDELs (only single allele is present in most of accessions) from hemi‐SNPs/INDELs and interhomoeologue polymorphisms (IHPs) (Trick et al ., ); (ii) the percentage of missing genotype of one variant is less than 60%. Finally, missing genotypes were imputed using Beagle software (version 3.3.2).…”

ddradseqtools: a software package for in silico simulation and testing of double‐digest RADseq experiments

“…) and oilseed rape ‐ Brassica napus ‐ (Wu et al . ) with input settings optimized through a series of runs of ddradseqtools was assessed to provide some guidance for running the software with reliable parameters. Experiments were selected to cover different features of ddRADseq experiments that have been parameterized in ddradseqtools , such as enzyme pair combination, range of selected fragment size, type of reads, type of library, length of insert and number of polymorphic loci (see the specific parameters for each experiment in Table ).…”