| Insertions/deletions(INDELs), is a type of abundant sequence and length polymorphisms in the plant genome, combine the characteristics of both simple sequence repeats(SSRs) and single nucleotide polymorphisms(SNPs), and thus can be developed as desire molecular markers for genetic studies and crop breeding. There has been no largescale characterization of INDEL variation in Brassica napus yet. In this study, we resequenced 23 inbred lines(ILs) from different geographic regions have different growth habit and obtained 351.2 Gb data. INDELs mined by aligning short paired end(PE) reads to the B. napus reference sequence(AC) and integrated ancestral reference genome(A+C). High sequencing depth 12.4x was obtained with AC reference genome as compared to 9.4x with A+C reference genome. Mapping rates varied from 61.1% to 89.1% in AC reference genome and 46% to 66.9% in A+C reference genome. Final outcomes of resequenced short PE reads alignment to reference genomes were as follows;A total of 538,691 INDEL variations that ranged from 1 to 10 bp in length were identified, and 104,190 INDELs were uniquely mapped on the pseudochromosomes of the reference genome. Of these INDELs, 135,596(25.17%) INDELs located in annotated genes. Single base INDELs(63.6%) dominated the other length of INDELs. Nucleotides A and T alone or in combination accounted for more than 65% of total INDELs. The average density in the whole genome of B. napus is 0.63 INDEL/kb DNA. The A subgenome has 311,927 INDELs with an average density of 0.99 INDELs/kb, while the C subgenome has 224,752 INDELs with a density of 0.43 INDELs/kb. C subgenome has higher number of INDELs in TE region than A subgenome. The number of INDELs on each chromosome was generally proportional to the length of chromosomes. However, in some cases such as chromosome C02, C05, C06, C08 and C09, the amount of INDELs was much lower than the average level. Hotspots of INDELs were identified on most of the B. napus chromosomes with up to 16.15-fold regional increase compared with the averages for the same chromosomes. A total of 38 and 335 hotspots were identified in the A and C subgenome respectively. The C subgenome has less INDELs and more hotspots than the A subgenome, suggesting that the distribution of INDELs in the C subgenome is uneven.A set of 595 INDELs of 2-5 bp in length was selected for experimental validation in 23 ILs. Of these INDELs, 530(89.01%) produced a single PCR product and thus are single-locus. A subset of 523(87.9%) INDEL markers were polymorphic among the 23 ILs. A genetic linkage map containing 108 single-locus INDEL and 89 anchor microsatellite markers was constructed using 188 recombinant inbred lines. The linkage map consisted of 19 linkage groups(LGs) and covered a total length of 1356.9 cM. The distribution of INDEL markers across the 19 chromosomes was uneven. Chromosome C03 had the maximum number of INDEL markers(22), followed by A08(8) and C04(8). C08 had the minimum number of INDEL markers(1). The majority of INDEL markers on the linkage map showed consistency with the pseudochromosmes of the B. napus cultivar ‘Darmor-bzh’. The INDEL variations and markers reported here will be the valuable resources for genetic studies and molecular breeding in oilseed rape. |
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