NGS-based Physical Mapping Of Rapeseed(Brassica Napus L.)Cultivar Zhongshuang 11

Rapeseed(Brassica napus L.) is an allotetraploid obtained by a hybridization event between ancestors of Brassica rapa and Brassica oleracea. Both B. rapa and B. oleracea genomes have experienced a WGT event. Genome research and MAS of rapeseed is often hampered by its complicated genome structure. The published reference of European winter oilseed cultivar Darmor-bzh only covered 79% of the estimated genome, which is not sufficient for researches on important agronomic traits using GWAS and map-based cloning. Therefore, a B. napus genome reference of high quality is urgently required. To achieve this goal, a high-quality physical map is the important foundation of a high-quality genome reference. A BAC library of ZS11 was constructed, fingerprinting was performed after BAC-pooling, fingerprints of each BAC were used in contig building. Physical mapping of ZS11 is finally obtained by integrating contigs with genetic maps.A total of 73,728 clones of ZS11 BAC library were available in 192 384-well plates. Every six 384-well plates were taken as a unit, 192 plates were divided into 32 units. Each unit was placed as 3×2 display, each row(48 BACs) and each column(48 BACs) were pooled, resulting in 96 pools per unit and 3,072 pools in total. Plasmids in these pools were extracted, digested by Sac I/MseI enzymes and amplified after barcoded adapters were ligated. Restriction fragments were sequenced from both SacI and Mse I sites, the length of PE reads are 150 bp. 389 million reads were deconvolutable, 23 million(5.90%) of them were from E. coli or vector.Deconvolution is the process of assigning tags to each BAC clone. PE reads were truncated by 50bp×2, 70bp×2, 90bp×2 and 110bp×2 tag length to form tags. Tags were assigned to 3,072 pools individually. Among them, tags that only exist in one row and one column pool were assigned to the corresponding BAC. Those tags are also called WGP tags, which are essential for contig building. The average tag number for each BAC in four lengths is 14.00, 14.58, 14.47 and 14.50, respectively.Eight WGP maps were generated by combining different tag lengths(50bp×2, 70bp×2, 90bp×2 and 110bp×2) and FPC cutoff values(10-10 and 10-15). With the same cutoff value, small differences in the map metrics were observed with varying tag length. Using of a lower cutoff value in FPC resulted in more contigs and smaller average contig sizes.In order to match the 90bp-sequence marker in genetic maps of NAM populations completely and to obtain a continuous map, we chose the tag length of 90bp×2 and a cutoff value of 10-10 as reference for our WGP maps. A total of 1.067 million WGP tags were assigned to 61,729(83.7%) BACs for the 90bp×2 set. Finally 3,278 contigs containing 44,903 BACs was built, with 16,826 BACs remained to be singleton. The mean contig size was 501 kb and the calculated genome coverage is 1,642 Mb, 137% of the estimated B. napus genome size.15 high density and high resolution genetic maps from NAM population were used to integrate with our physical map. 2,487 contigs were anchored on those genetic maps and the calculated genome coverage of these anchored contigs equals the estimated B. napus genome size. A high-quality physical map of B. napus is essential for applications of map-based cloning, fine-scale QTL mapping and biotechnology on genes/QTL responsible for important agronomic traits and also important for accelerating of rapeseed breeding progress.