Genetic Basis Analysis Of Brassica Napus L.Ecotype Differentiation

Oilseed rape（Rapeseed,Brassica napus L.）is the second most important oil crops in the world,which has a wide variety during the long-term domestication and cultivation process.According to the demand for low temperature during vernalization to flowering,rapeseed can be divided into three ecotypes:Winter Oilseed Rapes（WORs）,Spring Oilseed Rapes（SORs）and Semi-Winter Oilseed Rape（SWORs）.The basis of ecotype differentiation is still not clear.Oilseed rape is a heterotetraploid species with a complex genome.Although there have been several published rapeseed genome information,its quality and representativeness are not enough to meet the needs of functional genomic research and rapeseed variety improvement.Here we sequenced and de novo assembled 8 rapeseed accessions containing zhongshuang11（ZS11）,and obtained 8 high-quality reference genomes representing three different ecotypes B.napus.And then,the PAVs of flowering related genes were identified,and the whole genome association study（GWAS）of flowering time and the transcriptome sequencing of the leaves of 8 rapeseed accessions at 5 stages before and after vernalization were carried out.The key genes regulating flowering stage of rapeseed were identified,and the genetic basis of ecotype differentiation of B.napus was preliminarily revealed.The main results are as follows:1. Eight rapeseed lines including two SORs（Quinta and Tapidor）,four SWORs（ZS11,Zheyou7,Shengli,Gangan）and two SORs（Westar and No2127）.The eight rapeseed lines were sequenced on Illumina and Pac Bio platform and de novo assembled,after assisted assembly and rectification with Hi C sequencing data,eight high-quality genome were obtained.The eight assembled genome size were 1.001-1.003Gb,the Contig N50 were 2.1-3.1Mb,and the Scaffold N50 were50.90-57.88Mb.Compared with Darmor-bzh,the continuity and completeness of the genome were greatly improved.There are 1.87-3.94 million SNPs and0.97-1.48 million In Dels between the reference genome of ZS11 and the other 7rapeseed genomes.In addition,there were 18-34 thousand PAVs,with a total length of 77-150 Mb,and 1619-4810 genes related to PAVs.2. Totally 1,204 homologous genes were identified in the genome of SWOR ZS11 based on blast through 280 flowering genes in Arabidopsis,with an average of 4.3 homologous genes per Arabidopsis flowering gene in rapeseed.The difference of copy number of different flowering genes in B.napus shows that recombination and loss occur in homologous genes,and functional differentiation may happened.The expansion of copy number of key flowering regulatory factors and genes involved in vernalization pathway is conducive to the regulation of flowering time by integrating environmental factors in B.napus.The flowering genes in different chromosomes is uneven distributed.3. GWAS analysis was carried out about the flowering time of 210 inbred lines in two spring environments and three winter environments.Eight significant loci were identified in winter environment,and 38 significant loci in spring environment（in which three loci overlapped）,and 60 flowering genes were associated.There are 10 flowering related genes stably detected in more than two environments,including Bna A02.FT,Bna A05.FPA,Bna A10.FLC,etc.4. A total of 17,173 PAVs with the total length more than 16Mb（including redundancy）were obtained by comparing 1,204 flowering genes in ZS11 with corresponding genes in 7 other lines,the average length was about 1,083bp.Among them,there are 670 genes（de redundant）contain PAVs,while 149 genes contain PAVs in gene region.6 flowering genes（Bna A02.FLC,Bna A03.FLCb,Bna A07.AGL1,Bna A09.GA1,Bna A10.FLC,Bna C04.AAT）in two winter rape lines and 6 flowering genes（Bna A03FCA,Bna A03.VGT1,Bna A06.TEL2,Bna A08.EIF2,Bna C01.PBP1,Bna A03.CRP）in two spring rape lines.It may also be related to ecotype differentiation.5. By comparing sequences of 10 Bna. FLC copies and 6 Bna.FT copies in 8rapeseed lines,12 PAVs（mainly TEs）were identified in 5 genes（Bna A10.FLC,Bna A02.FLC,Bna A03.FLCa,Bna A03.FLCb and Bna C02.FLC）.PAVs in Bna A10.FLC,Bna A02.FLC,and Bna A03.FLCb are closely related to flowering time and ecotype of rapeseed.CNV also existed in Bna A02.FLC and Bna C02.FLC.There were many PAVs in the noncoding region of Bna A03.FLCa,Bna A02.FT,Bna A07.FTa and Bna C02.FT,but the gene structure had not changed,which may not be related to ecotype differentiation.6. The transcriptome analysis of the leaves of 8 rape lines in five periods before and after vernalization showed that 5 Bna.FLC（Bna A10.FLC,Bna A02.FLC,Bna A03.FLCa,Bna A03.FLCb and Bna C02.FLC）and three Bna.FT The genes（Bna A02.FT,Bna A07.FTa and Bna C06.FT）play an important role in the regulation of rapeseed flowering.Bna A03.FLCa may also participate in the regulation of flowering,without ecotype specificity,and has nothing to do with the ecotype differentiation of rape.There are four other flowering inhibitors Bna.FLC with significant different expression level in ecotype lines,which may be related to ecotype differentiation.Bna A10.FLC was not expressed in spring rape before flowering,and the expression level in winter rape was significantly higher than that in semi winter rape,which was the key gene to regulate the flowering and differentiation of different ecotypes of rapeseed.Bna A03.FLCb is also a gene regulating flowering and differentiation of different ecotypes in B.napus.Due to the relatively low expression,the function may be weakened.In addition,PAV and CNV of Bna A02.FLC and Bna C02.FLC fine tuned the flowering period of each ecological type of rape.7. The effects of Bna A02.FLC,Bna A10.FLC and Bnac02.FLC on flowering period were compared according to the flowering time of BN-NAM population in6 winter and 2 spring environments,The regulation ability of flowering inhibitors Bna A02.FLC^NS,Bna A10.FLC^SWand Bna C02.FLC controlling flowering time is quite different among different environments.In general,Bna A02.FLC^NScan inhibit flowering in winter and spring,and can cause variation of flowering period for more than 10 days,with the largest regulatory effect.Bna A10.FLC^SWonly inhibited flowering in spring environment,but delayed flowering was the most effective in the background of Bna A10.FLC^Sand no Bna C02.FLC,which could produce variation of about 5 days.The effect of Bna C02.FLC gene may be weaker than that of Bna A02.FLC^NS.In summary,5 Bna.FLCs（Bna A10.FLC、Bna A02.FLC、Bna A03.FLCa、Bna A03.FLCb和Bna C02.FLC）and 3 Bna.FTs（Bna A02.FT、Bna A07.FTa和Bna C06.FT）play an important role in the regulation of rape flowering.Among 5Bna.FLCs the genes,Bna A10.FLC was the key gene to regulate the flowering and differentiation of different ecotypes of rapeseed,the variation of PAV and CNV of4 genes（Bna A10.FLC,Bna A02.FLC,Bna A03.FLCb and Bna C02.FLC）have the largest influence on the flowering period of rapeed.It is of great significance to the genetic improvement,new accession selection and germplasm resource creation of rapeseed.