Study On Genetic Structure And Selection Of Domestication Of Brassica

Brassica napus (genome AnAnCnCn, rapeseed) was formed by allopolyploidy between ancestors of B. oleracea (Mediterranean cabbage, genome CoCo) and B. rapa (Asian cabbage or turnip, genome ArAr) at about 10,000 years ago. Rapeseed has a short domestication history of only 300-400 years. However, rapeseed shows broad ecophysiological adaption to different climatic zones and latitudes, being mainly classified into spring (S), winter (W) and semi-winter (SW) groups. With the rapid development of sequencing technologies and computational methods, population genetic analysis are now becoming a powerful tool for detecting natural variation and human selection underlying complex traits in crops. Owing to dramatically decreasing cost of genotyping technology, the emergence and application of next-generation sequencing technologies has been successfully applied in several crops. With the release of reference genome sequences of B. rapa, B. oleracea and B. napus, it is possible to dissect the genetic structure and selective domestication of B. napus.In this study, a panel of 327 rapeseed accessions from three ecotypes was genotyped using a 60K Brassica Infinium(?) SNP array which was recently developed by the international Brassica Illumina SNP consortium. In addition, a diversity panel comprising 101 B. napus lines (with 31 lines overlapped with the accessions tested by 60K SNP array) from Lu et al (2014) was used for population differentiation and selective domestication using RNA sequencing. The aims of this study were to investigate the genetic structure between winter, spring and Chinese semi-winter oilseed and to clarifiy the adaptation mechanism of rapeseed to different environments. This study will promote the uitilization of rapeseed germplasm and contribute to the adaptation ability of rapeseed. The main results were listed as follows.1. Genetic structure and the diversity in B. napusThe linkage disequilibrium (LD) and genetic structure analysis in the whole genome levelA total of 36,241 SNPs from the whole genome level were used for LD analysis. Result showed that the average distance over which LD decayed to half of its maximum value in rapeseed was-150 kb, being similar to that in other self-pollinated crops. In addition, when LD decayed to an average r2= 0.1, the decayed distance in A and C subgenomes of rapeseed was 0.35-0.4 and 3.5-4 Mb, respectively. This indicates that more recombination events might occur in the A subgenome than in the C subgenome. Most recombination events can be identified indirectly from patterns of LD. Here we defined whole-genome LD hotspots (10-kb adjacent windows, the least 5% of r2 and p< 0.01) and LD coldspots (10-kb adjacent windows, the largest 5% of r2 and P< 0.01). Based on the hotspots and coldspots analysis on the whole genomic level,78.16% of LD hotspots were identified from the A sub-genome of rapeseed, while 72.59% of LD coldspots were detected from the C sub-genome. Higher genetic density and GC content were detected from the hotspot regions than from the coldspot regions, while much more DNA transposons were found overrepresented in the LD coldspots, further suggesting a higer level of genetic variation in the A sub-genome of rapeseed.Population stucure and genetic diversity in rapeseedIn order to compare the three ecotypes of rapeseed from the whole genomic level, we further examined the nucleotide diversity (π) and the extent of LD (r2) of all the rapessed accessions using the SNP data. Three distinct groups, i.e. S, W and SW groups were easily distinguished from the population structure analysis and PCA model. Genetic introgressions were found among these groups, especially in semi-winter group which possessed great genomic components from the winter and the spring groups. A higher nucleotide diversity and a faster LD decay were detected from the A subgenome in SW than in W and S, This indicated that semi-winter group in China had induced wide genetic basis from B. rapa during its domestication, thus broadened the genetive diversity of the A subgenome of rapeseed.Population expansion analysisDuring the breeding process, degeneration of genetic diversity was found in rapeseed, however, the low- and moderate-frequency alleles were accumulated in the population. Therefore, the rare SNPs (MAF< 0.05) were investigated to study the expansion of populations in S, W and SW groups. The number of rare SNPs of each chromosome in SW group was significantly higher than that in S group and W group (T-test, p= 5.17E-/), and a negative Tajima’s D value was found in the SNP region of SW group, suggesting an ongoing population expansion of Chinese semi-winter lines after the introducing from Europe.2. Analysis of population differentiation (Fst) and selective signal in different ecotypes rapeseed.Population differentiation among three ecotypes of rapeseed In order to exploit differentiations in the three ecotypes, the divergence index Fst was calculated among the three ecotypes by using the overlapping 100-kb window approach by Genepop software (version 4.2.1). The differentiations were found unevenly distributed across the B. napus genome. The largest Fst value was identified between the W and SW groups (Fst= 0.235), while the smallest Fst value was detected between the S and SW groups (Fst= 0.173). Moreover, genes and QTLs associating with adaptation (such as flowering time, resistance to Leptosphaeria maculans and Scleratinia sclerotiorum) were identified from the top 5% FSt confidence interval region, providing clues for searching the genes involved in geographic adaptation.The differences of Chinese semi-winter rapeseed (SW) with European winter type and spring type (W&S) in North AmericanTo detect evidence of recent selective signal adaption to different environment, the genes that located in the top 5% Fst confidence intervals were analyzed using the agriGO software. Data showed that the genes which significantly differed from the SW type rapeseed with the winter and spring rapeseed were associated to vernalization response, indole glucosinolate catabolic process, regulation of lipid catabolic process, regulation of shoot apical meristem development, regulation of short-day photoperiodism, flowering, negative regulation of circadian rhythm, regulation of salicylic acid mediated signaling pathway and others.In order to identify the selection sweeps between SW groups and W&S group, we calculated the distribution of the π ratios (SW/W&S) and Fst values. We used an empirical procedure and selected windows simultaneously with significant low and high π ratios (the 5% left and right tails) and significant high Fst values (the 5% right tail) of the empirical distribution as regions with strong selective sweep signals along the genome. The largest selective sweep was identified at A09 and C06 for W&S group and SW groups, respectively. It was interesting that one QTL related to blackleg resistance and one QTL related to Sclerotinia resistance were identified fromthe selection sweep region on A09 and C06, respectively, indicating the different selection sweeps for disease resistance in the SW rapeseed and the winter and spring rapeseed.The genetic differences between winter and spring rapeseedEnrichment analysis was performed among the differentiated genes between winter and spring rapeseed by integratying the data of microarray and transcriptome. Three overlapping terms were detected, including response to stimulus, defense response and flowering behavior. This indicated that these differentiated genes between the winter and the spring rapeseed have mainly contributed to the adaptation of local environments.Genome wide association analysis (GWAS) for flowering time in winter and spring rapeseed In order to reveal the mechanism of flowering time (FT), we performed a genome-wide association study among 60 winter rapeseed and 71 spring rapeseed accessions with the PCA +K model in Tassel v5.0 for the GWAS.A total of 38 SNPs on chromosome A02, A03, A09 and C08 were identified significantly associated with FT, individually explained 5-28% of the phenotypic variations. Interestingly, the SNP association region on C08 (35.01-35.38 Mb) was found to be homologous with the association region on A09 (30.00-31.21 Mb). Among these 38 SNPs,37 were previously unidentified. Therefore, these SNPs may be associated with the adaptation to Chinese semi-winter environment. However, futher studies are needed for the identification of gene function.Gene conservative analysis of FT in different species The RNA-seq datas from B. napus and the Microarrays datas from Arabidopsis were used for biological pathway enrichment analysis between winter and spring groups. The result showed the H3K27me3 regulation mechanism is the main pathway and participates in plant vernalization response. In additons, related studies found that the H3K27me3 regulation FT gene. This study further found the gene conservative using express level and the three-dimensional sturcture of proteins among different species.