Genome-Wide Association Study Reveals The Genetic Archietecture Of Flowering Time In Rapeseed And The Funtional Analysis Of Arabidopsis Gene ECERIFERUM27

Association analysis become an effective method in QTL location and discovering important genes in recent years.Our study aimed to reveals the genetic architecture of flowering time in rapeseed by Genome-wide association analysis.We studied the function of CER27 and CER27 orthologous genes in Brassica napus by reverse genetics.The major points were described as follows:1)In this study,a diversity panel consisting of 523 B.napus cultivars and inbred lines was genotyped with the Brassica 60K Illumina Infinium SNP array.The probe sequences of the SNP array were used to perform a BLAST search against the Brassica napus Genomes Browser,They were regarded as nonspecific markers when BLAST matched to two or more locations in the reference genome.The standards of quality control for SNP data were as follows:call frequency≥0.8,MAF≥0.05,and homozygous genotype frequency cannot be zero.Specific markers that meet the standards of quality control were reserve.Finally,26,024 SNPs were selected for assessing population structure and relative kinship.21,117 of 26,024 SNPs had specific physical location information and were used for LD analysis and association analysis2)The population could be divided into two clusters,P1 and P2.P1 contained 96 lines,of which 25 lines were from Europe.P2 contained 424 lines,of which 403 lines were from China.The average relative kinship between any two inbred lines was 0.0443 in the panel.When r~2=0.1,the A subgenome and C subgenome were 1.2 Mb and 7.8 Mb,respectively.3)In two years,flowering times of the panel were investigated in five regions(Wuhan;Changsha;Nanjing;Ezhou;Chongqing)including eight different environments.The PCA+K model was eventually selected for association mapping A total of 41 SNPs distributed on 14 chromosomes were found to be associated with flowering time,and 12SNPs located in the confidence intervals of QTLs identified in previous researches based on linkage analyses.GWAS was performed on two derived traits,environment sensitivity and temperature sensitivity.The most significant SNPs were found near Bn-scaff＿16362＿1-p380982,just 13 kb away from Bna C09g41990D,which is orthologous to A.thaliana CONSTANS.B.napus genes orthologous to A.thaliana flowering time-related genes located within 300 kb of significant SNPs were treated as candidate genes.There were 25 candidate genes identified using this standard.4)Protein sequence analysis showed that At5g02890 is highly conserved in the Brassicaceae.q RT-PCR analysis shown that CER27 transcripts were detected at high levels in siliques。we fused the At5g02890 promoter to the Escherichia coliβ-glucuronidase(GUS).GUS signals were detected in seeds throughout the entire growth period and in the abscission zones at the bottoms of siliques as well as in buds,flowers,seedlings,stem tops,trichomes of stem bases,hydathodes of rosette leaves and cauline leaves.5)Overexpression of CER27 and CER27 orthologous genes Bn CER27-1 from Brassica napus in Arabidopsis causes glossy stems.Defective wax deposition and cuticle structure in OE-CER27 and OE-Bn CER27-1 inflorescence stems.The levels of wax monomers C28 or shorter were higher in the overexpression plants than in wild type.By contrast,reductions were observed in the levels of all wax monomers C29 and longer.CER27 is an ER-localized protein,which is consistent with its possible role in the biosynthesis of wax precursors.6)In total,3,945 significantly differentially expressed genes(DEGs)were isolated from the OE-CER27 and wild-type inflorescence stems by transcriptome analysis,which contained 1,434 upregulated genes and 2,511 downregulated genes.nine of 11 cuticular wax synthesis-related DEGs with different expression levels in OE-CER27 plants are involved in very-long-chain fatty acid elongation.Many DEGs involved in‘plant hormone signal transduction’.We measured the concentrations of endogenous indole-3-acetic acid(IAA),ABA,JA and jasmonoyl-isoleucine(JA-Ile)by HPLC-MS and found that overexpressing CER27 may influence ABA,JA and JA-Ile biosynthesis and block polar auxin transport.