| Plant height and the first branch height,related to thickness of pod canopy,are playing an essential role in yield of Brassica napus.To mine candidate genes of plant height and the first branch height,we utilized a high-density SNP genetic map to do quantitative traits locus(QTL)mapping for recombinant inbred lines(RIL)population,and used the resequencing data to do genome-wide association study(GWAS)for natural population in this study.Subsequently,we compared the RNA-seq data of extreme phenotype to determine the different expression genes.Then,we identified these genes by using quantitative real-time PCR and determined their basic biological characteristics.The main results were shown as follows.1.Phenotypic analysis of plant height and the first branch heightThe coefficient of variation of plant height in RIL population was 5.24% and 5.75% in 2016 and 2017,and the first branch height was 18.58% and 17.73% respectively.In natural population,the coefficient of variation of plant height was 8.57% and 10.16%,and the first branch height was 26.65% and 27.86%.In different environments,plant height and the first branch height were relatively stable in RIL population and natural population,and the coefficient of variation of plant height was lower than that of the first branch height.In RIL population,the broad heritability of plant height and the first branch height was 75.45% and 68.77%,and 87.42% and 82.21% in the natural population,respectively.In the same population,the heritability of plant height is higher than that of the first branch height.Both traits showed continuous normal distribution in each environment and were quantitative traits controlled by multiple genes.2.Correlation analysis and path analysis of the traitsIn RIL population and natural population of two years,plant height was significantly positive correlation with thickness of pod canopy and economic yield.There was a negative or non-significantly negative correlation between the first branch height and thickness of pod canopy,and there was an extremely significant positive correlation between the first branch height and the economic yield in the natural population,but no significant correlation in RIL population.Path analysis showed that plant height had a direct positive effect,the first branch height had a direct negative effect for economic yield in different environments.3.QTL mapping for plant height and the first branch height in RIL populationBased on the genetic linkage map,a total of 19 QTLs of plant height and the first branch height were detected using the 2016,2017,and BLUP values by utilizing composite interval mapping(CIM)method.The explained phenotypic variation of the individual QTL ranged from 4.60% to 19.10%.The additive effect of the QTLs locus on A01 and A09 were negative,which hinted that the increasing effective alleles were from the male parent,and the remaining QTLs were from the female parent.There were 10 QTLs of plant height distributed on chromosomes A03,A04 and A09,and the explained phenotypic variation of the individual QTL was between 4.60% and 13.29%,in which a QTL located on the A04 chromosome(A04:13373664-13531885)was detected using the 2017 and BLUP values simultaneously.Nine QTLs were detected for the first branch height and distributed on chromosomes A01,A02,A05,A09,C01 and C05,with the individual QTL explaining 5.12% to 19.10% of phenotypic variation.Among these QTLs,q-2017BH-A09-1(32375997-32618214),q-BLUP-BH-A09-2(32435925-32608817)and q-BLUP-BH-A09-3(32514368-32619386)were partially overlapped.4.Genome-wide association study for plant height and the first branch height in natural populationThere were 89 significant SNPs related to plant height and the first branch height under the optimal model.75 significant SNPs were detected for plant height,which were distributed on chromosomes A02,A03,A07,C02 and C06,with the SNPs accounting for 6.01% to 20.05% of phenotypic variation.And 42 SNPs among these SNPs were both detected using the 2017 and BLUP values,one was on the A02 chromosome,38 on the A07 chromosome,and three on the C06 chromosome.There were 14 significant SNPs related to the first branch height located on chromosomes A02,C02,C03,C04,C05 and C09.The contribution rate of phenotypic variation of the SNPs were between 6.23% and 18.36%.And 5 of these SNPs were detected simultaneously using the 2017 and BLUP values,with 3 SNPs on the A02 chromosome,1 SNPs on each of the C02 and C05 chromosomes.5.Transcriptome analysis of extreme phenotype accessionsA total of 7206 differentially expressed genes(DEGs)were detected in the stem apex of extreme plant height accessions,including 3475 significant up-regulated expression genes and 3731 significant down-regulated expression genes in stem apex of YC4 with the higher plant height.There were 5128 DEGs in stem apex of the extreme the first branch height accessions,including 2654 significant up-regulated expression genes and 2474 significant down-regulated expression genes in stem apex of YC11-the first branch height was higher.The results of GO and KEGG enrichment analyses for plant height and the first branch height were similar.In biological process,DEGs were mostly enriched in the terms of cellular process,single-organism process and metabolic process;in cellular component,DEGs were mostly enriched in cell,cell part and organelle;in molecular function,DEGs were mostly enriched in binding and catalytic activity.In KEGG enrichment analyses,DEGs were significantly enriched in ribosome,carbon metabolism,biosynthesis of amino acids and plant hormone signal transduction.6.Screening and expression verification of candidate genesThe candidate genes of plant height and the first branch height had been mined through QTL mapping,GWAS and RNA-seq.There were 13 candidate genes for plant height and 11 candidate genes for the first branch height.The Arabidopsis homologs of these genes are mainly involved in plant growth and development,cell proliferation and expansion,cell cycle,cell wall,the synthesis and signal transduction of gibberellin and spermidine,ribosome composition.The results of transcriptome data were verified by qRT-PCR,and the expression trends of each candidate gene among the different accessions were consistent.The candidate genes BnaA02g09200 D,BnaA04g17490D,BnaA07g27280 D,BnaA07g28720D,BnaA07g28820 D and BnaC02g35320 D were expressed much more in the shoot apex of the accessions with higher plant height,BnaA02g09270 D,BnaA03g47940D,BnaA04g17050 D,BnaA04g17120D,BnaC06g30260 D,BnaC06g30400D,BnaC06g30410 D had a lower expression level.Among the 11 candidate genes of the first branch height,BnaA01g25960 D,BnaA01g30830D,BnaA02g31980 D,BnaA02g37010D,BnaA09g49220 D,BnaC02g14570D,BnaC02g14600 D were highly expressed in the shoot apex of the higher accessions,BnaA01g27550 D,BnaA01g28120D,BnaA01g29630 D,BnaA09g48360D were contrary. |
