QTL Mapping And Molecular Mechanism Analysis Of Preharvest Sprouting In Brassica Napus

Brassica napus is one of the most important oilseed crops in China.During seed development,exposure to high temperature and high humidity could lead to preharvest sprouting（PHS）,resulting in a decrease in crop yield and quality.However,the mechanism underlying PHS in B.napus was poorly understood.It is important to analyse the molecular mechanism of PHS and to select and breed B.napus varieties that are resistant to PHS in order to improve oil yield of B.napus.To explore the mechanism of PHS,quantitative trait locus（QTL）mapping was performed on the PHS rate of the KN DH population.The candidate gene for PHS was verified in combination with the differentially expressed genes（DEG）between rapeseeds with high and low PHS rate.Further functional validation of the candidate gene in Arabidopsis and B.napus was carried out.The main findings of this study are as follows:（1）The oil content was decreased by 14.4%to 33.3%in PHS seeds.Compared to normal seeds,the relative content of C16:0 and C18:3 in the PHS seeds was increased,whereas the relative content of C18:0,C18:1 and C20:1 was decreased.The size of oil bodies and the proportion of small oil bodies in the cotyledon of PHS seeds was also decreased.The abscisic acid（ABA）content of the rapeseeds with high PHS rate was significantly lower than that of the rapeseeds with high PHS rate,while there was no significant difference in gibberellic acid（GA）content.（2）28 identified QTL controlling PHS were detected by QTL mapping,distributed in9 linkage groups,and the phenotypic variation ranged from 0.76%to 8.94%.After meta-analysis,25 consensus QTL were obtained,and 3 consensus QTL could be detected in multiple environments.Forty-three genes related to ABA synthesis and signal transduction were identified within the QTL.48 pairs of epistatic QTL were detected and they were distributed in all chromosomes of B.napus except for A4.Eight consensus QTL for PHS were collocated with QTL for thousand seed weight,oil content,oil yield,seed yield,seed number per silique and hemicellulose content.（3）Transcriptome sequencing was performed on the developing seeds of rapeseeds with high and low PHS rate from two genetical population.It was showed that the DEGs between the rapeseeds with high and low PHS rate were related to the processes of energy metabolism,signal transduction and carotenoid biosynthesis.The genes related to ABA degradation were highly expressed in the late seed development of rapeseeds with high PHS rate.A total of 1017 DEGs were common identified.KEGG enrichment results showed that these genes were involved in pyruvate metabolism,fatty acid degradation,limonene and pinene degradation,zeatin biosynthesis,α-linolenic acid metabolism,carotenoid biosynthesis,glycolysis and other biological processes（4）108 common genes were obtained by comparing candidate genes in QTL with DEGs in the transcriptome.Four genes involved in carotenoid metabolism and flavonoid metabolism（Bna A02g30320D,Bna C01g10770D,Bna C01g11550D and Bna C08g26020D）were significantly differentially expressed among the rapeseeds with high and low PHS rate.G₁₂₁₉ in Bna C01g10770D（Bn NCED2）was converted to T₁₂₁₉,resulting in the conversion of amino acids from valine to leucine.T₁₂₁₉ was significantly enriched in Asian rapeseeds and semi-winter rapeseeds.（5）Bn NCED2 could complement the phenotype of nced2 mutants in Arabidopsis and the allogeneic expression of Bn NCED2 in Arabidopsis could delay seed germination.RNAi of Bn NCED2 in rapeseed could lead to PHS,and the overexpression of Bn NCED2could delay seed germination.In summary,QTL mapping and transcriptome were combined to screen the candidate gene for PHS in rapeseed.Moreover,it has been demonstrated that Bn NCED2 could affect seed dormancy and germination in Arabidopsis and rapeseed,which lay the foundation for the improvement and cultivation of new rapeseed varieties with low PHS rate.