Fine Mapping And Candidate Gene Analysis Of Late Flowering QFT7.1 In Brassica Rapa

Brassica rapa is an important vegetable crop,which originated in China and has long cultivated in worldwide,mainly as vegetable food,such as Chinese cabbage and pakchoi,a small part for the production of feed and oil seeds.Flowering time is one of the most important trait because it affects seed yield and commercial quality,thus determined growing season and cultivation area.Flowering time is an important quantitative trait,because of regulation mechanism is complex and greatly affected by environmental condition,it is very important to explore the molecular mechanism of flowering time in B.rapa.In this study,the late flowering Chromosome segment substitution line CSSL16 and the early flowering line Rc Br as materials,constructed NIL-F₂ segregation population.BSR-seq combined with traditional QTL analysis to identified the candidate interval.Candidate gene analyzed and functional verified were carried out,and the main research results were as follows:1.QTL identification for late flowering in B.rapaFirstly,late flowering Chromosome segment substitution line CSSL16,the early flowering line Rc Br,and NIL-F₂ segregation population as materials.Phenotypic evaluate of parents under four different environment showed that significant difference in flowering related traits DE5,DE10,FT and BI.On this basis,the whole genome re-sequencing was used to identified the difference segment,it was found that the difference segment between parents mainly in the upper part of chromosome A02 and the middle part of chromosome A07.Statistical analysis showed that the difference segment was about 2.04Mb,accounting for about 0.6%of the whole genome.BSR-seq combined with traditional QTL analysis were used to identified the candidate region was the middle part of A07 chromosome by F₂ segregation population and named as qFT7.1.2.Fine mapping and candidate gene analysis of QTL qFT7.1 for late flowering in B.rapaMolecular markers were developed in A07 interval and the recombinants was screened.The self-crossing progeny of recombinants were planted and analyzed by genotype and flowering related phenotype.Finally the QTL was fine mapped to the 56.4Kb interval of chromosome A07,and candidate gene Bra A07g018240.3C was screened and annotation analyzed,it was homologous with Arabidopsis ATC gene AT2g27550.Phylogenetic tree analysis showed that this gene belonged to the PEBP family and was a flowering suppressor gene.Sequence analysis showed that there were many mutations in promoter sequence,and expression analyzed demonstrate the hypocotyl showed significant difference expression level.It was also found the expression level of ATC related gene AP1 and LFY were significant difference between two parents,and negatively correlated with Bra A07g018240.3C.3.Functional verification and expression analysis of candidate gene Bra A07g018240.3CTransient expression method was used to verify the function of candidate gene.By VIGS method silencing candidate gene,it was found that the expression level of ATC gene decreased after silencing,and the plants showed an early flowering phenotype,so VIGS demonstrate the function of Bra A07g018240.3C.Subcellular localization showed that the gene was mainly expressed in the nucleus and cytoplasm.Early bolting line Rc Br and late bolting line CSSL16 as material,according to candidate gene expression level,selected the hypocotyl and stem apex to proceed transcriptome analysis,by identified the transcriptome data accurate and reliable quality,further screening differentially expressed genes,the hypocotyl of the transcriptome data obtain 4 differentially expressed genes related to flowering,and thirty-eight differentially expressed genes related to flowering were screened from the stem apex part,including some key factors in flowering pathways,such as AP1,LFY and SPL.GO and KEGG enrichment pathway of all expressed genes were analyzed,some pathways related to flowering were identified,such as plant growth regulation pathway,REDOX pathway and plant signal transduction pathway.The SNP/In Del locus of Rc Br and CSSL16 were mainly on chromosome A02 and A07,which were consistent with the results of re-sequencing data,thus proving the accuracy of mapping interval.These results are helpful to understand the flowering mechanism and provide genetic resources for the improvement of B.rapa.