Studies On QTL Mapping And Gene Expression Regulation Of An Apetalous Trait In Brassica Napus

The apetalous trait has attracted much attention in Brassica napus because of its low-energy consumption,high photosynthetic efficiency and klendusity to Sclerotinia sclerotiorum,etc.However,very little is known regarding the molecular mechanism of the apetalous trait so far.In this study,the apetalous near-isogenic lines named APL01 and PL01,the AH population containing 189 recombinant inbred lines(RILs)derived from a cross between APL01 and another normally petalled variety ’Holly’,were used for the studies on QTL mapping and genes expression regulation,with a view to attempting to reveal the molecular mechanism of the apetalous trait in rapeseed.The main results were listed as follows:(1)High-density SNP map construction and QTL identification for the apetalous trait in rapeseed.The Brassica 60 K Infinium BeadChip Array was used to genotype 189 RIL individuals.A high-density genetic linkage map was constructed based on 2755 bins involving 11458 SNPs and 57 simple sequence repeats.The linkage map covered 2027.53 cM,with an average marker interval of 0.72 cM.Comparative anaylsis suggested that the genetic linkage map had good collinearity with the B.napus reference genome.Petalous degree(PD)was used to assess phenotypic variation of petals,and phenotypic data of PD in the AH population were obtained from the five different environments.Based on the high-density SNP map,QTL mapping for PD suggested that a total of 19 identified quantitative trait loci(QTLs)distributed across chromosomes A3,A5,A6,A9 and C8 were obtained,and these QTLs were integrated into nine QTLs by a meta-analysis.The major QTL qPD.C8-2 was consistently detected in all five environments,and explained 7.11%-11.29%of the estimated phenotypic variation(PV).The two QTLs,qPD.A9-2 and qPD.C8-3,were stably expressed in four environments,and explained 6.05%-7.06%and 6.12%-8.4%of PV,respectively.In addition,six QTLs were environment-specific QTLs detected only in one environment.(2)The abnormal growth stage of petals in apetalous rapeseed.The floral organ morphogenesis of the apetalous near-isogenic lines APL01 and PL01 in rapeseed were observed using paraffin sections.For the apetalous line APL01,sepal,stamen and gynoecium primordia successively appear in the young buds,while petal primordia don’t arise in the second whorl at later stage 5,and haven’t appeared in subsequent stages,suggesting that the apetalous characteristic of line APL01 is determined at later stage 5.(3)Transcriptome analysis of initial growth of petal primordium.Young buds in stage 3-5 collected from the apetalous near-isogenic lines APL01 and PL01 were used for high-throughput RNA sequencing(RNA-seq).In total,13205 differential expressed genes(DEGs)were detected,of which 6111 genes were significantly down-regulated,while 7094 genes were significantly up-regulated in line APL01 compared with PL01.GO enrichment analysis suggested that the down-regulated genes were significantly enriched in the 24 GO terms promoting a wide range of basic cellular processes,while the up-regulated genes were significantly enriched in the 7 GO terms preventing a wide range of basic cellular processes.These findings indicated that the basic cellular processes responsible for petal onset were potentially prevented,which leads to the abnormal growth of petal primordia of line APL01.A further analysis revealed that 36 petal regulators implicated in transcriptional regulation,epigenetic modification,protein ubiquitination and farnesylation were differentially expressed in line APL01 compared with PL01,suggesting that these regulators potentially participate in petal development of line APL01 probably by regulating the genes required for the basic cellular processes responsible for petal morphogenesis.RNA-seq data coupled with QTL mapping suggested that five,six and twelve genes in the mapped intervals of qPD.A9-2,qPD.C8-2 and qPD.C8-3,respectively,expressed differentially in line APL01 compared with line PL01.Subsequently,the expression patterns of these DEGs were analyzed between APL01 and another normally petalled variety ’Holly’ by using qRT-PCR.The dynamic expression levels of three,four and four DEGs in the mapped intervals of the three QTLs between lines APL01 and PL01 were same as between APL01 and ’Holly’.At last,eight genes(CG1-CG8)were considered as the potential candidate genes regulating the petal development of line APL01.(4)Construction of a hypothetical regulatory network involved in the apetalous trait.The expression levels of the 36 petal regulators and 1 candidate gene(CGI)regulating the petal development obtained from RNA-seq assay were analyzed in the young buds of APL01,PL01 and another normaly petalled variety ’Holly’ by using qRT-PCR.Total 13 petal regulators and CGI showed the same dynamic expression levels between lines APL01 and PL01 as between APL01 and ’Holly’.Thus,the 14 genes were chose as target genes(TGs)for the subsequent experiment.The expression levels of the 14 TGs in the AH population were analyzed in two environments by using qRT-PCR.For the quantitative trait transcript(QTT)-association analysis of PD,the 14 TGs expression levels were the genotypic data,while PD was the phenotypic data in each environment.QTT-association analysis of PD determined that PLURIPETALA(PLP)was significantly associated with PD with high heritability(h2=18.62%,h2=15.11%)in all two environments,suggested that PLP acted as the major QTT of PD(h2>10%).The effect of PLP on PD is-6.88 and-6.13 in all two environments,respectively,indicated that PLP negatively regulates petal development of line APL01.The ramaining TGs were not significantly associated with PD,implying that these TGs potentially participated in petal development probably by regulating PLP expression.The transcript-association mapping of PLP expression levels showed thatCHROMATIN-REMODELING PROTEIN 11(CHR11)(h2=17.28%)was significantly associated with PLP expression,and the effect of CHR11 on PLP is 46.77,suggested that CHR11 acted as the major and positive regulator of PLP expression.By that analogy,the relationships among the remaining TGs were revealed through QTT-association mapping for TGs’ expression levels.Based on the relationships among PD and TGs detected in the present study,we provide a hypothetical molecular regulatory network that 14 TGs potentially regulate the petal development of line APL01 mainly through the CHR11-PLP pathway.Obviously,PLP acts as the terminal signal integrator negatively regulating petal development in the CHR11-PLP pathway.