Molecular And Genetic Analysis Of Bolting And Flowering Time In Brassica Rapa

Brassica rapa vegetables are important crops cultivated in China.Bolting is one of the most important agronomic traits for production of Brassica rapa crops.Premature bolting triggered by low temperature leads to reduction of the yield and quality of the harvested products of B.rapa. Breeding for late-bolting variety is a critical step to resolve this problem.FLOWERING LOCUS C (FLC),a floral repressor,is a switch gene involved in vernalization pathway,which regulates the flowering time in Brassica.There are four FLC homologs and wide range of bolting/ flowering time variation in B.rapa.However,the ways of these homologs regulating the bolting and flowering and the relationships between these homologs and other flowering time-related genes are still unkonwn. Markers tightly linked to or co-segregated with these genes have not been reported.In order to utilize these genes to regulate bolting and flowering time in B.rapa,the relationship between sequence of BrFLCs and bolting time variation was investigated.A CAPS marker linked to bolting trait was developed,several important flowering related genes were mapped and the relationship between these genes and QTLs controlling bolting/ flowering time were analyzed.The main conclusions are as follows:1.Sequence variations in BrFLC1 homolog were associated with bolting time phenotype. Fragments of four FLC homologs(Exon 4-Exon 7)from 30 selected B.rapa accessions were amplified and sequenced.The analysis of sequence indicated that only the sequence variation of BrFLC1 was significantly associated with bolting time among amplified regions.No significant relationship between variation among the other three homologs and bolting time were detected.2.A mutation of base G-to-A at a 5' splice site in intron 6 of BrFLC1 altered splicing pattern. RT-PCR analysis of BrFLC1 revealed that base substitution of G-to-A at splicing site of BrFLC1 gene altered normal splicing and produced at least two different alternative transcripts.We discovered four alternative splicing patterns,SPA1- SPA4,in different BrFLC1 alleles,besides the normal one(SpG) after sequence analysis.Three of them partly retained regions of intron 6(SPA1- SPA3,intron retention) and one skipped entire region of exon 6(SPA4,exon skipping).Splicing patterns occurring in eight accessions showed that 5 accessions with G allele gave normal splicing pattern SpG,and other accessions with A allele,four alternative splicing patterns,SpA₁-SpA₄.Therefore,aberrant splicing of the pre-mRNA leaded to loss-of-function of BrFLC1.This finding will facilitate the understanding of bolting and flowering mechanism.3.Mutation at the splicing site affected bolting phenotype.A CAPS marker designated as FLC1-Mva I was developed for detecting alleles at the splicing site.The marker can distinguish G and A alleles at Pi6+1 site by digesting a PCR product covering the splicing site with Mva I.The genotype of this marker correlated significantly with bolting time in a diverse collection of 121 B.rapa accessions and a segregating DH population.Our data suggested that a naturally occurring splicing mutation in BrFLC1 gene contributed to bolting time variation in B.rapa. 4.Six important flowering time genes were mapped on a genetic linkage map of Chinese cabbage (B.rapa L.ssp.pekinensis).One hundred and eighty three doubled haploid(DH)lines derived from isolated microspore culture of a cross between two diverse Chinese cabbage DH lines,Y177-12 and Y195-93 were used to construct the map.The genetic linkage map comprises a total of 452 markers, including 139 SRAP,68 SSR,230 AFLP,6 STS,4 ESTP,1 CAPS and 3 morphological markers. Among them,47 common markers including 39 SSRs,4 ESTPs,2 STSs,1 CAPS and 1 SCAR provided anchors to previously published linkage map for B.rapa and B.napus and was followed as the referenced mapping of A01～A10.The total length of the linkage map was 1272cM with an average interval of 2.8 cM.Six important flowering time loci including three BrFLCs,two MAFs and one FT were mapped on the map.Three loci of FLC homologs represented the previously reported BrFLC-1,-2,-3(-3a and 3b)on A10,A02 and A03,respectively.One FT and two MAF loci were also integrated into the map and distributed on A07,A02 and A05,respectively.5.QTL associated with both bolting and flowering time were detected.QTL analysis were carried out for bolting and flowering time under multiple conditions(3 years and 2 locations)on genetic linkage map of Chinese cabbage.Thirteen QTLs for flowering time and 11 QTLs for bolting time were detected. These QTLs distributed over 18 putative genomic regions on 9 linkage groups.The percentages of variation explained varied between 2.4～28.8%.One of the most significant QTL associated with both bolting and flowering time was located at the same position on A02.It was detected under three years' data,which could explain 9.3～28.3%of phenotypic variation.Another QTL at the top of A10, co-segregating with BrFLC1,can be detected only under partly vernalized condition,indicating that the gene controlling this QTL loci was affected by vernalization status of plants.Furthermore,putative QTLs close to BrFLC3 gene on A03 and FT gene on A07 were also detected..