Mapping And Candidate Gene Analysis Of Bj.Pur,A Gene Controlling Purple Leaf In Brassica Juncea

The purple leaf trait,caused by accumulation of anthocyanin in plant tissue,can be used as a morphological marker in the utilization of heterosis,besides,anthocyanin has many kinds of biological and health care functions,like stress resistance,anti-oxidation,anti-proliferation,anti-mutation,inhibition of tumor cel s occurrence,prevention of cardiovascular and cerebrovascular diseases,etc.Thus,becoming a hot topic in vegetable genetic engineering research and new varieties breeding.Based on the six-generation genetic population constructed by Liu?2015?,in this study,we used the purple-leaf mustard inbred lines?ZT-15-P?and green-leaf mustard inbred lines?ZT-15-G?re-construct segregation populations,mapping and cloning a candidate gene for controlling of the purple leaf traits in Brassica juncea.The main findings are as follows: 1.Inheritance pattern analysis of the purple leaf traitTwo F₂ mapping populations?15-F₂ A and 16-F₂B?and BC5 near-isogenic lines?NIL?were reconstructed by using the sixth generation group.Genetic analysis showed that the separation ratio of purple leaf and green leaf traits of F₂ and BC population were consistent with 3:1 and 1:1,respectively.It was further confirmed that the purple leaf traits of musturd were controlled by a single dominant gene,named as Bj.Pur.2.Screening and mapping of linkage markersBased on the initial mapping results in Liu?2015?and genomic information of Brassica rapa as reference,by combined bulked segregant analysis with SSR,InDel and CAPS molecular marker technology,a total of 453 pairs of primers were developed and screened in 457 green individuals of 15-F₂ A mapping population,no polymorphic marker that showed stability differences was found.3.BSA-RNA-seq analysisThirty individuals of each group,purple leaf and green leaf homozygous F₂ plants,were selected and total RNA was extracted from the bulked samples to construct two extreme pools for sequencing.The transcriptome data were analyzed for twice:?1?Before the whole genome sequence of B.juncea inbred line ‘T84-66' was released,Brassica rapa?http://brassicadb.org/brad/?was used as the reference genome,analyzed and located the gene controlling the purple leaf traits at 19.5-21 M of A07 chromosome and a InDel marker,linked to purple leaf traits were found.?2?After genome information of Brassica juncea?https://www.ncbi.nlm.nih.gov/nuccore/LFQT00000000?was published,we performed a series of bioinformatics analysis,including mapped efficiency statistics,new genes identification,genes expression and differential expression genes analysis,differential expression genes functional annotation and enrichment,BSR correlation analysis,etc.Finally,selected six candidate areas on B2 and B6 chromosome: B2:645,778-822,189;B2:14,795,886-28,411,252;B2:30,951,237-33,131,066;B6:17,982,970-18,046,596;B6:20,817,254-20,923,707;B6:22,623,041-22,686,844.4.Identification,cloning and sequencing of candidate geneBy BLAST software,we re-position the previously linked markers and mapped Bj.Pur on the right side of the marker ID001?B2: 17.746M?,the genetic distance was 0.6cM.Then,combined with molecular marker localization result,45 differential expression genes related to anthocyanin synthase or light and temperature sensitive in the six candidate regions were selected and developed into 63 pairs of primers to screen in the parents.The result showed that,a marker,Bj-MYB90,developed by the Gglean060401 gene?annotated MYB90?were found to have fragment length polymorphisms among parents.Subsequently,the full length of gDNA and CDS of Gglean060401 was cloned and sequenced.The results showed that compared with the purple leaf parent,green leaf parent has a 1268 bp fragment insertion in the first intron region,while the CDS sequence has no difference.Suggesting that the Gglean060401 may be a candidate gene for controlling the purple leaf traits of mustard.5.Identification Gglean060401?MYB90?as the target gene Bj.PurThe Bj-MYB90 was identified in15-F₂ A and 16-F₂ B populations with a total of 2229 green individuals,the results showed that there was no exchange plant,indicating that the marker is co-segregation with the target trait.The expression analysis of Gglean060401 gene in purple and green leaf mustard showed that the expression level in purple leaf parent was 89.6 folds increase than that of green leaf parent.The above conclusions further confirmed that the Gglean060401 gene was the target gene Bj.Pur for controlling the purple leaf of mustard.