Gene Mining Of Glucosinolates Biosynthesis And Transport In Brassica Juncea

Allopolyploid Brassica juncea is an economically important crop in Brassicaceae.Glucosinolates(GSLs)and its hydrolysis,typically allyl-isothiocyanate is a characteristic determinant of the pungent flavor associated with mustard.However,the genetic mechanisms underlying GSLs accumulation and variation have yet to be elucidated.Furthermore,mustard(Brassica juncea var.gracilis),wasabi(Wasabia japonica)and horseradish(Armoracia rusticana)are popular pungent condimental crops favored by consumers for their specialized bioactive compounds of sinigrin.In this study,we resequenced 183 B.juncea accessions.Key genes associated with GSLs biosynthesis were firstly mined in B.juncea via SGS-GWAS(Scored genomic SNPbased GWAS).The structure variation between high-GSL and low-GSL accessions were accurately indentified via comparative genomics analysis and ONT(Oxford nanopore technologies)in B.juncea.Simultaneously,we de novo assembled the transcriptomes of mustard,wasabi,and horseradish in Brassicaceae.The convergent evolution mechanisms to accumulate sinigrin in these three species were revealed via comparative transcriptome analysis and gene mining of GSL transport was conducted.The main results in this study are as follow:1.Two major MYB28 orthologues associated with GSLs biosynthesis regulation were mined via SGS-GWAS.We conduct a systematic evaluation of seed GSLs in 183 B.juncea core accessions and resequenced them at 10 × coverage.A total of 689,411 high-quality SNPs located in gene region were retained after stringently filtering SGS(Scored genomic SNPs).Genome-wide association studies on seed GSLs accumulation was firstly conducted via SGS-GWAS in the re-sequenced B.juncea accessions panel.A total of 42 significant loci were associated with GSLs accumulation in 20.04-32.33 Mb region on A02 chromosome,and 3.44-4.44 Mb region on A09 chromosome in B.juncea.A total of 33 candidate genes were mined in the associated regions,including two major MYB28 orthologues(MYB28-A02 and MYB28-A09),in which MYB28-A 09 was firstly reported to be associated with the accumulation of GSLs in B.juncea.2.Copy number variations of MYB28 orthologues was confirmed to be associated with GSLs accumulation variation in B.juncea.A total of 6 and 4 copies of MYB28 orthologues were indentifed in a high-GSL vegetable B.juncea accession T84-66 and a low-GSL oilseed B.juncea accession AU213 based on their high-quality reference gneome,respectively.A total of one copy of MYB28 was lost both on A02 and A09 chromosomes of AU213 compared to that of T84-66.Furthermore,the copy number variations(CNVs)on MYB28 were validated to be associated with GSLs accumulation variation by ONT long-reads mapping between accessions with low and high GSLs in B.juncea.Total GSLs content of B.juncea accessions with one lost copy,and two lost copies of MYB28 on A02 or A09 chromosome was 146.54 μmol/g DW(Dry weight),and 34.32 μmol/g DW,respectively.Tissue expression analysis revealed that the expression level ofMYB28-A09 was higher than MYB28-A 02,and exhibited a highest expression level in silique wall.3.Convergent mechanisms underlying accumulation of sinigrin in mustard,wasabi and horseradish was revealed and a Glucosinolate transporter 4(GTR4)which belongs to the NPF gene family was preliminarily mined.We de novo assembled the transcriptomes of mustard(B.juncea),wasabi(W.japonica),and horseradish(A.rusticana).A total of 69,279,53,660,and 61,283 unigenes were generated.Some identical functional genes involved in the GSL biosynthesis and translocation such as IGMT1,FMOGS-OXI,MYB28,Dof1.1,and GTRs showed similar transcriptional patterns.A total of nine genes involved in the GSL metabolism exhibited convergent evolution via PCOC analysis.Simultaneously,a total of 478 genes were under positive selection in mustard,wasabi,and horseradish,in which a Glucosinolate transporter 4(GTR4)belonging to the NPF gene family was uncovered.4.GTR4 exhibited sinigrin uptake function in vitro.Phylogenetic analysis and protein structure prediction indicated that GTR4 is a homologue different from the reported Arabiopsis Glucosinolate Transporter gene 1/2/3(GTR1/2/3).Subcellular localization indicated that GTR4 was localized on plasma membrane.Tissue expression analysis revealed that the expression level of GTR4-B05 was higher than GTR4-A02,and exhibited a highest expression level in old leaf and silique wall.GSL uptake assay in Xenopus laevis oocytes indicated that GTR4 can transport sinigrin in vitro.