Genetic Analysis Of Silique And Quality Related Traits And Functional Characterization Of CqSL-C7 And GGSL-C2 In Brassica Napus L.

Rapeseed(Brassica napus)is an important oil crop,and its meal can be used as animal protein feed.The yield and quality related traits have always been the focus of breeders in rapeseed.As close relationship is existed between silique related traits and yield,mining QTLs of silique related traits,cloning the target genes of them and revealing their functions will be beneficial to high yield breeding.Meanwhile,increasing the oil content and reducing the glucosinolate content in seed can greatly enhance the economic value of rapeseed.At present,the research on quality related traits mainly stays at the stage of QTL mapping.Identifying new QTL loci for quality related traits,cloning important QTLs for glucosinolate content and developing corresponding functional markers can provide an efficient way for quality improvement in rapeseed.In this study,a DH population was developed with two parental lines ZS11 and G120,and QTL mapping for silique and quality related traits was conducted.Subsequently,two QTLs cq SL-C7 and q GSL-C2 for silique length and seed glucosinolate,respectively,were cloned by map-based cloning,and their functions were further characterized.The main results are as follows:1.QTL mapping for silique length(SL),seeds per silique(SPS),seed density per silique(SDPS),seed oil content(SOC),and seed glucosinolate content(SGC).QTL mapping for the five traits in seven environments was conducted.A total of 198 identified QTLs were obtained.After QTL meta-analysis,all the identified QTLs were integrated into 71 consensus QTLs,including 18,15,10,15,and 13 for SL,SPS,SDPS,SOC,and SGC,respectively.Among them,eight were major QTLs,i.e.,cq SL-A9-2,cq SL-C7,cq SGC-C2,cq SOC-A5-2,cq SOC-A5-3,cq SPS-A6-2,cq SPS-A7-2,and cq SDP-A9-2.2.Comparative analysis of QTLs for the five traits(SL,SPS,SDPS,SOC,and SGC).To integrate QTLs in the present and previous studies,QTL information of the five traits published in recent decades was collected,including mapping populations,the sizes of populations,QTL names,QTL positions on chromosomes,and the information of markers associated with QTLs.The comparative analysis showed that a total of 22consensus QTLs in this study were similar to those in previous studies,and the rest were novel QTLs.3.Prediction of candidate genes in the QTL intervals.A total of 21 overlapping QTLs and major QTLs were obtained in this study.To predict the candidate genes of these QTLs,sequences within the confidence interval of them were aligned to the Arabidopsis genome.Combining with the functional annotations of Arabidopsis genes for related traits,72 homologous genes in B.napus were considered as candidate genes for these QTLs.These candidate genes are involved in ovule development,growth hormone response,lipid transport,lipid metabolism,fatty acid biosynthesis and glucosinolate biosynthesis.4.Fine mapping of cq SL-C7 and prediction of the candidate genes.Analysis of a co-dominant markers in the peak region of cq SL-C7 showed that cq SL-C7 had a stable effect on SL.QTL analysis based on BC₃F₂ and BC₄F₂ populations showed that the cq SL-C7 locus explained 14.0-16.9%of phenotypic variance with additive effects ranging from 2.95 to 3.32.To narrow down the candidate region of cq SL-C7,a segregating population consisting 1,687 BC₃F₂ individuals was analyzed,and 149recombinants were identified.Based on this population,the QTL region was restricted to a 597-kb region on C7 chromosome.To further narrow down the candidate region,we continued to investigate the 4,948 BC₄F₂and 1,732 BC₅F₁ individuals,as a result,cq SL-C7 was restricted to a 135 kb region in the ZS11 genome.According to the gene annotations in the ZS11 reference genome,21 genes were predicted in the 135 kb candidate region of cq SL-C7.Based on the analysis of sequence polymorphisms of the candidate genes,a nucleotide deletion was present in Bna C07G0531500ZS at the fifth exon,resulting in a truncated protein in G120.Combining with the sequence variations and potential biological functions of candidate genes,Bna C07G0531500ZS is most likely to be the target gene of cq SL-C7.5.Cloning and functional verification of cq SL-C7.Bna C07G0531500ZS is homologous to the Arabidopsis ROT3 gene and is thus designated as Bna C7.ROT3.Both of Bna C7.ROT3^ZS11 and Bna C7.ROT3^G120 were transformed into Arabidopsis rot3-1 null mutant,the results showed that Bna C7.ROT3^ZS11 can restore the mutant phenotype while Bna C7.ROT3^G120 cannot,indicating that Bna C7.ROT3^G120 has lost its function,likely due to sequence variations.The further genetic complementation test in rapeseed confirmed that Bna C7.ROT3^ZS11 was the causal gene for the cq SL-C7 locus.6.Functional analysis of Bna C7.ROT3.The q PCR analysis and GUS activity detection showed that Bna C7.ROT3 was mainly expressed in leaves,flowers,and siliques.Subcellular localization showed that Bna C7.ROT3 was localized on the cell membrane.The haplotype analysis of Bna C7.ROT3 showed that the single nucleotide deletion in Bna C7.ROT3 was a rare allele.The haplotype analysis of the homologs of Bna C7.ROT3showed that the SL of several haplotypes of Bna A3.ROT3 and Bna A1.ROT3 were significantly reduced compared with other haplotypes.7.Sequence analysis of the candidate gene for q GSL-C2 locus.We aligned the candidate region of q GSL-C2 locus in B.rapa to the reference genome of B.napus ZS11,and obtained a physical region of 24.3 kb on the C2 chromosome.According to the gene annotation information,four predicted genes were existed in this candidate region.Among them,Bna C02G0527500ZS,named as Bna C2.MYB28,is homologous to At MYB28,which was the the predominant regulator of the biosynthesis of aliphatic glucosinolate.The great variations in the sequence of Bna C2.MYB28 between parents suggested that Bna C2.MYB28 was the most possible candidate gene for q GSL-C2.8.Functional verification of Bna C2.MYB28.The genetic complementation test showed that Bna C2.MYB28^G120 could significantly increase the seed glucosinolate content(SGC)in ZY50.Additionally,a significant decrease of SGC were observed in the mutant lines derived from CRISPR/Cas9-induced knockout of Bna C2.MYB28^G120.These results confirmed that Bna C2.MYB28 was the target gene for q GSL-C2.9.The regulation of Bna C2.MYB28 on SGC.The transformation of Bna C2.MYB28^ZY50 into G120 significantly increased its SGC,indicating that Bna C2.MYB28^ZY50 functions normally.Overexpression of Bna C2.MYB28 could significantly increase SGC,indicating that Bna C2.MYB28 positively regulates SGC.10.Functional analysis of Bna C2.MYB28.The q PCR analysis and GUS activity detection of Bna C2.MYB28 showed that it was mainly expressed in roots,cotyledons and mature leaves,and moderately expressed in siliques and buds,whereas nearly no expression in stems and seeds.The results of subcellular localization in tobacco and protoplasts consistently showed that the Bna C2.MYB28 proteins of both parents were localized in the nucleus.The Bna C2.MYB28 of both parents showed self-activation in yeast self-activation detection experiment.The protein truncation test showed that the self-activation region of Bna C2.MYB28 was located at the C-terminus.The results of yeast two-hybrid and SLC experiment consistently showed that Bna C2.MYB28 and Bna MYC3 interact specifically.The RNA-seq analysis on parental lines and its overexpression lines of Bna C2.MYB28 was performed,the results showed that the differentially expressed genes(DEGs)were mainly enriched in the pathways related to glucosinolate synthesis and metabolism.The results of q PCR confirmed the reliability of the RNA-seq analysis.