Preliminary Mapping Of Late Bolting Genes And Screening Of Candidate Genes In Leaf Mustard

Brassica juncea Coss.（Brassica juncea Coss.）is a characteristic vegetable in Guizhou Province.It has a large cultivation area and high prices for spring cultivation.Leaf mustard is a seed vernalization type plant,and it does not have strict requirements for low temperature.It is easy to bolt and bloom through vernalization,causing huge losses in production.Therefore,it is of great significance to study the regulation genes of leaf mustard late bolting.Bolting traits are quantitative traits controlled by multiple genes.At present,the mechanism of bolting and flowering regulation has been studied in cabbage and wild cabbage,but it is rarely reported in mustard.In this study,the bolting phenotype data of the F₂generation obtained by crossing the late bolting DH line’MN001’and the early bolting inbred line’MU056’of leaf mustard were used for statistical analysis of bolting phenotype data,combined with high-throughput sequencing Graded-seq technology,and bolting of leaf mustard the genome-wide analysis of QTL loci,and then based on the bioinformatics analysis of genes,realize the initial location of the main QTL for bolting traits,and discover candidate genes for controlling late bolting traits of leaf mustard.The main results of this study are as follows:1.Statistical analysis of bolting characters in F₂generation of leaf mustardThe F₂population was constructed with the late bolting DH line’MN001’of leaf mustard as the male parent and the early bolting inbred line’MU056’as the female parent,and bolting phenotype statistics were performed.The frequency distribution histogram was made according to the bolting time of the F₂generation population,The bolting data of leaf mustard conforms to the normal distribution,indicating that the bolting traits of leaf mustard are quantitative traits controlled by multiple genes.2.Determination of the candidate interval for late bolting of leaf mustard based on Graded-seq technologyAccording to the statistical results of the bolting situation of the F₂generation plants obtained from the cross between the parents and the two,the graded-seq correlation analysis method was used to locate the bolting traits studied,and four mixed pools were constructed based on the phenotypic traits,namely the late bolting pool.Early bolting pool,intermediate mixed pool 1 and intermediate mixed pool 2,mixed pool scale:98+106+99+97,combined with parental resequencing.After Ridit test and noise reduction treatment,the late bolting traits were located,and the candidate interval was located on chromosome 3 of the leaf mustard A genome,and the interval size was 1.1 Mb.3.Bioinformatics analysis of genes in the main QTL chromosome mapping segment for late bolting of leaf mustardA total of 178 genes were found within 1.1Mb of the main effect QTL mapping interval for late bolting of mustard,and 172 genes were annotated.Using the published genome information,BLAST software was used to GO annotate the coding genes in the candidate interval,and 172 were annotated.The genes are divided into 3categories,50 subcategories,biological function,cell component and molecular function.A large number of genes are related to biological functions such as cell nucleus,transcription,DNA templates,membrane components,and protein binding.4.Screening of candidate genes for late bolting of leaf mustardBy annotating the genes in the main QTL chromosome mapping segment for late bolting of leaf mustard,the function of each gene in the interval is known,and the candidate genes that affect the difference in bolting traits are finally determined.According to the reported six genetic pathways（photoperiod pathway,vernalization pathway,gibberellin pathway,temperature pathway,autonomous pathway and age pathway）that regulate bolting and flowering time in the model plant Arabidopsis thaliana,among the 172 functionally annotated genes Twenty candidate genes related to the regulation of bolting and flowering were screened out.There are 3 genes related to circadian rhythms,9 genes related to photoperiod and mosaic development,8 genes related to light stimulation and light intensity,and 5 genes related to gibberellin,and some genes are involved in multiple pathways.5.Combined q RT-PCR analysis to further screen the candidate genes for late bolting of leaf mustardIn order to compare the expression characteristics of these 20 candidate genes in their parents,real-time fluorescent quantitative PCR was used to analyze the expression changes of these genes in the late bolting parent MN001 and the early bolting parent MU056 on 7 days and 75 days after transplanting.The results showed that the genes Bju A001637,Bju A009062,Bju A009068,Bju A009109,Bju A009116,Bju A009119,Bju A009123,Bju A009174,Bju A009092,Bju A009108,Bju A009163,Bju A009164 and Bju A009205 were almost not expressed before and after bolting of MU056,and their expression levels were almost not expressed at the same time before and after the bolting of MU056.However,these genes are highly expressed during the growth of MN001,and these 13 genes are further predicted to be candidate genes related to late bolting of leaf mustard.The 13 genes have different functions in the 6 regulating bolting and flowering pathways,and they are all involved in regulating the bolting and flowering of plants to varying degrees.6.SSR mark verification of F_2:3familiesUsing SSR technology to analyze the bolting traits of F_2:3families of leaf mustard,using the sequencing results to develop 46 pairs of molecular marker primers,using two parents and F₁primers to screen,and screening a pair of molecular markers with polymorphism Primer.Using this pair of primers to verify the polymorphism of the bolting population of F_2:3families,the results showed:SSR-PCR amplified a total of 142 bands in the early bolting population,25with polymorphism,and the percentage of polymorphic bands was 17.60%;in the late bolting population,SSR-PCR amplified a total of 146 bands.There were 65 polymorphic bands,and the percentage of polymorphic bands was 44.52%.