Inheritance And Gene Identification Of Bolting Tolerance In Tiller Mustard

Mustard originated in West Asia and has a long history of cultivation.After long-term domestication and artificial selection,it has formed four major categories and16 varieties.Among them,leaf mustard varieties have the widest cultivation area and the strongest adaptability.Tiller mustard,commonly known as Xuelihong,is one of the most widely cultivated varieties of leaf mustard.During autumn and winter cultivation or overwintering in places such as the Yangtze River Basin in China,mustard is susceptible to factors such as high temperature,long sunlight,drought,low temperature,and insufficient fertility,leading to abnormal growth or early bolting and flowering,resulting in a significant decline in yield and quality.Therefore,tolerance to early bolting is one of the main breeding objectives for mustard vegetables.The main research results of this paper are as follows:1.Inheritance and gene mapping related to bolting tolerance in tiller mustard.This study used early maturing tiller mustard 21-410（male parent）and late maturing tiller mustard 21-419（female parent）as parents to construct an F₂isolation population.A trait investigation was conducted on the bolting date of F₂isolated populations based on the bolting criteria of bolting height up to 5 cm,and the results were in accordance with normal distribution,which indicates the bolting tolerance trait of tiller mustard was a quantitative trait controlled by multiple genes.The sample size of the early bolting pool and the late bolting pool used for BSA sequencing in this study is 20.Combining with sequencing results of parents,the major gene related to bolting tolerance in tiller mustard was located on chromosome 8 of the B genome,with a candidate interval size of 15.8 Mb.2.Sequence analysis and functional identification of candidate BjuCO gene.Through homologous sequence analysis and cloning,it was found that the three candidate BjuCO genes and At CO genes all have B-box and CCT conserved domains,with a protein sequence homology more than 64.6%.The overexpression vector 35S::BjuCO-Ps2300was constructed,the BjuCO gene was overexpressed in late maturing tiller mustard（Zhejiangxuecai）,with a positive rate more than 84.2%.Under long sunlight conditions,compared to transformed Ps2300 plants,the BjuCO.A10 and BjuCO.B02 overexpression positive T₀generation plants exhibited a very early bolting and flowering phenotype,which was similar to the function of the Arabidopsis At CO gene,while the BjuCO.B08overexpression positive T₀generation plants exhibited similar bolting time to the wild type plants.The results of qRT-PCR showed that the expression of BjuCO gene in overexpressed plants was upregulated by 500-7000 times compared to the transformed Ps2300 plants.3.Subcellular localization of the candidate BjuCO gene.The 35S::BjuCO-p RI101vector was constructed and subcellular localization of BjuCO.A10 and BjuCO.B02 genes was performed using the tobacco transient transformation system.The results showed that both of them were located in the nucleus,which was consistent with the subcellular localization of the At CO gene.Therefore,we speculate that BjuCO.A10 and BjuCO.B02genes are mustard homologous genes of At CO gene.4.Sequence analysis and functional identification of candidate BjuFLC genes.Through homologous sequence analysis and cloning,it was found that the eight candidate BjuFLC genes and At FLC genes all have MADS-box and k-box conserved domains,with a protein sequence homology more than 78.3%.The overexpression vector35S::BjuFLC-Ps2300 was constructed,the BjuFLC gene was overexpressed in Huajie 4,an early tiller mustard（Huajie 4）,with a positive rate more than 86.4%.The results of qRT-PCR showed that the expression of BjuFLC gene in overexpressed plants increased by 20-6000 times compared to the transformed Ps2300 plants.The specific phenotype needs to be observed in T₁and subsequent generations.5.The relative expression measurement of candidate BjuFLC genes during responsing vernalization.With the prolongation of vernalization time（0-28 d）,the relative expression of BjuFLC gene gradually decreased,which is similar to the results of At FLC gene research.However,due to the polyploidization of the mustard genome,the relative expression levels of each BjuFLC gene vary significantly,with BjuFLC.A02,BjuFLC.A03a,and BjuFLC.B08a having the highest expression levels,and BjuFLC.A10and BjuFLC.B02 having the lowest expression levels in 21-405（Xuelihong）.Moreover,there are significant differences in the speed at which different BjuFLCs respond to vernalization.However,no expression data of BjuFLC.A03b have been obtained,suggesting that it is not expressed or the expression level is extremely low in tiller mustard.6.Investigation on related traits of bolting tolerance in tiller mustard inbred lines.This study investigated the bolting stage,budding stage,and flowering stage of 42germplasm resources of tiller mustard inbred lines,and comprehensively evaluated the bolting tolerance of tiller mustard inbred lines through correlation analysis,membership function analysis,and systematic cluster analysis.The correlation analysis showed that there was a significant correlation between the bolting stage,budding stage,and flowering stage of tiller mustard.Through field investigation of phenotypes,membership function analysis,and systematic cluster analysis,the inbred lines of tiller mustard with different bolting tolerance were well distinguished,and two materials with strong bolting tolerance（22-413 and 22-416）,a material with extremely weak bolting tolerance（22-410）were selected from them.