Fine Mapping Of A Gene Controlling Purple Petiole In Brassica Campestris Ssp. Chinensis L.

Brassica campestris ssp.chinensis L.is the main variety of Chinese cabbage in South China.It has dark green leaves,tender white petioles and delicious sweetness.It is well received by consumers.In recent years,it has been widely introduced in other areas.Some purple petiole plants often appear in the seedling population of the dairy cabbage varieties planted in the north,which affects the uniformity of the varieties.The results showed that the purple petiole character was the result of anthocyanin accumulation in petiole.In this study,F₁,F₂ and BC1 populations were constructed by crossing the stable genetic green petiole inbred line（K6）and purple petiole inbred line（K19）.Based on the analysis and identification of the genetic characteristics of purple petiole traits,the purple petiole genes were precisely mapped using BSR-Seq and SSR methods in large-scale F₂ segregated populations.The main results are as follows.1.Genetic characteristics of purple petiole traitsBased on the identification results of petiole color of hybrid parents K6 and K19 and their F₁,F₂ and BC1 populations at seedling stage,it was proved that the purple petiole traits tested were controlled by dominant nuclear gene Ps.The genotype of K6 was psps,and the genotype of K19 was Ps Ps.2.Preliminary mapping of purple petiole gene based on BSR-Seq technologyFifty plants with purple petiole and fifty plants with green petiole phenotype were selected from F₂ isolation population.Total RNA was extracted to construct extreme phenotype pool for BSR analysis.Sequencing was used to obtain the sequence variation at transcriptional level between purple petiole and green petiole plants.The SNP loci related to purple petiole traits were determined.The control genes of purple petiole traits were initially located in the 19.9-26.1M interval of A07 chromosome.3.Fine mapping of purple petiole gene based on SSR linkage marker analysisBased on the results of BSR sequencing,SSR markers were developed and designed in the location area.After several screening and identification,the purple petiole gene was located between SSRL2 and SSRL8 markers on chromosome A07,with a physical distance of51.9 kb.Referring to the genome sequencing information of Chinese cabbage,there were 9genes in the localization region.Bra A07003126 was detected as differentially expressed gene in the localization region by BSR-Seq analysis,so it was predicted to be a candidate gene.4.Cloning and expression analysis of candidate genesFull-length cloning and sequencing of candidate genes revealed that there was a T-C SNPmutation in the first exon of Bra A07003126 in the purple petiole inbred line K19,which resulted in the wrong mutation of the genetic code and changed the amino acid sequence of protein from phenylalanine to serine.The expression level of Bra A07003126 in wild-type material（K6）was significantly higher than that in mutant material（K19）;the expression level of Bra A07003126 in petioles at different developmental stages of K6 was not significantly different;the expression level of Bra A07003126 in petioles at different developmental stages of K19 was different,with the highest at bolting stage and the lowest at seedling stage;the expression level in leaves and petioles of K6 and in K19 tissue.There was no significant difference in expression between leaf and petiole tissues.