Fine Mapping And Candidate Gene Analysis Of Yellow-green Leaf Genes BoYgl-1 And BoYgl-2 In Brassica Oleracea L.Var.Capitata

Cabbage（Brassica oleracea L.var.capitata）is an important vegetable crop,which belongs to the genus Brassica of Cruciferae family.The leaf color of cabbage is usually green,YL-1 and 4036Y are two yellow-green leaf mutants.In this study,the genetic analysis and gene fine mapping of the two mutants were performed,and the variations of the two candidate genes were analyzed,which laid the foundation for revealing the formation mechanism of yellow-green leaf and promoting the leaf color breeding of cabbage.The main results are as follows:1.The BoYgl-1 gene was fine mapped in the recombination-suppressed region of chromosome C01in YL-1,with a 6.3 Mb inversion in this region,and the candidate genes Bo1g078470 and Bo1g078800had mutations in the promoter or coding region of YL-1.With the BC₃-BC₅ segregation populations,which were constructed using cabbage yellow-green leaf mutant YL-1 and Chinese kale normal green leaf inbred line 11-192,the BoYgl-1 gene was finally mapped to a 392 kb interval on chromosome C01.A 6.3 Mb inversion occurred between markers B4-4 and T1-49 in YL-1,resulting in severe recombination suppression in the region.Among 32 genes in the located region,Bo1g078470 encoding a CLPC2/HSP93-III protein involved in chloroplast protein transport,and Bo1g078800 encoding a pentatricopeptide repeat protein（PPR）involved in RNA editing of mitochondrion and chloroplast,then these two genes were designated as the candidates for BoYgl-1.The expression level of Bo1g078470 in YL-1 was down-regulated nearly five times compared with GL-1（the near-isogenic line of YL-1）,and the expression level of Bo1g078800 was not different significantly between GL-1 and YL-1.Sequence analysis of YL-1 and GL-1 revealed no sequence variations in the coding region of Bo1g078470,whereas six SNPs were identified in the Bo1g078470 promoter region of YL-1.And eleven SNPs were identified in the Bo1g078800 exon region of YL-1,resulting in nine amino acids substitution.It was speculated that the promoter variations of Bo1g078470 or the coding region variations of Bo1g078800caused the yellow-green leaf phenotype of YL-1.2.The BoYgl-2 gene controlling the yellow-green leaf of 4036Y mutant was fine mapped,and the candidate gene Bo3g001140 was lost in 4036Y.The six generation population was constructed using cabbage yellow-green leaf mutant 4036Y and cabbage normal green leaf inbred line 01-20.Genetic analysis indicated that the yellow-green leaf was controlled by a single recessive gene,named BoYgl-2.The hybrid complementary test and BSA-seq analysis indicated that BoYgl-2 was a new gene different from BoYgl-1.The BoYgl-2 gene was finally mapped to a 142 kb interval on the left end of chromosome C03 and flanked by InDel marker B36-11,with genetic distances of 0.1 cM.Among 53 genes in the located region,Bo3g001140 encoding a pentatricopeptide repeat protein（PPR）involved in RNA editing and development of chloroplast,and Bo3g001370 encoding a histone-lysine N-methyltransferase ATXR3-like protein involved in chloroplast protein transport,then these two genes were designated as the candidates for BoYgl-2.Sequence analysis of 4036Y and 4036G（the near-isogenic line of 4036Y）revealed no sequence variations in the promoter and coding regions of Bo3g001370.The total length of Bo3g001140 gene was 1455 bp in 4036G,but no amplicon was found in 4036Y.Reads-IGV visual analysis and marker validation analysis were performed using the resequencing data.It was found that about 162 Kb region at the left end of chromosome C03 was lost in 4036Y,so we speculated that the lost of Bo3g001140 gene in the region might cause of the yellow-green leaf phenotype of 4036Y.