Fine Mapping Of The Etiolated Mutant Gene Brvyl In Chinese Cabbage (Brassica Campestris L. Ssp. Pekinensis)

Leaf color mutations are widespread in plant,and leaf color mutants have been identified in many plants such as rice,Arabidopsis thaliana and cucumber.The typical leaf color mutation is etiolation,and etiolation mutants have become important materials for study of photosynthetic mechanism,chlorophyll biosynthesis and chloroplast development in plants.Chinese cabbage?Brassica campestris L.ssp.pekenensis?is a kind of vegetable with leaf as the product organ.The variation of leaf color will affect photosynthesis and yield,which has been paid much attention by breeders.In this study,we used Chinese cabbage DH line ?FT? as the material and through EMS induced germinated seeds methrod to create a etiolation mutant vyl.On the basis of analyzing and identifying the phenotypic,photosynthetic physiological and genetic characteristics of mutant vyl,the mutated gene Brvyl was identified by using the map cloning combining with resequencing results of parent genome.The main research results are as follows:1.Phenotypic and genetic characteristics of etiolation mutant vyl in Chinese cabbage.The etiolation of mutant vyl in Chinese cabbage was aggravated at lower temperature,especially in new leaves.Mutant vyl and ?FT? were used as a female and male parent,for hybridization to produce progenies F₁,F₂ and BC₁.Genetic analysis,indicated that the etiolation leaves phenotype was controlled by a recessive nuclear gene.2.Mapping of mutant gene BrvylFlawering stalk DH line ?14S701? and mutant vyl were used as parents to produce F₂ mapping population.Fifty individuals plants with green and etiolation leaf phenotypes were selected to construct two RNA pools,respectively.By BSR-Seq analysis,the candidate regions linked to the target traits were obtained five segments on chr A06.The linkage between candidate intervals and target traits was verified by developing SSR markers on candidate regions.3.Fine mapping of mutant gene BrvylFor further narrowing the mapping region,SSR markers were screened from a large F₂ popualtion with 1,628 homozygous etiolation leaf individuals.The target gene was finally mapped between marker SSR5-21 and SSR4-24 on chr A06,with the genetic distance of 0.06 c M and 0.09 c M,respectively.Compared with Brassica rapa reference genome?v3.0?,25 genes were predicted in a region that physical distance was approximately 170 Kb.4.Candidate gene prediction based on whole genome resequencingDue to the low recombination rate in the interval,it is impossible to narrow the range of candidate interval through new molecular markers.Whole genome resequencing was performed on vyl and ?FT?.A total 72 homozygous SNP and InDel were detected in the 170 Kb candidate regions of the mutant vyl using the sequencing information,while only one homozygous non-synonymous SNP mutation occurred on exon region.The gene with this exon region,Bra A06g028040.3C,encoded AAA-type ATPase family protein.There is a conserved structural domain Fts H?Filamentation temperature-sensitive H?,which was predicted in the 295th-750 th amino acid sequence of the gene.AT5G64580?At Fts Hi4?,the homologous gene of Arabidopsis which is involved in embryonic development,and protein introduction into chloroplast matrix and proteolysis,In mutant vyl,a G?A base transition in the second exon of Bra A06g028040.3C,leads to the retention of the second intron,causes the 175 th amino acid Val?GTG?turns to Ser?AGT?and the translation terminates in the 198 th amino acid.