Mapping Of Yellowing Mutant Yl1 Gene In Brassica Napus

Brassic napus is one of the important oil crops in China.Increasing the production of rapeseed is of great strategic significance to safeguarding the safety of national edible oil supply.Photosynthesis is one of the key factors affecting crop yield.It is of great significance to study the molecular mechanism of photosynthesis,and leaf color mutants are ideal materials for the study of photosynthesis.In this study,using the EMS mutagenesis technology,in the mutagenic offspring of the double-low Brassica napus Ningyou 18（NY18）,a stable hereditary yellowing mutant yl1 was screened.On this basis,this study combined phenotypic investigation,genetic analysis,map-based cloning,transcriptome analysis,real-time fluorescence quantitative analysis and other methods to locate and analyze the functional genes of the mutant.The main results of this research are as follows:（1）The yellowing phenotype appeared in the seedling stage of the yellowing mutant yl1 obtained by EMS mutagenesis,the leaf color of mature leaves turned slightly green in the later stage of growth,and the stems,flower buds,young siliques and other organs and tissues appeared to varying degrees yellowing.The chlorophyll content of leaves and siliques wall at the seedling stage was detected.The chlorophyll content of yl1 leaves was significantly lower than that of NY18,and the chlorophyll b content decreased more;there was no significant difference in the chlorophyll a content of siliques,but the chlorophyll b content was significantly lower than NY18.The photosynthesis test results at the seedling stage showed that the photosynthetic rate of yl1 was significantly reduced,and F₁was between the two parents.The survey of agronomic traits at the maturity stage found that yl1was significantly weaker than NY18 in terms of plant height,branch height,number of grains per silique,and thousand-grain weight.The results of transmission electron microscopy showed that the yl1 basal granule thylakoids were less,and the normal development of the chloroplast structure was affected.（2）In this study,a population of six generations with yl1 and NY18 as parents was constructed,and the genetic law of the chlorophyll deficiency phenotype of Brassica napus was statistically analyzed.Through the measurement of SPAD values of the six generations,The genetic analysis of SPAD values in the six generations using the plant major gene+poly gene mixed inheritance model showed that the inheritance of the yl1 yellowing phenotype conforms to the genetic model of a pair of additive-dominant single genes.Using the method of BSA-seq,the leaf color control gene was initially located in the1499657-5194567 bp interval on the A03 chromosome,named Bn A03.chd.The expanded（Holly×yl1）F₂population combined with PARMS SNP marker technology was used to finely locate the Bn A03.chd locus,and locate it between the markers M5 and M6,corresponding to the 180.9 Kb interval of the reference genome.（3）In the fine mapping interval,there are 28 annotated genes in the reference genome"ZS11",among which Bna A03G0054400ZS encodes the large subunit CHLH of magnesium ion chelator,and its homologous gene in Arabidopsis is AT5G13630,which is involved in chlorophyll biosynthesis.RNA-seq results showed that a total of 374significantly differentially expressed genes were identified,of which 146 were significantly up-regulated and 228 were significantly down-regulated.The real-time fluorescent quantitative expression analysis results are consistent with the RNA-seq results,and there is no significant difference in expression of this gene between wild-type and mutant.The resequencing results showed that Bna A03G0054400ZS has a SNP between NY18 and yl1.Then we amplified the CDS sequence of the gene.The sequencing results showed that there was no sequence difference in the promoter,but a conversion of guanine G to adenine A occurred at position+4045,resulting in the conversion of glutamic acid at position 1349 to lysineand,and the mutation is located in the Cob N-Mg＿chel domain of the protein sequence.It is preliminarily determined that Bna A03G0054400ZS is a candidate gene for controlling plant yellowing.