Fine Mapping Of Py2, A Gene Referred To Yellow Leaf Mutant In Packoi (Brassica Campestris L. Ssp. Chinensis)

Chinese cabbage?Brassica rapa L.ssp.chinensis?is a variant of the genus Brassica?Cruciferae?Brassica campestris,a wide range of species,and a leafy vegetable crop of popular preference.As the main site of plant photosynthesis and transpiration,leaf blade directly influences the growth and yield of plants.Leaf color mutation is a common type of mutation,and the study of the yellow Mutant is concerned.The test materials used in this study were pylm mutants obtained in the process of free microspore culture,which originated from the green hybridization'Huaguan'.The F₁,F₂and BC₁ were prepared with mutant pylm and Chinese cabbage DH'FT'as parent.Genetic analysis indicated that the yellow mutation trait was controlled by 2 recessive nucleus genes,named py1and py2 respectively.BSR sequencing was performed using 2 extremist RNA mixtures constructed by the F₂ group,and the mutant gene alle were positioned respectively in A09 and A07 chromosomes.BSR-seq results combined with SSR linkage analysis,the yellow mutant gene py1 was mapped between Indelzk125 and SSRzk36 markers of A09 chromosome,and the gene Bra A09004189 of heme oxygenase was predicted as its candidate gene.In this experiment,the chlorophyll metabolism indexes of the yellow mutant were determined,the results showed that the chlorophyll content of mutant was significantly reduced,and the heme content was significantly higher than that of the control,and the yellowing was probably caused by the accumulation of heme in the chlorophyll precursor.The activity of heme oxygenase is significantly reduced,which may lead to the accumulation of heme,which leads to the decrease of chlorophyll content.According to BSR-seq results and further development of SSR markers,the py2 was mapped between SSR11 and SSR15 of A07 chromosome,with a physical distance of 310.8kb,which contained 42 genes.The differential expression SNV by parental weight sequencing was used to further narrow the mapping interval,and the py2 was eventually mapped in a section containing 5 genes.The above 5 genes were cloned and sequenced,compared with normal green plants,mutants had 1SNP on the 1th exon of Bra A07001774,and from A to G,this mutation was an amino acid mutation,changed from Met to Val.According to the gene annotation information,BraA07001774 is the embryo developmental defect gene.The expression of candidate gene was analyzed,and the expression of BraA07001774 in the mutant pylm leaf decreased significantly,the results of bioinformatics analysis of candidate genes show that the total length of CDS in BraA07001774 is 1149bp,the translation protein contains 382 amino acids,the protein relative mass is 43.5KD,the equal electricity point is 4.91,and the locus of signal peptide is located at the 23rd amino acid,no transmembrane region.