Cloning And Identification Of Heading Mutant Genes In Chinese Cabbage

Chinese cabbage（Brassica rapa L.ssp.pekinensis）is the main vegetable crop in China,with leafy head as the main product organ,occupying an important position in the annual supply of vegetables.The heading characteristics of Chinese cabbage determines its yield and product quality,and has always been highly valued by researchers.My team used the DH line‘FT’of Chinese cabbage as the test material and created a mutant library containing 718mutant materials using EMS treated germination of seeds.This study screened 11 low and flat type non-heading mutants from this mutant library.Based on phenotypic identification and genetic analysis,Mutmap method was used to mapping gene,combined with KASP genotyping verification and Sanger sequencing analysis,to locate the corresponding mutant genes.The gene function was verified through allelic mutant sequence variation analysis.The main research findings are as follows:1.It has been proven that the mutation traits of the 11 low and flat non-heading mutants screened from the mutant library are all controlled by a single recessive nuclear gene,resulting from mutations in 4 pairs of genesThe phenotypic characteristics of the 11 low and flat non-heading mutants in Chinese cabbage were similar,with plants growing on the ground during the entire growth period and unable to form leafy head.The genetic analysis results of the 6 generations have shown that their non-heading phenotype is controlled by a single recessive nuclear gene;The allelic testing results of the mutated genes confirmed that 11 mutants came from four pairs of allele variations:（1）The mutated gene Brlfm1,involving two mutants,lfm1-1 and lfm1-2;（2）The mutated gene Brlfm2 involves three mutants:lfm2-1,lfm2-2,and lfm2-3;（3）The mutated gene Brlfm3 involves four mutants:lfm3-1,lfm3-2,lfm3-3,and lfm3-4;（4）The mutated gene Brlfm4 involves two mutants,lfm4-1 and lfm4-2.2.Proved that the key genes for gibberellin synthesis in Chinese cabbage,Br KAO2,Br KS,and Br CPS,affect the formation of Chinese cabbage leafy head by regulating gibberellin biosynthesisUsing Mutmap method,the mutated genes Brlfm1,Brlfm2,and Brlfm3 were mapped on chromosomes A05,A07,and A09,respectively;KASP genotyping technology were used to perform co-segregation validation on candidate SNPs,it was demonstrated that Bra A05g012440.3C is the candidate gene for Brlfm1,Bra A07g043370.3.5C is the candidate gene for Brlfm2,and Bra A09g001510.3.5C is the candidate gene for Brlfm3.These three genes encode the three key enzymes KAO2,KS,and CPS in the gibberellin biosynthesis pathway.The cloning sequencing results showed that lfm1-1 had a base substitution from G to A at position 455 of the CDS sequence of Br KAO2,causing the amino acid to change from glycine（Gly,G）to glutamic acid（Glu,E）;lfm1-2 had a base substitution from C to T at position 389of the CDS sequence of Br KAO2,resulting in the amino acid being changed from serine（Ser,S）to leucine（Leu,L）.The last base of the ninth intron of Br KS in lfm2-1 changed from G to A,resulting in an 8-bp deletion at position 1424 of the CDS sequence,leading to frameshift mutations and early termination of protein translation;lfm2-2 changed from G to A at position1466 of the CDS in Br KS,resulting in the amino acid being changed from arginine（Arg,R）to lysine（K）;lfm2-3 changed from G to A at the 2032nd position of the CDS in Br KS,causing the amino acid to change from glutamic acid（Glu,E）to lysine（Lys,K）.The last base of Br CPS sixth intron in lfm3-1 changed from G to A,resulting in a 32bp deletion in the CDS sequence,leading to frameshift mutations and early termination of protein translation;lfm3-2mutated from C to T in the 799th position of the CDS sequence of Br CPS,resulting in a change in the amino acid from histidine（His,H）to tyrosine（Tyr,Y）;The last base of the third intron of Br CPS in lfm3-3 mutated from G to A,resulting in a 5bp deletion in the CDS sequence,leading to amino acid substitution and early termination of protein translation;The last base of the twelfth intron of Br CPS in lfm3-4 mutated from G to A,resulting in the deletion of G at position 1902 of the CDS sequence,resulting in amino acid substitution and premature termination of protein translation.The sequence analysis of three sets of allelic variant materials from the above nine mutants verified the functions of three candidate genes.The contents of gibberellin in the mutants were significantly reduced,and exogenous gibberellin can restore the mutants to the wild-type phenotype.3.It has been proven that the Brassica orphan gene BOG1 is involved in the formation of leafy head in Chinese cabbageUsing the non-heading mutant lfm4-1 and wild-type‘FT’to construct an F₂segregation population,the mutated gene Brlfm4 was located on a 13.75Mb region of A05 chromosome using Mutmap method.By screening and annotating differential SNPs within the localization region,two non-synonymous SNPs were identified,involving two genes.The results of KASP genotyping and Sanger sequencing analysis showed that only SNP A05 15078406 was co segregated with mutant phenotype in F₂plants.Therefore,it was determined that Bra A05g021450.3C where this SNP is located is the candidate gene for Brlfm4.The cloning sequencing results showed that the 271st base of Bra A05g021450.3C in lfm4-1 has a substitution from G to A,resulting in the amino acid being changed from alanine（Ala,A）to threonine（Thr,T）,resulting in changes in the secondary and three-dimensional structure of the protein.The allelic mutant lfm4-2 had a non-synonymous SNP（G to A）at the 266st base of Bra A05g021450.3C,causing the amino acid to change from arginine（Arg,R）to histidine（His,H）,and also causing changes in the secondary and three-dimensional structure of the protein.This result confirmed the function of the candidate gene.Bra A05g021450.3C encodes an unknown functional protein of 13.74 k Da.Homology search revealed that it only exists in the A and C genomes of Brassica genus and is an orphan gene of Brassica genus,named BOG1（Brassica Orphan Gene 1）.BOG1 has no transmembrane domain and no signal peptide.The subcellular localization results showed that BOG1 was located in the nucleus.The total active gibberellin content of mutant lfm4-1 was significantly reduced.Exogenous gibberellin can restore the mutant to the wild-type phenotype,while exogenous gibberellin inhibitors can transform the wild-type into the mutant phenotype,indicating that BOG1 may affect Chinese cabbage heading by regulating gibberellin biosynthesis.