Construction Of Mutant Library In Chinese Cabbage And Cloning Of Collapsed Non-heading Mutant Genes

Chinese cabbage?Brassica rapa L.ssp.pekinensis?is a widely cultivated vegetable crop in China,South Korea,Japan and other east Asia countries and regions,including heading,semi-heading and loose heading type.Heading cabbage is the most important variety type.The product organ of heading cabbage is the leafy head.Leafy head is a vital agronomic trait that facilitates the evaluation of the yield and quality of Chinese cabbage.Therefore,research on the formation and development of leafy head has always been paid more attention by researchers.In 2011,the sequencing information of Chinese cabbage genome was released,which laid a foundation for the functional genome research of Chinese cabbage.In view of the important role of mutants in functional genome studies,this study firstly used EMS to mutate Chinese cabbage microspore DH line?FT?and created a Chinese cabbage mutant library,containing 701 stably inherited mutant lines.Three collapsed non-heading mutants were screened from this mutant library,and the mutant genes were cloned and functionally identified by Mutmap and KASP genotyping.The main research results are as follows:1.EMS mutagenesis of Chinese cabbage microspore DH line‘FT'seeds to create mutant library7,800 germinated seeds of DH line?FT?were immersed in 0.8%EMS solution.1354mutant lines were identified and screened out of 3731 M1 lines with a mutation rate of36.29%.Since the M₂ generation,through further identification of mutant traits in the field,701 mutant plants with stable inheritance were obtained,and the mutation rate was 18.78%.The mutation types mainly include:plant shape variation,leaf shape variation,leaf color variation,fertility variation,bolting and heading variation and so on.2.The Mutmap technology combined with KASP genotyping verification was used to fine mapping the non-heading mutant gene Brnhm1The leaves of the mutant nhm1 exhibited geotropic growth throughout all stages of development,and leafy heads were not formed during the heading stage.Genetic analysis showed that the mutant trait was controlled by a single recessive nuclear gene.Using Mut Map and Kompetitive Allele-Specific PCR analyses,we demonstrate that Bra A07g042410.3C,which encodes an ent-kaurene synthase involved in the gibberellin biosynthesis pathway,was the nhm1 mutant candidate gene.Compared to the wild-type?FT?,Bra A07g042410.3C had lower levels of expression in the leaves of the mutant nhm1.In addition,gibberellin contents were lower in the mutant than in the wild-type,and the mutant plants could recover the wild-type phenotype after exogenous GA₃ treatment.We speculated that the mutation of Bra A07g042410.3C gene hindered the synthesis of gibberellin,which resulted in the inability of the leaf to form a leafy head.3.Allelic mutation was used to verify that the gibberellin synthase gene Bra A09g001440.3C has the function of regulating the leafy head formation in Chinese cabbageThe leaves of the mutant nhm2 and nhm3 exhibited similar phenotypes:geotropic growth throughout all stages of development,and leafy heads were not formed.The results of hybridization experiment proved that the mutated gene was an allele.Using Mut Map and Kompetitive Allele-Specific PCR analyses,we demonstrate that Bra A09g001440.3C,which encodes a CPS enzyme involved in the gibberellin biosynthesis pathway,was the candidate gene of Brnhm2.Cloning and sequencing of the candidate gene showed that a single base mutation from C to T occurred in the seventh exon of the gene in nhm2,resulting in the amino acid from lysine to phenylalanine;In nhm3,a single base from G to A mutation occurred on the fourth exon of the gene,resulting in the amino acid from temporinine to glycine.The q RT-PCR results showed that the candidate gene was expressed at different stages of leaf development,but the expressions in wild-type plants were higher than those in mutant plants.The determination of gibberellin content in leaves showed that the gibberellin content in wild-type leaves was significantly higher than that in mutant leaves.And the mutant plants could recover the wild-type phenotype after exogenous GA₃ treatment.These results suggested that the mutation of Bra A09g001440.3C might lead to the inhibition of gibberellin biosynthesis in the leaves of mutant plants,which led to the mutant plants could not to form the leafy heads.