Map-based Cloning And Functional Analysis Of The Glossy Gene In Brassica Napus

The waxy layer serves as a barrier for the direct contact between plants and the environment,and plays an important role in reducing plant water loss,resisting pests and diseases,and resisting ultraviolet rays;its visually distinguishable traits can be used as a morphological marker for breeding and it can be used as a breeding target for crop resistance breeding.However,the wide variety of waxy components and the complex synthesis regulation mechanism have brought challenges for us to serve breeding by improving waxes.In this study,we analyzed the phenotypic characteristics of the mutants Nilla glossy 2（ngl2）and zs11-e,successfully cloned the target gene and analyzed their basic functions.It can be beneficial to exploring the regulation mechanism of wax synthesis and resistance breeding.The main results were described as follows:1)The waxy phenotypes of the stems,leaves,flower buds,and siliques of the two mutants ngl2 and zs11-e are abnormal,and the phenotypes are stable throughout the growth period.Scanning electron microscopy results showed that the rods,tubules,and filaments waxy crystals were significantly reduced in mutant leaf surface,but platelets crystals increased,and the platelets crystals spread on the epidermis surface to form a glossy leaf phenotype.Analysis of leaf epidermal wax by gas chromatography-mass spectrometry showed that the content of branched primary alcohols in the ngl2 mutant was significantly reduced,and the content of branched alkanes was significantly increased.Therefore,we speculated that the mutant genes in ngl2 were related to fatty alcohol synthesis;in zs11-e mutants also found branched primary alcohols,branched alkanes and branched aldehydes,while the content of unbranched-chain waxes tended to increase,so we speculated that the mutant genes in zs11-e may be involved in wax de novo synthesis.2)Genetic analysis found that the phenotype of ngl2 glossy leaf is controlled by two pairs of recessive genes.Through traditional map-based cloning,the BC₃ population were used to locate the target gene in the A1 chromosome about 50 kb,and the other gene is located on the C1 chromosome 230 kb region.Then,by comparing sequencing and gene annotation,two genes Bn A1.CER4（Bna A01g03200D）and Bn C1.CER4（Bna C01g04460D）were screened.The transgene verified that these two genes can complement the ngl2 phenotype.Bn A1.CER4 and Bn C1.CER4 are both located in the endoplasmic reticulum,and are expressed in high amounts in leaves and stems,low in siliques and flower buds,and not expressed in roots.3)Yeast heterologous expression of Bn A1.CER4 and Bn C1.CER4 can detect C26fatty alcohol,but the content is very low,it is suggested that Bn A1.CER4 and Bn C1.CER4may have a preference for branched substrates.4)Detecting water loss rate and resistance to Sclerotinia sclerotiorum,we found that compared with the wild type,ngl2 mutant has a faster water loss rate,but the resistance to Sclerotinia sclerotiorum was enhanced.5)Genetic analysis found that the phenotype of the zs11-e mutant was controlled by a pair of recessive genes.The BC₅backcross population was constructed,and the target gene was located at the range of A08 chromosome 0-1M using the 60k rape chips combined with SSR markers analysis.Molecular marker analysis showed that there was a large fragment deletion in the target region;RNA-seq sequencing was performed to narrow the candidate genes to 46,and a gene Bn.BCDH1（Bna A08g00430D）was found based on the phenotype,which is a subunit of the branched-chain alpha-keto acid dehydrogenase complex,Transgenic verification found that it can complement the mutant phenotype.These results indicate that Bn A1.CER4,Bn C1.CER4 and Bn.BCDH1 are involved in the synthesis of branch chain wax in Brassica napus,which is very important to maintain the normal structure and function of cuticle wax.These studies laid a theoretical foundation for improving the stress resistance of Rapeseed by modifying waxy components with breeding strategies in the future.