Chromosome Mapping And Candidate Gene Analysis Of Mosaic-leaf Gene In Brassica Napus L.

Rapeseed that is one of the important oil crops in China has a very high economic value like fertilizer,honey and vegetables.At present,many reachers have more focused on leaf type that is an important morphological marker trait that can be used for identification of hybrid and pure specie.In this study,the F₂population was obtained from the F₁generation crossed between mosaic-leaf R01 and round-leaf R02.Then,two pools of mosaic-leaf and round-leaf progeny were constructed,based on the BSA analysis method.The whole genome re-sequence were conducted,and the candidate interval of mosaic-leaf gene were located on the chromosome using QTL-seq and QTL＿seq R method.SSR and In Del molecular markers closely linked to mosaic-leaf gene located the candidate interval were developed by MISA and IGV softwares.Some genes located in the candidate intervals were analyzed by GO enrichmentand KEGG enrichment and q RT-PCR,and the candidate genes related to mosaic-leaf were screened.Preliminary cloning candidate gene and bioinformatics analysis were also done.The main research results are as follows:1.Genetic analysis of mosaic-leafThe F₁generation crossed between mosaic-leaf（R01）and round-leaf（R02）were named 20W1,W398 and W400.These F₁lines were selfed to obtain three F₂populations.There were 247 round-leaf and 81 mosaic-leaf in 20W1 population,43round-leaf and 10 mosaic-leaf in W398 population,35 round-leaf and 11 mosaic-leaf in W400 population.The data showed that the separation ratio of round-leaf and mosaic-leaf plants were 3:1(χ_c²<χ_0.05²),which is consistent with Mendel’s law of genetic.We guess the mosaic-leaf gene is controlled by recessive gene.2.Mapping of mosaic-leaf geneBased BSA analysis,the F₂generation segregating population was constructed by the mosaic-leaf and round-leaf parents,which were extracted leaf DNA to construct a pool of mosaic-leaf offspring and a pool of round-leaf offspring,being re-sequenced at a depth of 30×.With Darmor-bzh being as the reference genome,the resequencing data of the mosaic-leaf parent R01,the round-leaf parent R02,mosaic-leaf offspring and round-leaf offspring pools were preprocessed and the mosaic-leaf gene was located in the 16.35-17.35 Mb of A10 chromosome using QTL-seq and QTL-seq R method in Brassica napus L.3.Molecular marker for In Del and SSRVisualize the In Del variation in the candidate interval between the parental pools and the mosaic-leaf and round-leaf offspring pools by IGV software,insertion and deletion sites larger than 5 bases were screened.The IGV software was applied to target the start and end positions of In Del in this interval and design In Del primers.A total 39pairs of In Del primers were designed.These In Del primers were used to amplify the DNA of mosaic-leaf and round-leaf plants by PCR respectively.After polyacrylamide gel electrophoresis and silver staining had been finished,two In Del markers closely linked to mosaic-leaf traits were obtained.MISA software was used to identify microsatellite and complex microsatellite sequences in the 16.35-17.35 Mb of A10 chromosome.Combining with the primer design software Primer3,a total of 43 pairs of SSR primers were designed rapidly and efficiently in the candidate interval.These SSR primers were used to amplify the DNA of mosaic-leaf and round-leaf plants by PCR respectively.When polyacrylamide gel electrophoresis and silver staining had been done,two SSR markers closely linked to mosaic-leaf trait were obtained.4.Analysis of candidate geneA total of 223 genes in the region of mosaic-leaf gene located in 16.35-17.35Mb of A10 chromosome were applied by QTL-seq and QTL-seq＿R pipelines.Two genes like Bna A10g26320D and Bna A10g27130D were enriched into pathways related to leaf formation by GO and KEGG enrichment analysis.We compared with homologous genes in Arabidopsis and analyzed functional annotation,the homologous gene of Bna A10g26320D is AT5G03790,which interacts with LYF to activate CAL expression and participate in leaf morphogenesis.Meanwhile,the homologous gene of Bna A10g27130D is AT5G02410,which is involved in encodingα1,2-glucosyltransferase（ALG10）being necessary for the biosynthesis of lipid related to oligosaccharides and leaf development.So,we speculated that Bna A10g26320D might be a candidate gene for mosaic-leaf trait.QRT-PCR analysis found that the expression levels of Bna A10g26320D in floral leaves were significantly higher than that in round leaves.Finally we inferred that Bna A10g26320D might be a candidate gene for mosaic-leaf trait.5.Cloning and bioinformatics analysis of Bna A10g26320DBna A10g26320D was cloned and done to multiple alignment with the two sequences between mosaic-leaf and round-leaf materials.There were 17 base differences being responsible for nine amino acid.Bioinformatics analysis showed that the proteins encoded by Bna A10g26320D in both mosaic-leaf and round-leaf were hydrophilic.The two proteins have either not membrane or secretory.Bna A10g26320D encodes 25 protein phosphorylation sites in mosaic-leaf and 23 protein phosphorylation sites in round-leaf respectively.The secondary structure of the protein encoded by this gene in mosaic-leaf includes 50.92%ofαSpiral structure,10.09%of extended chain structure,38.99%of the irregular curly structure.While the secondary structure of the protein encoded by this gene in round-leaf includes 52.05%ofαSpiral structure,10.05%of extension chain,37.90%of irregular crimp structures.The tertiary structure of Bna A10g26320D encoding protein are basically the same between mosaic-leaf and round-leaf.