Identification Of Cold Tolerance And Genome-wide Association Study In Brassica Napus Germplasm

Brassica napus L.is the main material source of edible vegetable oil in China,its planting area and yield are at the forefront of Chinese oil crops.In recent years,with the changes in environmental climate,the widespread use of late-live seeding methods,and the rapeseed northward,the disadvantages of cold resistance in B.napus L.must be better and the screening of cold-resistant B.napus L.germplasm need to be improved as soon as possible.The research of the cold resistance mechanism in B.napus L.will greatly promote the development of Chinese rapeseed industry and improve the self-sufficiency rate of edible vegetable oil in China.In this study,we set a rapid and feasible cold-tolerant identification methods of B.napus L.,which was used to identify the cold tolerance of 107 mini-core germplasm resources of B.napus L.to obtain excellent cold-tolerant germplasm.Then we used Genome-wide Association Study(GWAS)to screen cold-tolerant candidate genes in B.napus L.and combined with transcriptome sequencing to further screen cold-tolerant candidate genes,this study will provide a genetic and theoretical references for cold-tolerant breeding of B.napus L.The main research contents and results are as follows:1.Screening of excellent germplasm resources for cold tolerance of rapeseed.We carried out a germination experiments of 10 B.napus L.with three temperature of 5 °C,8 °C and 10 °C,and then analyzed the effects of different low temperature on the germination rate,germination index and vigor index of each variety.According to the result,we choose 5 °C and 8 °C as the experiments temperature and germination rate,germination index and vigor index as indicators.We identified the cold-tolerant of 107 B.napus L.by using the above established methods,3 strong cold-tolerant materials were selected,named: Huahuang 1,JANPOL and P27,which could provide resources and reference for cold-tolerant breeding and cold-tolerant mechanisms research.2.Exploring cold-tolerance candidate genes in B.napus L.by GWAS.Combined the cold-tolerant phenotypic data and re-sequencing genomic data of 107 B.napus L.,the cold-tolerant candidate genes were screened by GWAS.A total of 11 SNPs were significantly associated with the cold tolerance of B.napus L.and 16 candidate genes were selected by searching for gene-annotated information,and most of these genes were related to calciumion channel and oxidoreductase activity,and some candidate genes also related to cell structure.3.Transcriptome analysis of low temperature resistance in B.napus L.The cold-resistant materials as the experimental subject and 5 °C as the experimental temperature,transcriptome analysis on different stages of seed germination were implemented.The differentially expressed genes showed that there were 158 and 1630 differentially expressed genes during early germination periode and radicle elongation periode under control conditions and low-tempreture respectively,747 differentially expressed genes during early germination periode between low-tempreture and control conditions,and these differentially expressed genes were mainly divided into 6 categories.Enrichment analysis revealed that the functional annotations of differentially expressed genes during early germination periode and radicle elongation periode under low-tempreture were mainly related to response to auxin and response to endogenous stimulus,photosynthesis.KEGG pathway enrichment analysis showed photosynthesis-antenna proteins pathway is significantly correlated with seed germination and whitening at low temperatures.4.Evolutionary Analysis of COR413 Family Genes in B.napus L.The COR413 gene is a class of cold stress response gene family.The bioinformatics analysis of the retrieved COR413 family genes in B.napus L.showed that the COR413 family has a high conservation domain,and they are hydrophobic and stable,and contains the aromatic amino acid tryptophan(Trp),thus we speculated that the COR413 family gene is responsible for the plants growth under low temperature stress.In addition,most of the COR413 family genes are located on the membrane,we suspect that there is a close connection with the induction of low temperature stress signals.