Low-temperature Stress Caused Phenotypic Variation In Germination And Emergence And Screening Of Low-temperature Tolerance Candidate Genes For Rapeseed (Brassica Napus L.)

In recent years,direct seeding of rapeseed has developed rapidly.However,under the multi-ripening production mode,direct seeding date of rapeseed must be postponed,which decreases the probability of seeds to germinate at low temperature.How to make rapeseed seeds quickly germinate and establishment under low temperature stress is an urgent problem to be solved in rapeseed production.In this study,442 Brassica napus materials were used for the germination test under normal and low temperature conditions.The phenotypic differences among the materials were compared and the low temperature resistance evaluation was performed.The seed quality and thousand seed weight were measured.Ten materials with extremely different low temperature emergence characteristics were selected for pot experiment.Genome-wide association analysis(GWAS)was performed using more than 8 million resequencing SNPs and 442 material phenotypic indicators.The main findings are as follows:1.Comparison of phenotypic differences regarding to seed germination and seedling emergence of 442 different materials.There were significant differences in indexes during seed germination and emergence.The percentages of germination and emergence were reduced and the variation range was large under low temperature conditions of 10℃-15℃.The percentage of germination varied from 5.0% to 100.0%,and the percentage of emergence was 1.0% to 99.7%;low temperature prolonged the time of germination and emergence.The mean germination time and emergence time were 1.28 d and 5.59 d at 20℃-25℃,respectively,and the mean germination and emergence time were extended to 3.33 d and 11.73 d at low temperature of 10℃-15℃.Dry weight,root length and shoot length had similar changes with normal temperature,but low temperature stress significantly reduced the germination index and root growth rate,which were 63.9% and 53.7% lower than normal temperature,respectively.The broad-senseheritability of the low temperature stress tolerance index(STI)of all indicators were above 84%,and most of them were above 95%.Therefore,it was concluded that these indicators were mainly regulated by genetic factors.2.Evaluation of low temperature tolerance regarding to seed germination and seedling emergence from 442 different materials.Through principal component analysis,the STIs of 13 indicators at the stages of seed germination and seedling emergence were divided into five categories: seedling morphology,germination speed and germination rate,biomass,emergence speed and emergence rate,and shoot length.Principal component analysis of the low-temperature stress tolerance indices identified five principal components that captured 100% of the seedling response to low temperature.Through the classification of STI comprehensive scores,23 materials with strong low temperature resistance,396 materials with medium low temperature resistance and 23 low temperature sensitive materials were identified.According to the difference in characteristics of different resistant materials at normal temperature and low temperature,the STIs and the comprehensive scores of STI,it was found that compared with low temperature sensitive materials,low temperature resistant materials had stronger germination ability,higher emergence rate,and larger seedling biomass,and the seedlings were more vigorous.3.Correlation of seed thousand-seed weight,quality and low temperature stress tolerance indices.The content of erucic acid was significantly positively correlated with mean germination and emergence time,the percentages of germination and emergence,and other indicators of the STIs.The glucosinolate content had a very significant positive correlation with the STIs of the percentage of germination and mean emergence time.The oil content was significantly positively correlated with the STIs of mean germination time,germination index,mean germination time and the percentage of emergence.Thousand-grain weight was extremely significantly negatively correlated with the STIs of mean germination and emergence time,germination index and root length.The protein content was significantly negatively correlated with the STIs of mean emergence time and the percentage of emergence.Therefore,it was concluded that the speed of seed germination and seedling emergence was positively correlated with the content of erucic acid,glucosinolate,and oil,and negatively correlated with thousand-grain weight and the content of protein.4.Verification materials with extreme differences in emergence characteristics at low temperature in pot experiment under early-season seeding and late-season seeding.The order of mean emergence time of the 10 outdoor pot experiment and the indoor germination box experiment was basically same,and the correlation coefficient of the STI of mean emergence time of the two experiments was 0.86,showing a high significant positive correlation.The correlation coefficients between the STI of mean emergence time of potted plants,the yield per plant,and the biomass were all 0.83,and they all showed a high significant positive correlation.Therefore,it was concluded that faster emergence of seedlings might have a positive effect on the increase in yield and biomass.5.Screening and identifying 55 genes related to low temperature tolerance during seed germination.GWAS analysis of low temperature tolerance was carried out with 5 principal components,and 22 SNPs were screened to be significantly correlated with phenotypic indicators;of which 7 were related to seedling morphology(PC1),6 were related to germination speed and germination rate(PC2),5 are related to biomass(PC3),1 is related to emergence speed and emergence rate(PC4),and 3 are related to the traits of shoot length(PC5).Fifty-five candidate genes were screened to participate in the regulation of seed vigor under low temperature.Most of the functions can be divided into DNA repair and RNA translation,mitochondrial activation and energy production,ubiquitination and degradation,antioxidant system,plant hormones and signal transduction,etc.The effect of variations,the effect of haplotypes and the score of probability for gene function of 55 candidate genes were obtained,and 5 candidate genes with a comprehensive score of greater than 1 were screened out: BnaA03g40290D,BnaA06g07530D,BnaA09g06240D,BnaA09g06250D and BnaC02g10720D.