Gene Mining And Functional Analysis Of High Photosynthetic Efficiency In Rapeseed

Brassica napus is one of the important oil crops in the world and the main source of the vegetable oil in China.The yield of rapeseed is closely related to the photosynthesis of photosynthetic organs,and the photosynthesis of leaves can contribute at least 30%of the biomass.Therefore,it is of great significance to significantly increase the yield of rapeseed by improving the utilization rate of light energy and carrying out high photosynthetic efficiency breeding.In order to promote the progress of rapeseed breeding with high photosynthetic efficiency,the improvement of photosynthetic traits has become an urgent task and an important goal of rapeseed variety improvement.In this study,photosynthetic traits（photosynthetic rate,intercellular CO₂ concentration,stomatal conductance and transpiration rate）of 560natural population materials were measured under low light intensity and high light intensity conditions,and re-sequencing data was used for genome-wide association analysis（GWAS）.Meanwhile,transcriptome data of extreme materials were combined to screen candidate genes for target trait differences,the hub genes related to traits were identified through weighted gene co-expression network analysis（WGCNA）,and the correlation between them and different candidate genes was determined.The main results are as follows:1.Phenotypic variation of photosynthetic traitsUnder the conditions of low light intensity and high light intensity,we measured four photosynthetic traits（photosynthetic rate,intercellular CO₂ concentration,stomatal conductance and transpiration rate）of 560 Brassica napus.All of the four traits had extensive phenotypic variation and presented continuous normal distribution,indicating that they were all typical quantitative traits and controlled by multiple genes.The positive correlation between each trait reaches a very significant level.Among them,the correlation coefficient between photosynthetic rate and transpiration rate under high light in 2019 was the largest,followed by stomatal conductance and transpiration rate under low light,intercellular CO₂ concentration and transpiration rate under high light,and the correlation coefficients between traits were 0.997,0.994 and 0.990,respectively.2.Genome-wide association analysis of photosynthetic traitsUnder the best model K+Q model,a total of 34 significant SNPs sites were detected in the photosynthetic rate of the natural population,mainly distributed on chromosomes A02,A07,A08,A09,A10,C03,C05,C08 and C09,among which the most sites were detected on chromosome A10,with a total of 8 sites.One of these SNP markers located on C03 chromosome was detected in 2018 and 2019 under low light conditions and its physical location was relatively close,294kb apart.Under high light conditions,one SNP marker was detected in both 2018 and BLUP values,and it was located on the A10 chromosome.The range of explainable phenotypic variation of these loci under two different light conditions over a two-year period ranged from 7.48%to15.46%.Intercellular CO₂ concentration detected 25 significant sites under two light conditions,distributed on 13 chromosomes,which were A03,A04,A05,A07,A08,A09,C02,C03,C04,C05,C06,C07 and C09 chromosome,the explainable phenotypic variation was 7.63%～8.41%.Six SNP markers associated with stomatal conductance were located on chromosomes A03,A07,A10 and C06,the contribution rate of phenotypic variation was 8.13%～14.47%.For transpiration rate,the K+PCA model was used to detect fewer sites under the two light conditions,one for each of the C02and C04 chromosomes,and the single site contribution rates were 8.08%and 10.59%.3.Transcriptome analysis of extreme phenotypic materialsA total of 872 co-differentially expressed genes were screened from the photosynthetic rate materials under low light conditions,including 458 co-up-regulated genes and 301 co-down-regulated genes in the four extreme materials.Under high light conditions,1237 common genes were differentially expressed,of which 525 were up-regulated genes and 553 were down-regulated genes in the four extreme materials.A total of 6463 differentially expressed genes were obtained from intercellular CO₂concentration materials under low light conditions,including 3091 significantly up-regulated genes and 3372 significantly down-regulated genes.There were 7731differentially expressed genes under high light conditions,of which 4099 were significantly up-regulated genes and 3632 were significantly down-regulated genes.For stomatal conductance,8450 significantly differentially expressed genes contained 3903significantly up-regulated genes and 4547 significantly down-regulated genes respectively.There were 2047 up-regulated genes and 2999 down-regulated genes among the 5046 differentially expressed genes under high light conditions.11005differentially expressed genes were detected in the transpiration rate materials under low light conditions,of which 5176 were up-regulated genes and 5829 were down-regulated genes.A total of 11984 differentially expressed genes under high light conditions were obtained 6907 significantly up-regulated genes and 5077 significantly down-regulated genes.By comparing the biological trends and metabolic pathways of different traits in GO analysis and KEGG analysis under two light conditions,it was found that the biological functional trends of the four traits were consistent in the GO functional enrichment analysis.The GO terms involved in differentially expressed genes were mainly cellular processes,metabolic processes,single biological processes and other biological processes.Participate in cell components,cells,organelles and other cell components;Molecular functional pathways such as response binding and catalysis.In KEGG analysis,the most enriched pathways were ribosomes,amino acid biosynthesis and carbon metabolism.In addition,the photosynthetic rate was enriched to starch and sucrose metabolic pathways under high light conditions.Intercellular CO₂ concentration was enriched to glycerol metabolic pathway under low light conditions and to sulfur metabolic pathway under high light conditions.In the extreme materials of stomatal conductance with low light intensity,the differential genes were significantly enriched to the metabolism of glycolysis/gluconeogenesis,pentose phosphate pathway,cysteine and methionine.Transpiration rates were significantly enriched into the carbon fixation pathway in photosynthetic organisms under low light conditions.4.Analysis of weighted gene co-expression networkIn this study,WGCNA was used to excavate the site of high photosynthetic effect on the photosynthetic traits of Brassica napus under low light intensity and high light intensity,a total of 25 co-expression modules were obtained from low-light materials,and 31 co-expression modules were obtained from high-light materials.The correlation analysis between modules and traits shows that blue module is the core module under two light conditions.Blue module was correlated with intercellular CO₂ concentration in low light materials（r=0.69,p=2e-04）.Blue module in high light materials is closely related to stomatal conductance and transpiration rate（r=0.52,p=0.009;r=0.58,p=0.003）.The candidate genes in the module are mostly involved in chloroplast biosynthesis and stress response to abiotic stress.Including three phytohormones-related RD20,CBF2 and ERD15,five chloroplast biosynthesis-related CPSRP54,RPI2,FTRA1,CRR23,CRR3,and signal transduction four genes CIPK21,EXL2,GRF2,VHA-A.The Arabidopsis gene AtCPSRP54 was also detected in the results of GWAS analysis using high-intensity population materials,while AtEXL2 and AtCRR23 were detected in the results of differential gene function annotation analysis of high-intensity extreme materials.Other genes were only mined through WGCNA.