Genotypic Differences And Mechanisms Of Chlorophyll Content And Fluorescence Characteristics In Rice

Rice is one of the most important crops for maintaining world food security,providing the staple food for about 50% of the world’s population.In recent years,with the double pressure of the continuous decline of rice planting area and the growing demand for food,increasing the yield per unit of rice has become an important way to solve the problem of food security.More than 90% of the dry weight of crops is directly determined by photosynthesis.Increasing the photosynthetic rate of leaves and promoting the accumulation of plant biomass has become the key to improving rice yield and an important goal of high-yield and high-efficiency breeding in the future.Chlorophyll is one of the most important pigments in plant photosynthesis and one of the main indicators for evaluating the photosynthetic rate of leaves.Chlorophyll fluorescence analysis technology has a unique role in measuring the absorption,transmission,dissipation and distribution of light energy in plant leaves.Therefore,studying the photosynthetic characteristics of leaves is of great significance for improving rice yield,and it is also the focus of rice research.Previous studies in this area have mainly focused on the effects of environmental or management factors such as leaf structure,gas exchange,nutrient elements,Rubisco enzyme activity,fluctuating light,and various stress responses,while the genetic resources mining and genetic resources of rice chlorophyll content and fluorescence parameters regulated genes Mechanistic research,related research reports are relatively rare;in addition,convenient and efficient measurement techniques,stable,accurate,and highthroughput characterization parameters have attracted more and more attention in photosynthesis research under field conditions.In this study,173 rice varieties with similar growth stages were selected from the historical cultivars of the Crop Physiology and Cultivation Research Center(CPPC)of Huazhong Agricultural University,the Mini Core Variety Bank of Huazhong Agricultural University and the 3K RGP varieties(Table 2.1),According to the 3K RGP classification,it is divided into 4 subpopulation: Indica,Japonica,Aus/boro and Intermediate.The subpopulation with unknown information is represented by Unknow.The field planting experiment was carried out for two consecutive years in2018,and the relative chlorophyll content(RC)and chlorophyll fluorescence parameters(NPQt,Phi2,Phi NO,Phi NPQ,LEF,Fv P/Fm P,G,q L and q P)of flag leaves of each variety at full heading stage were determined.,and analyzed the genotypic differences of parameters among and within varieties,as well as the correlation and clustering characteristics between parameters.In this study,NGS high-throughput sequencing technology and genome-wide association(GWAS)analysis were used to screen and identify candidate genes for relative chlorophyll content(RC)and nonphotochemical quenching(NPQt)traits.Two cultivars specific for relative chlorophyll content(RC)and non-photochemical quenching(NPQt)traits were selected,and the differentially expressed genes were screened and identified by transcriptomics(RNAseq),and verified by q PCR experiments.Factors carry out gene family identification and analysis,and further analyze the structure and function of key candidate genes.The main conclusions of this study are as follows:(1)The genotypes of chlorophyll content and fluorescence parameters of different rice varieties were significantly different.The results of this study show that there are extensive genotypic differences in the distribution of rice chlorophyll content and fluorescence parameters between different tested rice varieties or within the same variety,which is an important resource for mining candidate genes for chlorophyll content and fluorescence parameters in rice.In addition,there are extensive correlations between phenotypic traits,among which NPQt has a significant positive correlation with Phi NPQ,NPQt has a significant negative correlation with Phi2,Phi NO and Phi NPQ have a significant negative correlation,and RC has a significant positive correlation with Phi2 relationship,indicating that the measured parameters can be applied to multivariate and multidimensional research and analysis.The cluster analysis of chlorophyll and fluorescence parameters can be divided into three groups,the first group has low NPQt,low Phi2,high Phi NO,low Phi NPQ and high RC value,the second group has low NPQt,High Phi2,low Phi NO,low Phi NPQ and low RC,the third category has high NPQt,low Phi2,low Phi NO,high Phi NPQ,high RC.(2)Screening and identification of candidate genes for relative chlorophyll content(RC)and non-photochemical quenching(NPQt)in rice.In this study,gene regions(4498kb – 4 698 kp)related to NPQt traits were mapped on rice chromosome 2,and 29 candidate genes were screened;gene regions related to RC traits were mapped on chromosome 4(32 158 kb – 32 427 kp),60 candidate genes were screened.Through the gene expression analysis of the Rice Anther Net database(rice anther development gene co-expression network),the range of candidate genes was further narrowed,including 29 candidate genes for the NPQt trait,Os02g0182100(RR24)and Os02g0182800(HOS58)are highly expressed in leaves,while Os02g0181900(CLPBM)and Os02g0181300(WRKY71)are lowly expressed,and Os02g0181300(WRKY71)is significantly different in different plant tissues.Among the 60 candidate genes for RC traits,Os04g0634500(RLCK166),Os04g0635900(DNA repair),Os04g0636100(Glycosylhydrolase),Os04g0631600(bHLH),Os04g0637000(BZIP37)high expression in leaf,Os04g0632100(RLCK163),Os04g0632300(RLCK379),Os04g0632300(RLCK165)low expression.(3)Screening,identification and validation of differentially expressed genes and transcription factors of rice varieties with relative chlorophyll content(RC)and nonphotochemical quenching(NPQt)traits.2366 differentially expressed genes were screened,of which the up-regulated and down-regulated genes were 1434 and 932,respectively.The GO function enrichment analysis of differential genes showed that the number of GO items reached 3467,accounting for 26.91%,62.47% and 10.62% in MF,BP and CC,respectively.KEGG pathway analysis of differentially expressed genes,a total of 393 differentially expressed genes were annotated,and 11 metabolic pathways were significantly enriched.Five candidate transcription factors,bHLH,ERF,MYB,NAC and C2H2,were screened to mediate the specific phenotype of relative chlorophyll content(RT)and non-photochemical quenching(NPQt)in rice.Among them,there are 149 genes in bHLH transcription factors,and ERF transcription factors There are 121 factors,112 MYB transcription factors,101 NAC transcription factors,and 97 C2H2 transcription factors.In this study,four key candidate genes,Os04g0634900,Os04g0632000,Os04g0636400,Os04g0631600,which regulate relative chlorophyll content(RC)in rice,were jointly screened through the combined analysis of GWAS and RNA-seq results,and were verified by q PCR to have high expression consistency.The Os04g0631600 gene belongs to the bHLH transcription factor gene family,and this gene and the bHLH gene family have not been systematically identified and analyzed in rice.(4)The rice bHLH transcription factor gene family can be subdivided into 4subfamilies with a total of 169 genes.The bHLH transcription factor gene family can be subdivided into 4 subfamilies: G-box-binding,Non-G-box,Non-E-box,Non-DNAbinders,with 97,17,19 and 36,respectively This family of genes is closely related to the regulation of plant photosynthetic traits.And previous studies have shown that Os04g0631600 has a high expression in rice leaves.In summary,there were significant genotype differences in the relative chlorophyll content(RC)and fluorescence parameters(NPQt,Phi2,Phi NO and Phi NPQ)in rice.There was a significant correlation between the parameters,and it was basically consistent with the trend shown by clustering and difference analysis,indicating that the relative chlorophyll content and fluorescence parameters of rice had rich genetic variation.The candidate genes and transcription factors that regulate RC traits and NPQt traits can provide genetic resources and research guidance for the selection and cultivation management of high-photosynthesis-efficiency rice varieties.