| Soybean [Glycine Max(L.)Merr.] is one of the important food crops and oil crops in the world,which is rich in high quality vegetable protein and oil,and plays an important role in the development of human life and industry.Although China is the origin country of soybean,its domestic soybean production is far from meeting people’s demand,and it can only rely heavily on imported soybean.For soybean breeding,increasing yield is particularly important.Since photosynthate is the basis of crop biomass and yield,it is an effective and feasible strategy to increase crop yield by improving the utilization rate of light energy.Photosynthesis-related traits are complex quantitative traits controlled by multiple genes and easily affected by environmental factors,which makes genetic analysis of photosynthesisrelated traits difficult.In recent years,with the rapid development of modern molecular biology and sequencing technology,traditional breeding based on the combination of high density single nucleotide polymorphism(SNP)markers and transgenic technology can effectively achieve the analysis of complex quantitative trait control loci and genetic basis,greatly shortening the breeding process.In this study,the phenotypic data of photosynthetic related traits(net photosynthetic rate,stomatal conductance,intercellular carbon dioxide concentration and transpiration rate,etc.)of soybean at seedling and bulking stages were collected from a recombinant inbred line population and a natural population.Linkage analysis and genome-wide association analysis(GWAS)were carried out to identify quantitative trait loci(QTLs)related to soybean photosynthetic traits,and further combined with bioinformatics and molecular biology techniques to screen candidate genes for photosynthesis.The biological functions of the candidate genes were verified in order to analyze the genetic variation of photosynthetic related traits in soybean,so as to provide ideas for improving the utilization efficiency of soybean photosynthesis to increase yield.The main results obtained are as follows:1.Three photosynthetic traits(net photosynthetic rate,stomatal conductance and intercellular carbon dioxide concentration)of soybean at bulking stage were measured in pot experiment and field experiment with the hybrid population material of NN94-156 and Bogao under four different environments.Statistical analysis of the phenotypic data showed that the data distribution of the three photosynthetic indicators showed continuous or approximate normal distribution,and showed a wide range of genetic variation,and there was a significant interaction between genotype and environment.The generalized heritability of net photosynthetic rate,stomatal conductance and intercellular carbon dioxide concentration was 64.56%,73.1% and 74.68% respectively.In addition,there were differences in the three photosynthetic gas exchange parameters,especially in the net photosynthetic rate.These results indicated that photosynthetic related traits were complex quantitative traits,and the recombinant inbred population was suitable for linkage analysis of photosynthetic related traits in soybean.After linkage analysis of phenotypic data,a total of 26 photosynthetic related QTLs were identified on 17 chromosomes except chromosome10,12 and 20,with phenotypic variation ranging from 1.15% to 14.20%.Among them,19 QTLs were detected only in a single environment,while QPH-9 and QPH-18-3 were repeatedly detected in three environments,and QPH-18-3 was the only one that could be simultaneously located by different photosynthetic related traits in three environments.Therefore,these two QTLs were regarded as the main effect QTLs related to photosynthesis.2.The photosynthetic gas exchange parameters of 219 soybean materials at seedling and bulking stage were collected in pot experiment and field experiment under multiple different environments,and GWAS was performed on them respectively.Analysis of photosynthetic related indexes at seedling stage showed that: A total of 12 SNPs significantly associated with photosynthesis were detected.According to the LD decay distance of this population,the 130 Kb range before and after the SNP marker was defined as a QTL,and a total of 12 photosynthetic related QTLS were distributed on chromosomes 1,2,6,7,9,11,12,13,15,16,18 and 19.Two QTLs(q P1 and q P16)on chromosome 1 and 16 were detected in two or more environments using different photosynthetic traits,with phenotypic explanation rates of 16.89% and 16.30%,respectively.According to the gene annotation of the soybean genome public database and the related annotation function of homologous genes in Arabidopsis,two candidate genes were screened in q P1 and q P16,respectively.Candidate genes on chromosome 1 were Glyma.01G158000 and Glyma.01G159800,and candidate genes on chromosome 16 were Glyma.16G141100 and Glyma.16G141700.The expression levels of these four candidate genes showed significant differences in photosynthetic extreme materials,but their potential biological functions in regulating photosynthesis need to be further explored.The analysis of photosynthetic related indexes at the bulking stage showed that: a total of 12 significant SNP markers were associated,among which SNP marker AX-93870020 on chromosome 18 explained 30.15% of phenotypic variation and could be detected in multiple traits under multiple environments.The genome region containing 130 kb before and after it was defined as a candidate region for photosynthesis.By comparing the linkage analysis results,it was found that this segment was located within the QTL q PH-18-3 located on chromosome 18.Therefore,the combination of linkage analysis and genome-wide association analysis could improve the accuracy of QTL mapping and facilitate the screening of candidate genes.Although the candidate range was narrowed,the region contained 29 genes,and other analysis methods were needed to identify candidate genes for photosynthesis.3.The whole genome of the parents of the recombinant inbred line population was resequenced to compare the different SNPs of Bogao and NN94-156 in the target region of chromosome 18,excluding those SNPs that were within the gene sequence but did not produce non-synonymous mutations.SNPs with non-synonymous mutations in the coding region and promoter region(2.0 kb region before the start codon)were retained and the 29 genes screening was reduced to 7 genes.Combined with real-time quantitative PCR,only the expression level of Glyma.18G065600 gene was significantly different between parents,so it was selected as a candidate gene related to soybean photosynthesis.According to the gene annotation information published in the soybean genome public database,Glyma.18g065600 encodes an ATP-dependent zinc ion-binding metalloprotease.Bioinformatics analysis showed that this gene belongs to the FtsH subfamily in the AAA gene family,which contains AAA domain and Peptidase＿M41 domain.The FtsH gene family in soybean contains 34 members.According to their chromosomal locations,Glyma.18g065600 was named GmFtsH25.Compared with the FtsH gene family in species such as Arabidopsis,rice and maize,it is evolutionarily conserved,of which GmFtsH25 is the closest relative to At FtsH9 in Arabidopsis.4.Further analysis of GmFtsH25 showed that it was expressed in all soybean tissues,with the highest expression in leaves,followed by flower.Subcellular localization showed that GmFtsH25 was localized in chloroplasts.Using the genetic transformation technology of soybean cotyledon nodes mediated by Agrobacterium tumefaciens,transgenic soybean lines with overexpression and stable transformation of GmFtsH25 were developed.The photosynthesis-related traits and yield-related traits of the three stably overexpressed transgenic lines were analyzed,and it was found that the net photosynthetic rate of GmFtsH25 overexpressed plants was significantly higher than that of WT;In terms of yieldrelated traits such as pod number per plant,grain number per plant,and yield per plant,the transgenic material was significantly improved compared to the WT.The molecular mechanism exploration of GmFtsH25 found that there was an interaction between GmFtsH25 and the light-harvesting antenna protein Gm LHCa2.The expression level of Gm LHCa2 increased with the increase of GmFtsH25 expression,and the expression levels of other photosynthesis-related genes in GmFtsH25 overexpressed transgenic plants were also significantly increased.These results suggest that GmFtsH25 may be positively regulating the photosynthetic efficiency and contribute to the improvement of soybean yield. |
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