| Rice(Oryza sativa L.)is the most important food crop in China and the cornerstone of national food security.With the economic development and the increasing number of people,the demand for food is increasing.The reduction of cultivated land area and the increase of urban land make saline land an important reserve land resource in China.Rice is a salt-sensitive crop.The salt tolerance of rice is a quantitative trait controlled by multiple genes and has a complicated genetic process.Therefore,excavating the major QTL for salt tolerance in rice and cloning key genes of salt tolerance play an important role in promoting the breeding of salt-tolerant rice.The predecessors had conducted a lot of research work on salt tolerance gene mining in rice seedling stage,and there were few reports on QTL mapping and candidate gene mining in the main reproductive growth period of rice.In this study,the recombinant inbred line(RIL)population was construct for the experimental material by the high-yield and high-quality japonica rice variety"Dongnong 425(DN425)"used as the female parent,and the salt-tolerant japonica rice variety"Changbai 10(CB10)"used as the male parent.From 2015 to 2017,under normal and salt stress treatment conditions,five yield-related traits,such as seed setting rate,grains per panicle,effective panicle number,panicle length,and thousand-grain weight,were investigated and analyzed.The relative value of each trait was used as the salt tolerance evaluation index.QTL analysis was performed by Inclusive Composite Interval Mapping(ICIM)of ICIMapping V4.0 software.The relative grains per panicle was used as the evaluation index to screen individuals with extreme traits in the RIL population for construct mixed pools for BSA sequencing analysis,and employed the?SNP-index method to identify the major salt-tolerance QTL.Combining the results of QTL mapping and BSA sequencing analysis,the QTL co-localization intervals detected by the two methods were used as candidate regions for salt tolerance,and candidate-gene mining was performed through gene function annotation.At the same time,natural populations composed of170 germplasm resources from different countries and regions were used as test materials for sequencing and sequence polymorphism analysis of candidate genes.Combined with the yield-related traits and physiological traits related to salt tolerance,candidate-gene association analysis was conducted to discover SNP and In Del loci that were significantly associated with trait variation,which provided a theoretical basis for improving salt tolerance by molecular breeding techniques in the reproductive growth period of rice.The main findings were as follows:(1)A survey of 5 yield traits,such as seed setting rate,grain number per panicle,effective panicle number,panicle length and 1000-grain weight,of the RIL population for three consecutive years found that after salt treatment,the trait value of each trait had been reduced.The decrease of Dongnong 425 was significantly greater than that of salt-tolerant variety Changbai 10.According to the relative values of various traits,the effective panicle number of Dongnong 425 decreased the most,followed by the thousand-grain weight and seed setting rate;Changbai 10 had a greater impact on the number of grains per panicle after salt stress treatment,followed by the thousand-grain weight and effective panicle.The relative values of the traits in the RIL population were continuously variable and there was a certain degree of superparent separation.The frequency distribution of each trait basically conformed to the characteristics of normal distribution and was suitable for QTL analysis.(2)QTL mapping was performed by Inclusive Composite Interval Mapping(ICIM)of ICIMapping V4.0 software according to the five salt-tolerance-related traits in the reproductive growth period,such as relative seed setting rate,relative grain number per panicle,relative effective panicle number,relative panicle length and relative 1000-grain weight.A total of 13 salt-tolerant QTLs were mapped on chromosomes 2,3,5,6,8,9,and 12 of rice.Among them,QTL q GP2-1related to the relative number of grains per panicle was detected in three years and located in the Indel18-RM1342 interval of rice chromosome 2.The three-year LOD values were 3.9801,5.2717and 4.7121,respectively,and the contribution rates were greater than 12%.The QTL was the major QTL for salt tolerance in the reproductive growth period of rice.(3)Taking the relative number of grains per ear as an evaluation index,20 strains with extremely high and extremely low phenotypes were screened from the RIL population to construct salt tolerance bulk(ST)and salt sensitive bulk(SS)for BSA sequencing analysis.A total of 496,333high-quality SNP sites were detected.Using the?SNP-index calculation method,a total of 7candidate regions were located,located on chromosomes 2,5,7 and 12,respectively,with a total length of 10.07 Mb and a total of 1465 genes.(4)Combining the results of QTL mapping and BSA sequencing analysis,q GP2-1 was located in the 25,830,000bp-28,159,456bp interval of rice chromosome 2,the interval length was 2.329Mb,and contained a total of 327 genes.LOC?Os02g44230(Os TPP1)was selected as the candidate gene through gene function annotation.Os TPP1 encoded rice trehalose-6-phosphate phosphatase,was a member of the rice TPP family.(5)The Os TPP1 gene of 170 germplasm resources were amplified and spliced to obtain a full-length 4031bp,including 10 exons.Through sequence alignment and polymorphism analysis,a total of 35 SNP polymorphism sites and 56 In Del sites were detected,most of the mutations occurred in the 5'UTR region,and the average nucleotide difference(K)was 1.32633.(6)Haplotype analysis of candidate genes.35 SNPs divided 170 materials into 21 haplotypes,of which 11 haplotypes contained only one material,haplotype Hap?9 contained more than half of the materials;and 35 SNP sites and 56 In Del site divided the materials into 33 haplotypes,of which21 haplotypes contained only one material,and 40%of the materials exhibited Hap?18 haplotype.(7)The population structure of 170 natural populations was analyzed using 85 pairs of SSR markers evenly distributed on 12 chromosomes.According to the results of STRUCTURE V2.3.4software and STRUCTURE HARVESTER website,the natural population could be divided into 3subpopulations,represented by P1,P2 and P3 respectively.The three subpopulations respectively contain 66,23 and 58 materials,and the remaining 23 materials were divided into Mix population.(8)Candidate-gene association analysis was carried out using the detected various yield-related traits and physiological traits.The results showed that 8 trait variations were significantly correlated with 10 SNP sites and 3 In Del sites of Os TPP1 gene(p<0.05).They were relative leaf peroxidase content,relative leaf proline content,relative root proline content,relative leaf soluble sugar content,relative root soluble sugar content,relative Na~+/K~+in stem,relative effective panicle number and relative thousand-grain weight.The maximum contribution rate(R~2%)was 9.23%.These polymorphisms were not involved in the coding of amino acids,so the protein structure of Os TPP1was not altered. |
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