QTL Mapping Of Cold-tolerance And Functional Characterization Of The Cold-responsive Gene ShCIGT In Tomato

The tomato belongs to the high-temperature plant,and its growth and development are affected by the low temperature stress at every stage.As a model plant in modern molecular biology and genetics and breeding research,it has important practical value to study the regulation mechanism of tomato in low temperature response,to excavate QTL_S and genetic genes related to cold tolerance,and to develop molecular marker-assisted breeding.In this project,a cold-related gene ShCIGT is positively regulated by low temperature discovered in the microarrays related with cold tolerance.The trihelix gene family were performed a comprehensive bioinformatic analysis in tomato and the function of ShCIGT was studied by genetic transformation.288 re-sequencing core germplasms and the recombinant inbred lines derived from the cross of cold-tolerant toamto LA3969 and cold-sensitive tomato LA4024 were treated at low temperature in the seedling stage,and the QTLs related to the cold tolerance were excavated by GWAS and BSA.It has played a certain role in the cold-tolerant genetic breeding and the study of mechanism of response to low temperature in tomato.The main findings are as follows:1.Isolation and functional characterization of ShCIGT,a trihelix transcription factor from S.habrochaites.We found a new gene CIGT belonging to the GT-1 subfamily of the trihelix gene family by using microarrays to identify differentially expressed genes between the cold-tolerant tomato（LA1777 and LA3969）and cold-sensitive tomato LA4024 under cold stress.Under cold stress,it was positively regulated by low temperature and its expression was more strongly induced in cold-tolerant tomato than in a cold-sensitive tomato.There are few reports about the functions of GT family in tomato,especially in the stress response pathway.In the present study,the comprehensive analysis of 36 putative GT factors was performed in tomato.SlGT members can be classified into six subgroups（GT-1,GT-2,SH4,SIP1,GT-γand GT-δ）.Expression analysis of SlGT gene transcripts showed the distinct expression patterns of SlGT genes in various tomato organs.All the SlGT genes were regulated in response to various abiotic stresses and hormone treatments by the quantitative real-time PCR（qRT-PCR）analysis.Several SlGT genes,including SlGT-24,SlGT-27 and SlGT-34,were highly regulated by multiple abiotic stresses or phytohormone treatments.Taken together,our results presented here would be providing a useful platform for molecular clone and functional identification of SlGT genes in tomato.We isolated ShCIGT from the wild tomato species Solanum habrochaites.It was constitutively expressed in various tissues and induced by cold,drought,salt and ABA treatments.The results of functional analysis in transgenic plants showed that overexpressing ShCIGT enhanced the tolerance of transgenic plants to cold and drought stress,and reduced their sensitivity to ABA during post-germination growth.Further,we found that ShCIGT can directly interact with Sn RK1.We speculated that interactions between ShCIGT and Sn RK1 promote abiotic stress tolerance in tomato.2.Establishment of recombinant inbred lines and identification the cold-tolerant phenotype.In this study,we established a stable system evaluating the cold-tolerant phenotype.Because the cold-tolerant phenotype is quantitative traits,the phenotypic identification of strains is more reliable.First,we constructed a stable genetic population of F₇ recombinant inbred lines（more than 500 strains）derived from the cross of cold-tolerant tomato LA3969and cold-sensitive tomato LA4024.The seedlings of F₁,RIL and 288 accessions were treated with 4℃,and the index of chilling injury（CI）was calculated according to the grade of leaf wilting.The CI of F₁ can be seen that its cold-tolerant phenotype is between LA3969and LA4024,and it belongs to quantitative characters and there should be multiple sites controlling cold-tolerant phenotype.The re-sequencing core germplasms and RIL population have a diversity of the cold-tolerant phenotype and were screened extremely cold-tolerant and extremely cold-sensitive materials,respectively.3.GWAS and BSA analysis.The genome-wide scan using the SNP-based genotype and chilling indices of 288 accessions by GWAS,we observed 21 highly significant SNPs associated with the cold-tolerant phenotype on chromosome 1,3,4,5,7,8,9,10,and 12respectively.Two bulks of DNA samples from the screening of extreme tomato materials from the RILs were sequenced and identified by bulked segregant analysis,and found markers linked to a genomic region on chromosome 1.The CAPS markers exploited on mutation sites were used to validate the sequencing material,and the results showed that the markers were closely linked with the phenotype,and there might be two major effect QTLs regulating the cold-tolerant phenotype of tomato,which coincided with the two major effect SNPs on the first chromosome identified by GWAS.In order to convert multiple QTLs into single-site regulation and do the fine-mapping,we constructed subpopulation of this population,and applied the cold-tolerant linkage marker to breed new tomato varieties of cold-tolerant.The polymorphic markers could be used as a way of indirectly selecting the plant target traits and would promote developing new tomato variety by marker-assisted selection.