QTL Mapping For Cold Tolerance And Differentially Expressed Genes Analysis Under Low Temperature Stress In Rice(Oryza Sativa L.)

Rice is a thermophilic crop,which is sensitive to temperature in the whole growth and development process.Low temperature injury is one of the main limiting factors of rice production,and it is also a common problem in the main rice producing areas in the world.It is very important to study the genetic mechanism of cold tolerance in rice.The cold tolerance of rice is a complex quantitative trait,which is specific in every stage.A set of chromosome segment substitution lines(CSSLs)derived from Sasanishiki(japonica ssp.cold tolerant)and Habataki(indica spp.cold susceptible)were used for analysis low temperature tolerance in this study.QTL localization analysis of low temperature tolerance of 10 days at 5 ℃ at budding stage,cold tolerant QTL location analysis at booting stage under natural low temperature conditions,mechanism analysis of abnormal seed-setting under different temperature gradient low temperature stress in late booting stage for 7 days,as well as comparative analysis of differentially expressed genes for low temperature(20℃)and 7 days treated rice materials at the booting stage by using RNA-sequencing technology were carried out.The following main findings were obtained.1.qCTP4,qCTP10,and qCTP11 for cold tolerance at budding stage were identified on chromosome 4,10,and 11,respectively.The range of QTL additive effect values detected was-14.3~-30.3%,and the phenotypic variation explained by each QTL ranged from 15.4% to 32.5%.Among them,qCTP11 had the largest additive effect(a=-30.3 %),which could be repeatedly detected in duplicate testsand explained 32.5% of the phenotypic variation.qCTP10 may be a newly discovered QTL.All alleles for cold tolerance were contributed by Sasanishiki.The main reason leading to the decrease of cold tolerance during the budding stage was the introduction of the chromosome fragment from the indica donor parent Habataki.2.Eight quantitative trait loci(QTLs)for low temperature tolerance at booting stage were detected,of which,2 QTLs qCTSF3.1 and qCTSF3.2 were located on chromosomes3,the other qCTSF4,qCTSF5,qCTSF6,qCTSF7,qCTSF8,and qCTSF9 on chromosomes4,5,6,7,8,and 9,respectively.The phenotypic variation explained by each QTL ranged from 5.4% to 25.3%.Of the 8 QTLs,6(qCTSF3.2,qCTSF5,qCTSF6,qCTSF7,qCTSF8,and qCTSF9)were repeatedly detected in two environments.qCTSF3.1,qCTSF7,and qCTSF9 were overlapped with previously reported QTLs.All alleles for cold tolerance were contributed by Sasanishiki.3.Compared with japonica rice variety Sasanishiki,indica rice variety Habataki showed obvious sensitive to low temperature under low temperature gradient stress at booting stage for 7 days.The seed setting rate at 23℃ was significantly different from thatat normal natural condition(28℃),and reached a very significant level at 22℃.However,the seed setting rate of japonica rice variety Sasanishiki at 20℃ was significantly different from that under normal growth condition at 5% level,and reached 1% at 19℃.Under low temperature stress,the vigor of stigma of female organs was normal,but pollen of male organs could not germinate normally to complete fertilization.4.A total of 133 million high quality reads(50 bp)were used for transcriptional analysis analysis after screening.1472 differentially expressed genes(DEGs)were found in at least 1 comparison groups among the 3 experimental comparison groups.These suggest that metabolic pathways may be inhibited or activated in response to low temperature stress.149 DEGs were found under low temperature stress.5.The results of GO enrichment analysis,KEGG database analysis and STRING database analysis show that low temperature stress can lead to changes in a series of gene expression in the process of photosynthesis and metabolism of precursor substances,particularly the obvious interaction between genes which involved in photosynthesis process(LOC_Os03g07300,LOC_Os03g39610,LOC_Os12g17600)and the genes of precursors in the process of energy metabolism(LOC_Os08g01380,LOC_Os11g07020,LOC_Os12g19381)have interaction obviously.All of these lead to the nutrients or energies needed in the development of pollen are not supplied enough,resulting in the microspore not fully developed or the accumulation of nutrients in pollen is insufficient.As a result,pollen germination was affected and fertilization was not able to be completed normally,which leads to the significant decrease in seed setting rate.