| Rice is one of the most important food crops in the world,and it is also a cold sensitive crop.Cold injury in booting stage will affect the formation of rice panicle morphology and is one of the main factors restricting the high and stable yield of rice.High latitude and high altitude are the main areas of cold injury in booting stage in China,especially in the three provinces of Northeast China,which are the main producing areas of japonica rice.However,the research progress is slow because the cold tolerance in booting stage of rice is controlled by micro-effect polygenes and influenced by environment.rice was sensitive to environment and could stably inherit less QTL of cold tolerance.Therefore,the study of cold tolerance QTL at booting stage is the basis of mapping cold tolerance genes,and it also plays an important role in the selection of cold tolerant varieties by molecular marker assisted selection.In this study,the recombinant inbred lines derived from Dongnong422 and Kongyu131 were used to identify the cold tolerance at booting stage in paddy field for three consecutive years.The phenotypic values of panicle length,grain number,the number of primary branches,the number of secondary branches,grain yield,panicle number,percent seed set,heading stage under natural and cold water irrigation were analyzed by QTL and QTL×environment.On the one hand,we combine QTL results with meta analysis to find out the important chromosomal regions to control cold tolerance in rice,and to mine the candidate genes for cold tolerance.On the other hand,through the identification of phenotypes under many years,the DNA pool was constructed with the percent seed set of booting stage as the target trait,and the correlation of cold-tolerant QTL interval was carried out with the next generation sequencing technique.By comparing the results of QTL analysis and BSA analysis,the cold-tolerance candidate genes of booting stage were mined in the common interval of the two analysis methods,so as to mine the cold tolerance candidate genes.The main results were as follows:1.Eight traits of 190 RIL and parents were identified in 2015-2017.The results showed that percent seed set,primary branch number,secondary branch number,grain yield,panicle number and heading date were sensitive to cold water stress.It can be used as an index to evaluate cold tolerance in booting stage.Correlation analysis showed that the correlation among other characters was significant except percent seed set.The phenotypes of panicle length,grain number,grain yield and heading date were mainly determined by genotype,while the primary branch number,secondary branch number,panicle number and percent seed set were significantly affected by environment.QTL analysis showed that there were differences in genetic patterns between percent seed set and other cold-tolerant traits under cold water stress.Correlation analysis and hypergeometric probability function analysis confirmed this conclusion.2.A genetic linkage map containing 158 pairs of SSR markers was constructed.The genotypes of the two parents showed normal distribution in the RIL,indicating that the RILs were suitable for genetic mapping and was an ideal population for genetic research.3.QTL analysis showed that the number of QTL detected under control and cold water stress was 32 and 29,respectively,and 8 QTLs were shared under control and treatment,respectively.The genetic contribution ratio of 20 QTLs was more than 10% under control environment,the alleles of 18 QTLs were from Dongnong422.The genetic contribution ratio of 16 QTLs was more than 10% under cold water environment,the alleles of 13 QTLs were from Dongnong422.There were 28 QTLs related to cold water reaction index,the genetic contribution ratio of 15 QTLs more than 10% to cold water reaction index,the alleles of 9 QTLs were from Kongyu 131.In this study,17 QTLs related to cold tolerance of booting stage were identified,which were qPL7-1,qGN7-3,qPBN7-1,qPBN7-2,qSBN7-1,qGY11-1,qGY2-2,qGY3-1,qGY5-1,qGY7-2,qPN7-1,qPN7-2,qPSS6-2,qPSS7-1,qHD6-1,qHD7-1 and qHD7-3,respectively.Among them,qGY2-2,qPSS6-2,qHD7-3,and qGY11-1 are 4 newly discovered QTL.A pair of extreme lines was used to verify the main effect of QTL qPSS6-2,and 9 lines with cold tolerance sites were found to be able to be used for genetic improvement.4.18 QTLs were detected by single environment analysis,single treatment combined analysis and combined analysis of all environments,which were qPL7-1,qGN1-2,qGN1-3,qGN6-2,qGN7-2,qGN7-1,qGN11-1,qPBN7-1,qGY1-1,qGY1-2,qGY6-3,qGY7-2,qGY7-1?qPN7-1,qPN11-1,qPSS6-2,qHD7-1 and qHD11-1,respectively,all of which were significant QTLs.Among them,qGN7-2,qGN11-1,qGN6-2,qPBN7-1,qGY7-2,qGY6-3,qPN7-1,qPSS6-2 and qHD7-1 were considered to be environmentally stable QTLs.In addition,17,16 and 14 polymorphic QTL regions were detected in three analysis models,which were distributed in 33 regions on 11 chromosomes.5.QTL × environmental interaction(QEI)analysis showed that qPL7-1,qSBN7-1,qPN6-1,and qPN6-2 had a significant QEI under the control condition.qGY1-1,qGY7-2,qPN7-1,qHD1-2,qHD6-2,and qHD7-1 had significant QEI under cold water treatment.Moreover,significant QEI was detected in 19 QTLs under the combined analysis of all environments.It can be seen that the role of QTLs × environmental variance in the total variance of the population changes with different environments.Environmental factors play an important role in trait inheritance.6.QTL correlation analysis showed that cold water stress could significantly improve the correlation of QTL compared with the control environment,and the more the environment,the closer the correlation was,which indirectly reflected the genetic correlation of traits under cold water stress.This may be a genetic association in a particular environment,or cold water stress may change the genetic factors of the traits and make them have genetic commonness.The correlation between QTL and phenotypic correlation was highly consistent,which revealed that the genetic factors of cold tolerance caused the variation of characters.7.Meta analysis showed that a total of 189 rice cold tolerance QTLs were integrated into the physical map,covering the length of rice genome 370.26 Mb.A total of 47 MCqtls related to rice cold tolerance were found on 12 chromosomes.By comparison,8 reported cold response genes were found in the physical interval of 7 MCqtls,and 20 QTLs related to cold tolerance at booting stage were found in 11 of them.Through bioinformatics analysis,seven candidate genes related to cold tolerance were identified,which were LOC_Os08g38560,LOC_Os09g30190,LOC_Os01g07560,LOC_Os06g36920,LOC_Os02g10150,LOC_Os03g17220 and LOC_Os02g09740.8.Based on the results of cold resistance identification and QTL-mapping,it was determined that qPSS6-2 was the main cold tolerance QTL.by constructing the DNA cell with extreme percent seed set,the amount of data of 70.55 Gbp was obtained by sequencing.The clean reads was 69.61 Gbp,Q30 was 85%,and the average sequencing depth of each sample was 41.44 X,the average ratio of sample to reference genome was 99.18 X,the average coverage depth was 39 X,and the genomic coverage was 98.29 X.The overlapping interval BSA-Seq based on ED and SNP-index algorithm annotates 12 candidate genes related to cold response.After fitting,the correlation interval was reduced to 3.68 Mb,and four cold-response genes were annotated.Comparing the interval detected by BSA-Seq and QTL-mapping,we found that the results of 9 QTL intervals and BSA-Seq overlapped.And the region of 1.81 Mb on chromosome 6 was co-located,which contained 212 genes successfully annotated,of which only two genes,LOC_Os06g39740 and LOC_Os06g39750,were related to cold stress response,The only 13 nonsynonymous mutations in this region contain LOCOs06g39750.The amino acid variation of the gene was found between the parents and the DNA pool.The results of qRT-PCR showed that LOC_Os06g39750 was strongly induced by cold stress in cold tolerant parent 131. |
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