| Rice chilling is an irreversible metabolic and physiological injury that caused by the environmental temperature below optimum growth temperature. Rice seedling chilling, which result in plant growth delay and death, has beacome a natural disaster that affecting stable food production around the world. Therefore, mining of cold-tolerant germplasm, identifying of chilling molecular mechanism and providing of reliable quantitative trait loci (QTLs) for molecular marker-assisted selection are important for enhancing rice tolerance and ensuring food safe production.In this study, a mapping population consisting of 151 BC2F1 plants was constructed by using chilling-tolerant Dongxiang wild rice (Oryza rufipogon Griff.) as a donor parent and chilling-sensitive indica as a recurrent parent. With leaf osmotic potential, plant survival rate and root conductivity as chilling-tolerant trait indexes, seven chilling tolerant QTLs were detetered on 2,5,8,10 chromosomes by composite interval mapping method. Two QTLs related to plant survival rate, qPSR2-1(LOD= 3.3) and qPSR2-2 (LOD=3.1), were detected on the long arm of chromosome 2, which explained 12.3% and 9.8% of the phenotypic variation, respectively. For leaf osmotic potential, qLOP2, qLOP5 and qLOP8 were identified on 2,5,8 chromosomes with 3.8,4.3,5.1 of LOD value, and explained 10.1%,8.8% and 6.9% of the phenotypic variation, respectively. Two QTLs (qRC10-1 with LOD=3.1 and qRC10-2 with LOD=6.1) related to root conductivity were harbored at 10 chromosome, which explain 9.4% and 32.1% of phenotype variation. qPSR2-1 and qLOP2 were located within the same region, molecular markers closely linked with them were RM318 and RM106.To further study the loci showed more than 10% of phenotype contribution, two fine mapping populations were constructed respectively to fine mapping of qPSR2-1, qLOP2 and qRC10-2. Through development of polymorphic molecular markers (RM3793, RM3508, RM3512, RM221, RS3, RS8, RS11) covering qPSR2-1, qLOP2 interval and two large segregating populations consisting of 11,326 BC4F2 and 8,642 BC4F3, qLOP2 and qPSR2-1 were dissected to a 39.3-kb candidate region between RM221 and RS8. Based on Nipponbare physical map in the RAP 1.0 database, two CBF3 genes (Os02g0676800、Os02g0677300) existing in this candidate region. Real-time PCR result indicated that the expression level of Os02g0677300 was significantly increased after 3h chillibg stress (P<0.01), while Os02g0676800 was not show a significantly increased tendency during the whole chilling stress. Therefore, Os02g0676800 may be the best candidate gene for qPSR2-1, qLOP2.To fine mapping qRC10-2, a large BC5F2 fine-mapping population with 13,324 progenies was constructed and eleven polymorphic molecular markers (qc8, qcl8, qc19, qc25, qc27, qc45, qc47, qc48, qc55, RM25628, RM25681) was developped in this region. Lastly, qRC10-2 was delimited to a 22.5-kb region between qc47 and qc48. After sequenced genomic sequence of qRC10-2 interval in Dongxiang wild rice, only one open reading frame was identified in this region, which contains 830bp of full-length cDNA with 624bp of coding region,21 bp of 5’UTR and 167 bp of 3’UTR. Compared with Nanjing 11, two base mutations located at the 343th and 524th site of the encoding frame, ’G’ and ’A’ showing in Dongxiang wild rice while ’A’,’G’ in Nanjingl 1, which caused the corresponding amino acid (Gly and Asp) in Dongxiang wild rice have changed into Ser, Gly in Nnajingll, respectively. Further analyzed sequence various among 38 japonica varieties and 50 indica varieties, the result showed that all varities could be assigned into three groups (G1, G2 and G3), the root conductivity value of Dongxiang wild rice and japonica group G1 was lower than Nanjing 11 and group G2, G3 (P<0.01). qRC 10-2 was highly homologous with Os10g0490100 gene (pectin lyase fold family protein) from Nipponbare. Real-time PCR results indicated that its expression level was up-regulated by chilling stress in the leaf of the cold-tolerant (Dongxiang wild rice, L188). Such tendency was not showed in root, but a significant expressional difference between the cold-tolerant and cold-sensitive (Nanjingl 1) was present before chilling stress (P<0.01).In order to verify the physiological function of qRC10-2 gene, the code frame of qRC10-2 was connected with UBI promoter and transformed into the cold-sensitive indica cultivar (R6547). Total 23 positive To transgenic seedlings were acquired. Seeds from these To plants were harvested and used to identify cold characterization. After 4℃/48h chilling stress and recovered 2 days at room temperature, most of the wild-type plants (R6547) were completely withered while the survival rate of the above transgenic strains was from 25% to 95%, the transgenic plants of S23 strain could maintain normal growth. Besides S1, S2 showed 20% and 25% of survival rate, that of other strains were more 60%. So over expression of qRC10-2 could enhance the ability of withstand chilling injury.In addition, BC5F3 near-isogenic lines (R1-2 and R2-2) with qRC10-2 present cold-tolerant charactation under cold water irrigation (5℃) and natural field identification (night average temperature of 12～15℃) at mature stage. It was suggested that flag leaf chlorophyll content of R1-2 and R2-2 was significantly higher than that of the plants without qRC10-2 (Nanjingll, R1-1 and R2-1) after suffered cold stress, their matured grain weight was lower than R1-2 and R2-2. Therefore, qRC10-2 was a locus controlling rice cold tolerance at seedling and mature stages, it could reduce chilling injury of root cell membrance at seedling stage and guarantee grain filling at mature stage. This study provide a basis for revealing the molecular mechanism of rice cold tolerance and some stable loci for improving rice cold tolerance by marker-assisted selection... |
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