Evaluation And Mapping Of Drought-tolerance Traits Of O.sativa L. And Its Application In Breeding

Drought is one of the most serious world-wide problems in agriculture, rice is the largest water consumer in crops. Development of drought tolerant varieties is largely based on the quick and precise screening of germplasm and breeding materials in water-limited environments, mapping and cloning the drought-tolerance gene, incorporating the gene of high-yield, good-quality and drought tolerance by using MAS and transgenic technology. In this study, we analysed the characteristics of drought tolerance including panicle neck diameter and panicle water potential; mapped QTLs affecting drought tolerance in a RIL population; developed four sets of QTL-NILs by MAS and evaluated their DT ability; cloned a DT gene named OsGRAS by candidate-gene strategy; and applied QTL mapping result to the breeding program by introgressing the target region from chromosome 4 into upland rice CMS maintainer line "Huhan1B". Main results of the present study are as following:1. Study on panicle water potential. Two upland varieties (IRAT109, IAPAR9) and one lowland variety (Zhenshan 97B) were grown in summer season and treated by drought stress and normal condition in reproductive stage. Panicle water potential (PWP) and leaf water potential (LWP) were measured every l-1.5h in 24h in sunny days. Both PWP and LWP of upland varieties started to decreased later in the morning, maintained in a higher level at solar noon and recover more quickly in the evening than that of lowland variety. The results showed that PWP can be used as an indicator of plant water status based on the parallel daily changes, and the high correlation between PWP and LWP. Similar correlations were also observed between PWP, LWP and seven traits about plant growth and grain yield formation. PWP seemed to be more effective to distinguish the upland rice varieties with different drought tolerant ability. Difference of PWP and LWP between upland and lowland rice varieties were also observed in solar noon even under normal water condition.2. Study on panicle neck diameter and DT target interval introgressed into "Huhan1B" by MAS. The panicle neck diameter was measured in a rice RIL population of 187 lines from the cross "Zhenshan97B/IRAT109" under different water conditions in 2003 and 2004. Panicle neck diameter was found to be significantly correlated with many agronomical traits, especially with the panicle size. It was found by path analysis that panicle neck diameter has effect on yield by increasing the spikelet number and grain number. Based on a linkage map of 213 SSR markers, three main effect QTLs were detected for PND with contribution rates varied from 5.03% to 11.01%, including one locus detected under both water conditions (qrPND-4). The chromosomal region on chromosome 4 (RM241-RM349) also hosted QTLs for seven panicle traits, including panicle number, panicle length, primary branch number, secondary branch number, spikelet number per panicle, spikelet density and grain number per panicle. Seventeen epistatic QTLs were detected for PND under well-watered and stress conditions. The contribution rate of each pair of epistatic effect varied from 2.58% to 5.64%.During three times backcross procedure, positive individuals were selected in the progeny by foreground markers for further crossing or selfing. The target interval of chromosome 4 from IRAT109 was introgressed into an upland maintainer line "Huhan1B". It was found that introgressed lines have more grain number, higher yield and thicker panicle neck diameter but less panicle number than "Huhan1B" under drought stress.3. NIL Development and gene cloning of OsGRAS by candidate-gene strategy. Five QTLs controlling several DT traits was selected according to the mapping results for two years. NILs was developed by crossing and continuous backcross, and progeny selection by both foreground and background markers. The drought tolerance ability was evaluated for four populations of QTL-NILs. The NILs has higher yield and spikelet fertility than Zhenshan97B as the recurrent parent. Fine mapping was done to narrow down the interval by adding more SSR and ILP markers for the interval RM273-RM255 on chromosome 4 and RM134-RM420 on chromosome 7. For the target interval of chromosome 4, QTLs of both grain number and grain yield located in the interval RM17355-RM17352 (95kb), QTL of panicle number and spikelet number located in the interval RM5320-RM17355 (609kb), QTL of spikelet fertility located in the interval RM17183-RM17190 (227kb). For the region RM134-RM420 of chromosome 7, we found that QTLs influencing both grain number and spikelet fertility were located in RM134-RI04226 (83kb), spikelet number and grain yield located within RM1306-RI04046 (446 kb).Based on the sequence of candidate gene, full-length cDNA and promotor sequnece was obtained by primer design and PCR amplification. OsGRAS is a member of the GRAS Family of transcriptional regulators. OsGRAS has the typical domain of GRAS family accoding to the conserved domain alignment.Three expression vectors of OsGRAS were constructed by enzyme digestion and vector ligation.