| Hot disaster appears more and more frequently with the rapid development of global economy and the increasing of carbon dioxide emission year by year, leading to the decline of crop production and product quality when it becomes more serious. An urgent and significant research issue is put into place on how to stabilize and improve the yield of crop, and how to ensure agricultural economy and grain security in agricultural science domain in the face of such cultivation adversity. Rice is the most important crop in the word as it is the major source of food for more than half of the word population. At present, there are a wide range of researches on high temperature-stress responses and heat tolerance characteristics of rice, including the variation of the protein expression profile, the physiological and biochemical reactions, location of Quantitative Trait Locus (QTLs) responsible for high temperature tolerance, et al. However there is no report on map-based cloning of heat tolerance so far. The reported genes, related with heat tolerance, are homology-based cloning method to get and most are heat shock proteins.The plant materials used in the present study include HT54, a heat tolerance cultivar, and HT13, a heat sensitive cultivar, which were provided and identified by Xuhua Zhong a research fellow from Rice Research Institute of Guangdong Academy of Agricultural Science. We attempted to establish high temperature resistance evaluation procedures as well as its assesment criteria at seedling stage, and on this basis, to further conduct inheritance analysis, gene mapping and physiological index assessment of high temperature resistance present in HT54. The main results detained are as follows:1. The establishment of high temperature resistance evaluation procedures and its assessment criteriaHT54and HT13, both cultivated by nutrition solution or soil, were removed into growth cabinet and treated by42℃,45℃or48℃at two to three leaves or three to four leaves. The relative humidity was set at75%and the other cultural conditions were kept on the same level, and after that, the response on high temperature was observed at a certain period of time. The results showed that HT54, cultivated by nutrition solution, were all survive and HT13were all dead after84h48℃high temperature treatment at two to three leaves, but it needed only79h to achieve the same effect for soil culture. Both two culturing methods could be used in the high temperature resistance evaluation procedures, but the latter was highly efficient as compared with the former one. The obvious differences were observed after72h45℃high temperature treatment at the tillering stage. Although it solved the limits of sample for DNA isolation because of the death, more time and more work were needed to select and cultivate tillerings. At the heading stage, seed setting rate reduced vigorously after48hours under36℃high temperature and the varying degree of variance reached5%significance between HT54and HT13cultivars, but the rate of HT54after high temperature was significantly lower than that under natural condition. Therefore heading stage was not the optimal stage for high temperature resistance evaluation of HT54. According to these results, we established the high temperature procedures at seedling stage:two to three leaves, cultivated by soil or nutrition solution,84h or79h48℃high temperature treatment with75%air relative humidity, evaluation after5day recovery after treatment as the assessment criteria of high temperature resistance.2. Genetic analysis of high temperature resistanceF2population was made by pollination of HT54with HT13. Regarding to the assessment criterion, the resistance response of F1plants was consistent with high temperature resistance cultivar HT54and the segregation of F2progeny was consistent with3:1ratio after high temperature treatment. These results thus demonstated that the high temperature resistance presence in HT54at seedling stage was controlled by complete dominance of monogenic allele, named OsHTAS (Oryza saliva heat tolerance at seedling stage).3. Mapping of high temperature resistance geneThe mapping population was constructed with131sensitive plants randomly selected from F2generation according to the bulked recessive extreme segregats strategy. At the same time, two DNA pools for primary linkge analysis were also constructed with DNA samples extacted from10extreme sensitive and resisant plants, respectively, which were randomly selected from the same F2population. The high temperature resistance gene OsHTAS was then mapped using these populations. The results showed that the marker RM444, position on chromosome9, had the polymorphism between two DNA pools. This result was preliminarily confirmed by marker screening of61high temperature sensitive plants. Further marker density increasing analysis with61sensitive plants and an enlarged F2population containing131recessive extreme segregants demonstrated that the OsHTAS is located between markers InDel5and RM7364. The map distance between OsHTAS and the two closely linked markers is2.5cM/3.2cM and1.7cM/1.2cM, respectively. The physical distance between the two markers InDe15and RM7364is420kb. Two genes were selected and consequently sequenced according to deduced function analysis of cadidate genes within the interval of these two markers as well as previously reported chip expression data. Two single base pair (SNP) differences on the resultant sequences of of one gene between HT54and HT13were detected and one of these SNPs was developed into CAPS marker. Further analysis revealed that this CAPS marker was cosegregated with OsHTAS gene.3. High temperature resistance related physiological index SOD and POD assayThe SOD and POD activity in the seedling samples of HT54, HT13were assayed at different time points after high temperature treatment at stage of two to three leaves and the currently well-known high temperature resistance cultivar Huanghuazhan was used as control. The result showed that the SOD and POD activity detected at different time points was stimulated to increase during heat stresses. But in case of activity detection value, the HT54was in the middl among three cultivars. In addition, these results also showed that under heat stress conditions, the relative degree of variation of the POD and SOD activity in Huang Huazhan and HT13was inconsistency. The SOD activity of HT13was the lowest but its POD activity appeared the highest among three cultivars except one case lower than Huanghuazhan at48℃. Therefore, all these results indicated that the high temperature resistance of the tested varieties could not be evaluated by simply detecting the SOD and POD activity. |
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