Identification And Pyramiding Of QTLs For Cold Tolerance By Use Of Single Segment Substitution Lines In Rice (Oryza Sativa L.)

Low temperature injury on rice occurs in many countries across the world. Cold stress may occur at different developmental stages in rice production. Although use of cold tolerant variety has been considered the most effective way to prevent cold damage in rice,it is very difficult to breed rice variety with cold tolerance by using the phenotype-based conventional breeding methods because of the complex genetic basis of cold tolerance. It can be expected that the break through in cold tolerant rice breeding can be achieved only after fully understanding the genetic basis of cold tolerance and incorporating different favorable QTLs into one variety. In this study, for developing cold tolerant cultivar, the parents of single segment substitution line(SSSL) library with genetic background of“Huajingxian74” were used for detecting cold tolerant germplasms; then the SSSLs derived from cold tolerant donors were used for identification of stable cold tolerant QTLs; finally,pyramiding breeding were performed by using these favorable cold tolerant QTLs in rice.The main results were described as follows.1. Thirty-four varieties from 11 countries were used to screen for rice germplasm with cold tolerance at different growth stages. japonica “Katy” with the highest low temperature relatively germination rate 90.96% at the germination stage; Four japonica“Lijiangxintuanheigu”, “kongyu131”,“IR65598-112-2” and “IR66897B” with survival rate above 90.00% at the bud bursting stage; japonica “Nanyangzhan” with the highest survival rate, and two japonica “Lijiangxintuanheigu” and “kongyu131” with survival rate 81.3%,78.1 % respectively, at the seedling stage; japonica “Kyeema” with the highest cold tolerance index 0.95, indica “IR58025B” with high cold tolerance index 0.69 also at the flowering stage. Cold tolerance has no significant correlation between germination stage and bud bursting, seedling, flowering stage respectively. Cold tolerance showed a significant positive correlation between bud bursting stage and seedling stage, but no significant correlation between bud bursting stage and flowering stage; the correlation between seedling stage and flowering stage of cold tolerance is not significant.2. Five independent experiments were conducted in detection of cold tolerant QTLs at the bud bursting stage by using thirty SSSLs derived from cold tolerant donors. A total of15 QTLs were mapped on chromosome 1, 2, 3, 5, 6, 9, 10 and 12. Among them, five QTLs(q Ctp-1, q Ctp-3, q Ctp-5-3, q Ctp-6-1, q Ctp-10) had significant cold tolerant effect by t test across the five independent experiments at P<0.05; At four QTLs(q Ctp-1, q Ctp-3,q Ctp-5-3, q Ctp-6-1) the alleles derived from the donor parents increased cold tolerant effect.The QTL q Ctp-6-1 had the highest positive additive effect 17.98%.3. Five independent experiments were conducted in detection of cold tolerant QTLs at the seedling stage by using twenty eight SSSLs derived from cold tolerant donors. Two cold tolerant indicators, the degree of cold injury(DCI) under cold treatment and survival rate(SR) under normal conditions after cold treatment were used to evaluate the cold tolerance of the SSSLs. Six QTLs for DCI were mapped on chromosome 4, 6 and 12. Among them, 3QTLs(q Ctd-4-2, q Ctd-6-2, q Ctd-12) had significant positive cold tolerant effect by t test across the five independent experiments at P<0.05; The QTL q Ctp-6-2 had the highest positive additive effect 1.13; Nine QTLs for SR were mapped on chromosome 3, 4, 5, 6, 9and 12. Among them, 3 QTLs(q Cts-3, q Cts-6-1, q Cts-12) had significant positive cold tolerant effect by t test across the five independent experiments at P<0.05; The QTL q Cts-3had the highest positive additive effect 13.00%.4. Three independent experiments were conducted in detection of cold tolerant QTLs at the flowering stage by using twenty one SSSLs derived from cold tolerant donors. Six QTLs were mapped on chromosome 1, 3, 6 and 12. Among them, 3 QTLs(q Cth-6-1,q Cth-6-2, q Cth-12) had significant cold tolerant effect by t test across the three independent experiments at P<0.05; The QTL q Cth-6-1 had positive additive effect; The QTL q Cth-6-2and q Cth-12 had negative additive effect. The absolute values of additive effect of them were above 10.00%. Further, the candidate region of QTL q Cth-6-1 was narrowed down to a 45 kb DNA fragment by fine-mapping, and in this region eight putative genes were predicted.5. The SSSLs carrying cold tolerant QTL were used for pyramiding breeding. Thirty two pyramiding lines were selected by MAS. The target traits of pyramiding lines were involved cold tolerance at the germination stage, bud bursting stage, seedling stage and flowering stage. The results of cold tolerance phenotype evaluation demonstrated that the combinations of q Ctp-4/q Ctp-6-1 and q Ctp-3/q Ctp-6-1 could significantly enhance the cold tolerance at the bud bursting stage; and the combinations of q Cts-6-1/q Cts-12 and q Cts-6-1/q Cts-12 //q Cts-3 could significantly enhance the cold tolerance at the seedling stage.In this study, some cold tolerant strains were identified, the stably expressed QTLs for cold tolerance were mapped by use of SSSLs derived form cold tolerant donors. And then,the multi-QTLs pyramiding lines were developed. The results not only verified the existingof these QTLs, but also indicated that cold tolerant rice varieties could be developed through the pyramiding of cold tolerant QTLs in different growth stages. These cold tolerant QTLs detected in this study enriched the genetic basis of cold tolerance of rice.It can be expected that elite varieties with strong cold tolerance could be developed through MAS by use these stable expression cold tolerant QTLs.