Genetic Analysis Of Heading Date Of Japonica Rice Cultivars In China,Map-based Cloning And Functional Analysis Of OsHAP3H For Heading Date And Yield Traits In Rice (Oryza Sativa L.)

Heading date （HD） determine the grown regions of rice cultivars. The yield of rice is the most concerned trait by people. Therefore, high-yield cultivars combining with moderate growth duration is a major target in rice breeding. Heading date is mainly affected by three factors, i.e. duration of the basic vegetative growth （BVG）, photoperiod-sensitivity （PS） and temperature-sensitivity （TS）. Diversification of heading date resulting from the complexity of the genetic mechanism of heading date provides abundant resources for rice breeding in different habitats. However, it also synchronously enhances difficulty in breeding rice cultivars with optimum growth duration. Rice yield is mainly determined by effective panicles, grain number per spike and grain weight. The traits about plant type, such as plant height, tiller number also very important for rice yield. In the present study, the BVG, PS and TS of HD of Japonica rice cultivars of different region of China were investigated. Genetic analyses were conducted on these cultivars using a set of heading date tester lines. QTL analysis of heading date was conducted using a recombinant inbred line （RIL） population derived from the cross of Asominori （japonica） and IR24 （indica）. The qDTH-8 was fine mapped and OsHAP3H, the candidate gene of qDTH-8 was cloned. The results showed that OsHAP3H was very important for the heading date, plant height and yield. At last, we also discussed how to breed optimum heading date for rice cultivars using known genotypes or QTL information. The main conclusions are as follows:1. The 83 Japonica cultivars were grown under artificial short-day （9h light/15h dark, SD） and natural long-day （14h light/10h dark, ND） conditions in the field near Nanjing, and in greenhouse （high temperature, HT） and under natural low temperature condition （LT） during the winter at a site in Hainan province. The BVG, PS, TS of these cultivars were investigated. The results showed that there was a significant positive linear correlation between HD and PS or BVG. The diversification of heading date of Japonica rice cultivars in China was determined by the PS and BVG of cultivars.2.83 Japonica rice cultivars grown in different ecological regions in China were genotyped for heading date using a series of heading date tester lines-EGO, EG1, ER, LR, T65m and T65Ebm, NIL（Hdl） and NIL（Hd2）. The results showed that the different genotypes on E1, Ef-1 played an important role in the diversification of heading date of Japonica rice cultivars. When cultivars carried the recessive photoperiod insensitivity allele e1, their PS and BVG were weaker, and their heading date was shorter. When cultivars carried the photoperiod sensitivity allele E1 or E1t, their PS and BVG were stronger, and their heading date was longer. The BVG of cultivars were weaker and their heading date of were shorter, when these cultivars carried Ef-1, while the BVG of cultivars were stronger and heading date were longer, when these cultivars carried Ef-1’or ef-1.The distribution of different genotypes of every major heading date gene in different ecological regions in China were different. At E1 locus, the cultivars from northeast region of China mainly carried the e1; the cultivars from northwest, north, southwest, southeast regions of China mainly carried the alleles E1 or E1t. At Se-1 locus, most cultivars in China ware Se-1e, small part cultivars carried the Se-1n or Se-1u. At Ef-1 locus, most cultivars in China carried the dominant early heading allele Ef-1, while, some cultivars from southwest region carried the Ef-1t. At Hd2 locus, most cultivars from southwest region were hd2, the cultivars of other regions, some were hd2, and some were Hd2.3. Heading date QTL were mapped in a recombinant inbred line （RIL） derived from the cross Asominori （japonica）×IR24 （indica） for five years in Nanjing. At total,7 QTLs were detected on chromosome2,3,6,8 and 12. The qDTH-8 which values of LOD and contribution rate were biggest, on the short arm of Chr.8 can be detected in the five years. We believe it was a stable expression QTL. In Nanjing nature long day condition, the heading date of CSSL61, which beared the IR24 allele at qDTH-8, was shorter than Asominori. The plant height of CSSL61 also was lower and the yield of CSSL61 also was poorer than Asominori.4. Based on F₂ populations derived from the cross of CSSL61 and Asonimori, qDTH-8 was narrowed down to a candidate genomic region of 47 kb long defined by Indel markers Ind8-47 and Ind8-15 on chromosome 8. Base on the sequence difference, we believed that the gene OsHAP3H in this chromosome region was the Candidate gene for qDTH-8. OsHAP3H showed high homology to a CCAAT-box-binding transcription factor subunit. in IR24 the function of OsHAP 3H was loss, because of deletion of 1bp combined with a frame shift and premature termination. Then some expression vectors were constructed for functional verification of OsHAP3H.5. Semi-quantitative RT-PCR analysis showed that OsHAP3H can expression in various tissues or organs. Transient expression in onion epidermal cell showed that GFP-OsHAP3H was localized in the cell nuclear. The expressions of OsHAP3H present a circadian rhythmicity and the expression of OsHAP3H was much higher at dusk under LD condition. The expression of OsHAP3H in Asominori and CSSL61 was not different. The OsHAP3H had a considerable effect on the expression of Ehdl and Hd3a. OsHAP3H suppressed the expression of Ehdl in LD and SD conditions, but the expression of Hd3a was almost completely suppressed under LD.6. When the function of OsHAP3H was loss, the rice plant height was lower. The length of each internodes of CSSL61 was shorter than Asominori because of the cell length of internodes of CSSL61 was shorter than Asominori. When the function of OsHAP3H was loss, the rice yield also becomes lower because of the grains per spike and grain weight was reduced.7. In this study, we also report a conventional and marker-assisted breeding program using known gene information for selecting suitable heading date, where the breeding objectives were （1） to avoid the transgressive delayed heading in indica-japonica hybrids, （2） to increase the efficiency in selecting hybrid rice combinations with suitable growth duration to fit different habitats and regions, and （3） to develop cultivars with target growth duration using the QTL information. The results present in this study demonstrate the usefulness of known gene information in rice breeding.