Map-based Cloning And Functional Analysis Of DTH7 For Grain Yield And Heading Date In Rice (Oryza Sativa L.)

The yield of rice is the agronomic trait that most concerned by people, and optimal flowering time (or heading date in cereal) is an important index which determines the high and stable yield of rice, so high-yield cultivars combining with precocious trait is a major target by rice breeders. This study first reported an association analysis between the flowering time and PS (photoperiod sensitivity) and the trait of yield and the flowering time in 91 accessions of the rice germplasm collected from 17 Asian countries and regions, and used one near isogenic line (NIL) named QTL-qDTH7 among the 38 lines which carrying chromosome segments of PA64S in Kitaake background to further examine this notion. Then fine-mapping of QTL-qDTH7 simultaneously controling heading date, plant height and yield potential of rice was carried out using map-based cloning and its candidate gene OsPRR37 was characterized. The results showed that OsPRR37 was very important for the heading date, plant height and yield potential of rice. Moreover, the relationship between the different allelic combinations of the three major flowering QTL loci Ghd7, DTH8 and DTH7 which reported simultaneously regulate heading date, plant height and yield potential of rice were analyzed and a strong correlation were found between their different functional combinations and heading date of rice. The main conclusions are as follows:1.91 rice varieties gathered from 17 Asian countries and regions were planted in four different locations (Sanya, Nanjing, Beijing and Harbin) across the country in a suitable local time respectively and investigated the days to heading of each variety and the grain number of the main panicle (an index of rice yield) as well. Through statistical analysis, with the extension of the daylength, the heading date of rice gradually delayed, and the number of variety never heading is increasing with the latitude gradually become higher, and there is a noticeable correlation between the yield of rice and the heading date.2. Heading date QTL-qDTH7 was mapped in a near isogenic line (ML) carrying 1.05-cM chromosome segment containing DTH7 of PA64S in Kitaake background. With the extension of the daylength, the heading time of Kitaake and NIL-DTH7 plants becomes longer, but the changes between NIL-DTH7 plants are greater than that of Kitaake; and both of the yield of NIL-DTH7 and Kitaake becomes higher and higher, but compared to Kitaake, the difference between the NIL-DTH7 lines is greater.3. Based on the F2 and subsequent F2.3 populations derived from the cross of NIL-DTH7 plants and Kitaake, qDTH7 was narrowed down to a candidate genomic region of 114 kb between molecular marker IND-36 and the end of the long arm of chromosome 7. When compared the full genomic sequence of OsPRR37 between NIL-DTH7 and Kitaake, a total of ten amino acid mutations in which the amino acid L changed into P in the conserved CCT domain was found. So OsPRR37 was believed to be the candidate gene for qDTH7, and the amino acid change in the CCT domain of Kitaake be the main cause of the phenotypic differences between NIL-DTH7 and Kitaake.4. To further confirm the function of OsPRR37, three types of vectors were constructed as follow: a full-length genomic sequence of OsPRR37 for complementary, its own promoter plus the CDS of OsPRR37 which were transformed into rice variety Kitaake and the CaMV35S over-expressions OsPRR37 which was transformed into Arabidopsis col-0 respectively. The results showed that the full length genomic fragments can make Kitaake obtain the phenotype of grain yield, plant height and heading time of the NIL-DTH7 plants but the differences of these traits between the transplants with its own promoter plus the CDS of OsPRR37 and Kitaake are not significant; meanwhile, over expression of OsPRR37 transgenic Arabidopsis plants showed earlier flowering phenotype both under long day and short day conditions than the wild type plants.5. Real-time PCR analysis showed that, DTH7 is expressed in various tissues or organs, and its transcript level is most active in young leaves with a light dependent manner, and is regulated by the circadian clock with an expression peak after 8 hours of light both under long day and short day conditions; transient expression in rice protoplasts showed that DTH7 was localized in the nucleus and DTH7 exhibited transcriptional repression activity in yeast. The expression level of DTH7 in Kitaake is higher than that in NIL-DTH7 plants, and the former is almost twice of the latter. DTH7 mediated floral repression signal from OsphyB to the ’florigen’ genes’ up-regulator, Ehdl, to delay flowering and increase grains per panicle under long day conditions, and its expression was induced by OsPhyB under short-day conditions. All the results above showed that DTH7 played a very important role in the photoperiodic flowering pathway in rice.6. DTH7 played an important role on the panicle and stem development in rice. DTH7 increased yield of rice through increasing the primary and secondary branches of rice. Weakening or loss-of-function of DTH7 will make the stem of the rice plants smaller and each section significantly shorter, and cytological observations suggest that the shorten cells between internodes is the direct cause of the dwarf phenotype of Kitaake. The weakening or absent function of DTH7 leads to rice production declined, mainly as a sharp reduction in grain number per panicle with grain weight not changed.7. Kitaake and NIL-DTH7 plants were sowed under different controlled day length conditions, and the downstream genes in the photoperiodic flowering pathway of DTH7 in rice was analysed using Real-time quantitative PCR. The results showed that, with the extension of the daylength, downstream genes of DTH7 promoting flowering in rice were inhibited by DTH7, which was correlated to the phenotypic variation under different natural conditions.8. DTH7 is one of the five members of the rice PRRs gene family, and the expression of Arabidopsis PRRs gene family existed the expression order patterns as follow:PRR9→PRR7→ PRR5→PRR3→PRR1. After the continuous light or continuous dark treatment of Kitaake and NIL-DTH7 plants, there also existed the expression order patterns in rice PRRs gene family as follow: OsPRR73→OsPRR37→OsPRR95(OsPRR59)→OsPRR1.9. The relationship between the different allelic combinations of three major flowering QTL loci Ghd7 and DTH8 which simultaneously regulated rice yield, plant height and heading date and DTH7 which exhibited the same function on these traits showed that the functional haplotype of the three genes had a good correlation with the heading phenotype, and there had the same trend in the four different locations nationwide; meanwhile, the analysis showed that they all had large contribution to the heading date in rice. The contribution became larger with the number of the functional ones among the three genes increased, indicating that they had a certain synergy in the photoperiodic heading date regulation in rice.