Mapping Of High-Tillering And Dwarf Mutant Htd1-2 In Rice And Characterization Of HTD1 Gene Promoter In Transgenic Arabidopsis

Rice is one of the most important cereal crops and about harf of the world population depends on rice as a staple food.Tillering and plant height are two major agronomic traits affecting its architecture and further determine grain yield.In order to explore the molecular mechanism of rice tillering,in the present study,a high-tillering dwarf 1-2(htd1-2) mutant was isolated from the offspring of the indica rice(Oryza sativa L.ssp.indica) variety 9311 treated with 350Gy ⁶⁰Coγ-radiation.Compared with wild-type phenotype,lower height,more tillering,thinner and smaller leaves and seeds,the delay of maturity period are all the obvious characteristics of the htd1-2 mutant.By using the method of the classical genetics,the plant heights and tiller numbers of F₁,F₂ of three different crosses(htd1-2/9311,htd1-2/tp309 and tp309/htd1-2) were studied.The F₁ generations showed wild-type phenotype,whereas,the F₂ population was segregated into wild-type plants and mutant plants.x² test indicated that the segregation followed the expected proportion of 3:1.Genetic analysis showed that both high tillering and dwarf phenotypes of htd1-2 were controlled by a single recessive nuclear gene.To primarily mapping the HIGH-TILLERING DWARF1-2(HTD1-2),116 plants,F₂ population of htd1-2/tp309,with high-tillering and dwarf and other mutant characteristics were used in the research.By linkage analysis with molecular markers,the HTD1-2 gene was firstly mapped between the simple sequence repeat(SSR) markers RM17303 and RM17321 on chromosome 4,with genetic distance of 3.1cM and 0.4cM,respectively.To make a fine mapping of the HIGH-TILLERING DWARF1-2(HTD1-2),Cleaved Amplified Polymorphic Sequence(CAPS) markers designed by Zou(2005) was consulted. By using these markers,HTD1-2 was finally mapped accurately in a DNA region of 116 kilobases which is between the CAPS A6 and E2.Within this region,there is a cloned gene HIGH-TILLERING DWARF1(HTD1),which controls rice tillering.By comparing sequences of HTD1 between htd1-2 and 9311,in combination with the results from dCAPS analysis,we believed that HTD1 is an orthologue of the gene HTD1-2.By comparing the phenotypes of htd1 mutant and htd1-2 mutant,it showed that the tiller number of htd1 is more than htd1-2 and the plant height of htd1 is also higher than htd1-2. Although htd1 and htd1-2 are the allelic mutants,htd1 and htd1-2 have different phenotypes because of different genetic backgrounds.Furthermore,removal of axillary buds proved that dwarfism of htd1-2 mutant is partly attributed to its excessive tillers.Moreover,the expression pattern of the promoter of rice HTD1 gene in transgenic Arabidopsis was studied according to the analysis ofβ-glucuronidase(GUS) reporter gene. GUS histochemical assay showed that GUS expression under HTD1 promoter occurred mainly in the vascular tissues of leaves,petioles,hypocotyls and basal main roots in the seedling stages of transgenic Arabidopsis.