Map-based Cloning And Functional Analysis Of The Dwarf Gene D62 And Glabrous Gene Gl1 In Rice

Dwarfism is one of the most important agronomic traits in crop breeding because dwarf cultivars are more resistant to lodging. During the famous "green revolution", dwarfism was adopted to improve crop architecture which significantly increased the grain yield worldwide. In rice, more than 60 dwarf mutants have been identified up to now and several of them have been characterized thoroughly. There are various factors responsible for dwarfism in plants, but gibberellin (GA) and brassinosteroid (BR) are the most intensely studied factors in determining plant height, especially the GA which control diverse biological processes including seed germination, stem elongation, leaf expansion, trichome development, pollen maturation, induction of flowering and fruit development, ect., is considered the most important bioactive growth regulator in plants. The identification and characterization of a dwarf gene, d62 (dwarf62), which controlled several growth phenotypes and affected the GA pathways were reported in present experiment. The main results are the following.1. Under normal growth conditions, the d62 mutant had multiple abnormal phenotypes such as dwarfism with wide and dark-green leaf blades.2. The d62 mutant exhibited decreased levels of the endogenous GA1, and reduced activity of a-amylase in embryoless half-seeds.3. Genetic analysis indicated that the d62 mutation was controlled by a single recessive nuclear gene. The D62 gene was primarily mapped to the short arm of chromosome 6 by SSR markers RM19289 and RM19320 with the genetic distances of 0.7 and 1.1 cM respectively, and was subsequently fine-mapped to a region of 131 kb using newly developed molecular markers. Within the 131-kb region, a putative gene (IRGSP loucs:Os06g0127800; TIGR locus:Os06g03710) that encoded a gibberellin response modulator of GRAS transcription factor was considered as the optimal candidate for D62 gene. Sequence analyses revealed a 2-bp deletion of GC in the open reading frame (ORF) region of the D62 gene that caused frame shift mutation in the d62 mutant. Transformation of D62 gene into d62 mutant rescued the dwarf phenotype of this mutant.4. The D62 gene encoded a protein of 617 amino acid which contained conserved motifs shared among the GRAS proteins and showed homology (33～37% identity and 49～53% similarity) with DELLA, DELLA-like and SCR subfamily of GRAS superfamily.5. RT-PCR analysis and promoter activity analysis all indicated that the D62 gene was expressed in all tested tissues including roots, stems, leaves and panicles of rice plant. 6. The results from RT-PCR and Real-time qPCR analyses indicated that the expression levels of GA biosynthetic genes including OsCPS1, OsKS1, OsKO1, OsKAO and OsGA20ox2/SD1 were up-regulated in the d62 mutant, further indicating that the D62 gene involved the GA pathways.In present experiment, characterization and fine mapping of the gl1 (glabrous leaf and hull-1) gene in rice mutants were studied. Glabrous leaf and hull in rice were useful agronomic traits in facilitation of harvest and follow-up process. So these traits were well utilized in rice breeding programs. Although the gene(s) controlling glabrous leaf and hull phenotypes in glabrous varieties had been named gl1 and primarily mapped on the short arm of chromosome 5 in the previous genetic studies, it has not been fine mapped or cloned till now. A map-based cloning strategy was applied to identify the target gene and the gl1 gene had been isolated and sequenced in present experiment. The main results are the following.1. The gl1 mutant produced smooth leaves and hairless glumes under normal growth conditions. By analyzing through scanning electron mic roscope, it was revealed that the leaf trichomes, including macro and micro hairs, were deficient in the mutant.2. Genetic analysis indicated that the gl1 mutation was controlled by a single recessive gene. Using nine SSR markers and one InDel marker, the gl1 gene was mapped between RM1200 and RM2010 at the short arm of chromosome 5, with genetic distances of 1.0 and 1.0 cM respectively. This mapping result was consistent with the mapping of gl1 in previous studies. To facilitate the map-based cloning of the gl1 gene,12 new InDel markers were developed. A high-resolution genetic and physical map was constructed by using 1,396 mutant individuals of F2 mapping population. Finally, the gl1 gene was fine mapped in 54-kb region containing ten annotated genes.3. The sequencing of the target region from 4 gl1 mutants (gl1-1, gl1-2, gl1-3 and gl1-4) and 4 glabrous rice varieties (Jackson, Jefferson, Katy and Lemont) all showed that the same single point mutation (A→T) occurred in the 5'-untranslated region (UTR) of the locus Os05g0118900 (corresponding to the 3'-UTR of STAR2).4. RT-PCR analysis of the locus Os05g0118900 revealed that its mRNA expression level was normal in the gl1 mutant. RNA secondary structure prediction showed that the single point mutation resulted in a striking RNA conformational change.