| The Setaria genus is increasingly of interest to researchers, as its two species, S. viridis and S.italica, are being developed as models for understanding C4photosynthesis and plant functionalgenomics with the release of the whole genomic sequences of the two S. italica accessions, however,there is no reports about gene clone and functional analyses in S. italica.Plant height, one important agronomic trait, has a large effect on yields of S. italica. Output can beimproved by reducing the plant height, due to enhanced lodging-resistant,“The green revolution” haslargely improved the yield of wheat and rice successively by breeding for ideal dwarfness. Characterand isolation of dwarf genes in foxtail millet can contribute to understand mechanism of dwarf andprovide useful dwarf genes for the green revolution of foxtail millet.84133is a spontaneous mutant detected in the field in1979with a phenotype of severely shorteninternodes, dark green leaf, and intensive to exogenous GA treatment. Genetic analysis revealed that themutant84133contains a semidominant dwarf gene, we narrowed the dwarf gene to the region flankedby the maker Si332and Si188, and then isolated the dwarf gene, designated it as SiDw1-2. SiDw1-2is ahomologous gene of GAI/RGA in Arabidopsis, SLR1in rice, D8in maize, Rht in wheat and belongs toDELLA gene in GRAS family. Sequencing analysis showed that the coding region of SiDw1-2wasinserted by an about5.5kb copia retrotransposon, the transcript of SiDw1-2was break up into twosegments, designated as SiDw1-c2and SiDw1-c3, SiDw1-c2was a chimeric transcript including the5′terminal of DELLA gene and partial LTR region of copia, the transcript SiDw1-c3was a truncated onewith the deletion of5′terminal of DELLA gene.The PCR amplification and Southern blot showed that the mutant84133genome also contained anintact DELLA gene locus with no insert or deletion except for SiDw1-2，we designated it as SiDw1-1.Linkage analysis revealed that SiDw1-1was linked with SiDw1-2, and then we confirmed that SiDw1-1carried at least10kb original sequences including5.5kb promoter sequences and2.5kb3′terminalsequences by nesting PCR. We used Southern walking to confirm the presence of the two copiesinvolving DELLA gene and infer one possible breakpoint between region probe8/9to probe7. Inaddtion, we have identified two recombination hot-spots in the putative breakpoint interval withsequence CCTCCCT (ch9:7637259-7637265), CCCCTGCCCGC (ch9:7637667-7637677). Combinedthose datas, it is very possible that the novel locus involving SiDw1-1was generated through themechanism of non-allelic homologous recombination due to misalignment after copia insertion.We detected the transcript of SiDw1-1and named SiDw1-c1, the expression of SiDw1-c1wasobviously reduced compared to SiDw1-c3and specifically expressed in elongated internodes and leaf.Furthuremore, the mRNA level of SiDw1-c1was obviously up-regulated after exogenous GA3treatment, indicating it also repressed GA response. We performed functional analysis byoverexpression the three transcripts in rice and foxtail millet, plant overproducing intact SiDw1-c1exhibited a mild dwarf phenotype, yet introduction of the truncated one SiDw1-c3resulted in severe and representative dwarf plants with the phenotype of shorten internodes and dark green leaf as those of84133, the chimeric transcript SiDw1-c2was functionless. All these datas demonstrated that SiDw1-c3and SiDw1-c1co-underlied the plant height of mutant84133and the former played a major role. |
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