Functional Characterization And Candidate Gene Analysis Of Rice Semidwarf Gene Sd12

Dwarfism is one of the most important agronomic traits in crop breeding programs.Dwarf cultivars are not only with improved lodging resistance and harvest index,but also have better responses to fertilizers（Khush,2001）.The introduction of two well-known dwarf genes,semi-dwarf1（sd1）in rice（Oryza sativa L.）and reduced height1（Rht1）in wheat（Triticum aestivum）,to create semi-dwarf varieties has greatly increased crop yields and initiated the“Green Revolution”in the 1960s.So far,sd1 is the only semi-dwarf gene used in rice breeding,and its risks are also of concern to breeders.Therefore,the exploitation,identification and utilization of new dwarf genes are the most concerned issues of breeders.In this paper,a natural dwarf mutant M22-3-2 was obtained from japonica-compatible indica line M22-3-1,containting sd1 and a new semi-dwarf gene sd12,and a series of experiments were carried out.The research results are as follows:1.Mutant M22-3-2 has phenotypes such as dwarfness,shorter leaves,increased tiller number,and reduced panicle number,which belonged to dn type.Cytological analysis showed that the dwarfism of the plant was caused by the shortening of the length of the slitted cells and the reduction of the number of cells.2.Mutant M22-3-2 was used to study the endogenous hormone levels and stress responses to exogenous GA₃and BL.GA analysis revealed different concentrations of GA₃induced plant stem elongation,but no differences were detected among wild-type,mutant,and dwarf lines.Meanwhile,all three materials were able to produceα-amylase under GA₃conditions,making potato starch degradate.Endogenous GA₃levels were comparable among three tested materials,indicating that the dwarfism in M22-3-2 was not caused by abnormal GA biosynthesis and signal transduction pathways.BR analysis demonstrated that different concentrations of BL induced leaf angle response in wild-type,mutant,and dwarf lines at the same level,however,the endogenous BL test showed that the the content was low in materials containing sd12 gene,manifesting that the dwarfism was not related with BR signal transduction pathway but may be caused by the abnormal BR biosynthesis pathway.3.The offspring population generated by the cross between mutant M22-3-2 and MH63 was used to finely map sd12.sd12 was initially located between makers RM101 and RM511,with genetic distances of 8.3 c M and 8.6 c M,respectively.By expanding the population,sd12 was mapped between the markers RIDM196818 and IND13,with physical distance of around 2000 Kb including 60 ORFs.Since sd12 was near the centromere region,it was difficult to shorten the mapping region to a smaller interval.Quantitative expression analysis of 60 ORFs in the interval showed significant differences between mutant and wild type in 7 ORFs.Preliminary sequencing analysis of these 7 ORFs revealed that ORF3 and ORF4 had base sequence deletion in the mutant,resulting in amino acid alteration,which might affected its function.So we thought these two genes as the candidate genes.4.Using wild type and mutant to perform transcriptome analysis at the jointing stage,the results showed that a total of 27,695 genes were screened,and 1,207 genes were differentially expressed,of which 779 genes were up-regulated and 428 genes were down-regulated.Functional classification by GO analysis showed that up-regulated genes were divided into 431 functional groups and down-regulated genes were divided into 468functional groups.394 differential genes were involved in KEGG pathway enrichment,belonging to 87 pathways.These provide valuable information for future rice research and analysis of the role of sd12 in biological processes,functions and pathways.5.In order to further determine the application value of sd12 in breeding,the yield traits of sd12 and sd1 were compared.It was found that there were no significant differences in thousand-seed weight and grain numbers,but the effective tiller number of sd12 was significantly higher than that of sd1,which resulted in a significantly higher yield per unit than sd1,demonstrating the application value of sd12 in breeding.In this research,cytological analysis,hormonal responses,candidate genes and transcriptome analysis were performed to study semi-dwarf gene sd12.The knowledge we gained in this study would shed light on the significance in rice breeding,enrich the understanding of dwarfing mechanism in rice,and bring potential semi-dwarf resources for plant type improvement.