Map-based Cloning Of The Dwarf Gene D1-a In Rice

Plant height is a key and crucial agronomic trait, which is directly bound up with grain production, photosynthetic efficiency and lodging resistance of rice(Oryza Sativa.L). Therefore, identification and function analyze of new dwarf gene is significant to reveal the dwarf mechanism and increase the yield of rice.A dwarf mutant which has the similar phenotype as the brassinosteroid-deficient mutant or brassinosteroid-insensitive mutant, including specific shortening of the second internode, dark green leaves and small round grains was indicated in this study. Genetic analysis carried out on the cross combination between the mutant and rice Japonica cv. Nipponbare showed the dwarf phenotype was controlled by a single recessive gene. Map-based cloning result shows that the dwarf gene was located on chromosome 5, between two SSR markers RM289 and RM18565, and co-segregation with the SSR marker RM18453.According to related references, we found it that there is a recessive gene DWARF 1 (D1),which controlled plant height and located in this region had cloned. With sequencing result indicated that this mutant has 102bp deletion, exactly the whole exon5, in D1 cDNA, we confirmed that this gene is a allele of D1 and we named it dl-a.To clone the whole dl-a gene and reveal the allelic relationship between Dl and dl-a, we designed 14 primers according to the Dl sequence from Gramene database, then amplification and sequencing results confirmed that dl-a is new allele of Dl whose mutation pattern has never reported.First, both D1 gene in wild-type and d1-a mutant has inserted a short sequence, including the 3’splice site of the third intron, the whole fourth exon, the fourth intron and the fifth exon of D1 gene. Though the repeated sequence has 35bp deletion and change the base GCCATT to GCTGATT in the fourth intron, this insertion had no effect to transcript Dl gene to a normal D1 mRNA in wild-type. Besides, dl-a gene changes 3’ss ’AG’ to ’AC’ of the third intron from the insertion.Second, in wild-type we can clone 2 complete copies of fifth exon from both the insertion and the D1 sequence, and in d1-a mutant we only clone 1 complete copy of fifth exon from the insertion, and part of fifth exon near the 5’ss of the D1 sequence. Due to failing to do the amplification, it became difficult to obtain the whole sequence of dl-a gene.Third, comparing to D1 cDNA sequence from Gramene database, d1-a has the whole fifth exon was deleted in cDNA sequence, while the DNA d1-a has 1 complete copy and 1 incomplete copy of the fifth exon. These result imply that the mutation is not caused by deletion of the fifth exon in DNA sequence.According to these result of the above, we inferred the major reason for the mutant mutate which combine to delete the fifth exon in cDNA from dl-a. it associates with the region which we fail to amplification, for example, a long insertion in the fifth exon cause abnormal amplification and splicing.A hypothetical model of D1 gene in WT and d1-a has given in this research, but the splicing mechanism still needs further research.The expression between dl-a and wild-type in the leaf indicate that the mutation does not cause a significant effect the expression in dl-a mutant.Interaction analysis, among three grain length gene Dl, GS3 and qGL3, carried out on the cross combination between the dl-a and rice indica cv.LPBG08 showed that gs3 is epistatic to dl-a and there may be some other major gene which control grain length in F2 population.