MAPPING AND MOLECULAR CHARACTERIZATION OF DWARF (OsGAIISd) INSERTIONAL MUTANT IN BASMATI

Abstract

Rice (Oryza sativa L.) is a staple food for half of the world population and its production was almost doubled due to cultivation of semi-dwarf cultivars containing sdl gene form Dee-Geo-Woo-Gen (DGWG) source. Besides its commercial importance, rice is a model monocot for plant biotechnologists due to the availability of its complete genome sequence, its small genome size (-389 Mb) and its efficient tissue culture response. Basmati 370 is the first wonder Basmati variety which was released during preindependence era and continues to reign till today. But it lodges due to its tall stature, resulting in its poor grain yield. In the present study, a T-DNA insertional dwarfmutant (OsGAI/Sd) of Basmati 370 with approximately half the plant height as compared to Basmati 370 was isolated by T-DNA transformation containing HmRDs cassette. It was found to be insensitive to exogenous Gibberellic acid (GA3) application. About 55-60% of the plants carrying the hpt gene showed dwarfand GA-insensitive phenotype which can be due the presence of incomplete penetrance and variable expressivity of the mutant. Incomplete penetrance in this context refers to the presence of dwarf phenotype in 55- 60% of the plants carrying the hpt gene in homozygous condition while variable plant height from severe-dwarf, dwarf, semi-dwarf to tall individual plants in the selfed progenies and GA3 sensitivity from 2-30% in between mutant and Basmati 370 indicated variable expressivity. The dwarfing gene OsGAI/Sd had pleiotropic effects for reduced seed size, tillering, panicle length and fertility. Another insertional semi-dwarf mutant OsGAS/Sd was found to be sensitive to exogenous GA3 application at 120 ppm concentration. Presence of T-DNA in the dwarf, semi-dwarf and another insertional mutant B-3-1 was confirmed by PCR amplification of hpt gene used as the selectable marker in HmRD5 construct of the T-DNA. The seeds of mutants were also resistant to hygromycin at 80 ppm concentration during germination in petri-plates. Single copy insertion of T-DNA in OsGAI/Sd was confirmed by Southern hybridization and 3:1 segregation of hpt gene in 164 F2 plants obtained from OsGAI/Sd X PR106 in PCR amplification and germination on hygromycin at 80 ppm. Total leaf chlorophyll content was found to be more in the dwarf mutant. In histological studies, the cell size of the OsGAI/Sd was found to be reduced due to loss-of-function of the gene in the mutant. 164 F2 progenies of OsGAI/Sd X PR106 cross segregated in 3:1 ratio for GA-insensitive vs. GA-sensitive phenotype, whereas 107 F2 plants of OsGAI/Sd X Basmati 370 segregated in 3:1 ratio for dwarfvs. tall phenotype. Out of 213 rice mapped SSR markers, 95 showed parental polymorphism between Basmati 370 and PR106. Based on the data of recombination frequency in the mapping population of OsGAI/Sd X PR106 cross, the anchored SSR markers RM14645 and RM14667 of chromosome 3 mapped at distance of 1.21cM and 6.49cM, respectively from the T-DNA insertion containing hpt gene. The TDNA flanking region isolated through TAIL-PCR showed a single hit on chromosome 3 in BLASTN with the total japonica cv. Nipponbare genome sequence present in NCBI database. Using PCR genome walking, the T-DNA flanking region of the OsGAI/Sd could not be amplified due to lack of appropriate restriction sites near the T-DNA insertion. The T-DNA insertion was found at the second exonic region of a gene which encodes for sixth subunit of Anaphase Promoting Complex/Cyclosome (APC/C). The APC/C plays role in the protein degradation of several proteins through ubiquitinproteasome mediated proteolysis pathway. The 8.6 kb of the candidate gene encodes 728 amino acid protein containing a conserved Tetra Tricopeptide Repeat (TPR) domain. The only paralog of APC/C in rice, isopencillin N-synthase family protein on chromosome 3, was without the TPR domain. The ortholog of Anaphase Promoting Complex (APC) n component 6 in Arabidopsis thaliana showed highest identity with the candidate gene. In the different models of GA-signaling proposed so far in rice, the SCFGID2 complex mediates the degradation of a DELLA protein SLR1 which in turn switches on the GAdependent growth. The present study reports a novel function of APC/C holo-enzyme in Basmati rice which may degrade the SLR1 protein via ubiquitin mediated proteolysis pathway. Through RT-PCR, the expression of wild type gene was found in both roots and shoots. Four Flanking Sequence Tags (FSTs) of the candidate gene OsGAI/Sd were found in OryGeneDB, whereas two FSTs were found for OsGAS/Sd in RiceGE database. FST along with RNAi mediated gene silencing approach can be used for the functional validation of the target gene in both the GA-insensitive and sensitive mutants of Basmati