MOLECULAR MAPPING AND CLONING OF POLYEMBRYONY (OsPE) INSERTIONAL MUTANT IN BASMATI RICE

Abstract

In plant functional genomics, mutants play an important role in studying gene functions. Rice (Oryza sativa L.) is recognized as an important model plant among cereals because of its small genome size and the development of efficient transformation system. T-DNA insertional mutagenesis is one of the most important approaches of finding and cloning new genes. It is believed that the T-DNA insertions in the genome are random, and that the insertions can be stably transmitted. Analysis of the flanking sequences of three T-DNA/Zte insertional mutants of Oryza sativa, cultivar Basmati 370: B-4-1 (Polyembryony; OsPE), B-2-2 and B-8-7 has been done. Sequence analysis showed that the T-DNA insertion was in the promoter region in OsPE, in the coding sequence in B-2-2 and in the junk region of B-8-7. PCR amplification with 950bp hpt gene amplification (used as selectable marker during transformation) confirmed T-DNA insertions in all the three mutants and Southern blot analysis showed that the mutant phenotype in polyembryony is due to single copy T-DNA insertion. The present investigation aims at mapping and cloning the candidate gene for multiple embryo formation in one of the independent T-DNA insertional mutants. OsPE mutant represents the first reported example of high frequency and heritable polyembryony in rice caused by insertional mutagenesis. Polyembryony has been described previously in several crops and cereal plants, but the underlying genetic causes of twinning in such cases have been complex and difficult to resolve. This homozygous and fertile polyembryonic mutant showed resistance to hygromycin VI and the twin/triplet seedlings at afrequency of 15-20* Multiple embryos with independent root and shoot axis were attached to single scutellum. Amapping population was developed by crossing OsPE with anon-Basmati fine rice cultivar PR106 and alarge F2 was obtained. Morphological data for plant height, number of tillers per plant and hpt gene amplification was collected for F2 population and for hpt gene tf. „56 at ,dfand p< 0,5, The ^ phenotypic ^ for polyembryony unlike hpt data did not segregate in the expected 3:1 ratio. Out of 175 F2 plants only 14 showed polyembryony (twin/triplet) phenotype. All the 14 plants which showed polyembryonic phenotype gave PCR amplification for hpt gene as well. This may be due to incomplete penetrance and variable expressivity in the inheritance of polyembryonic phenotype in F2 population. Penetrance here refers to the presence of polyembryony while expressivity as single, twin, triplet and quadruplet embryos. The OsPE mutant was highly fertile with somatic chromosomal number 2n= 24 and normal 12 lis at meiotic metaphase I. Aset of 98 markers showing polymorphism between parental lines, Basmati 370 and PR106 were used in the OsPE/PRl06 mapping population for identifying markers associated with the OsPE gene through Bulk Segregant Analysis. RM14645 (5.79cM) and RM14667 (2.17cM) markers showed linkage with OsPE gene on chromosome 3. The mapping of OsPE on rice chromosome 3using BSA has been further confirmed with other reverse genetic approaches including genome walking and TAIL-PCR involving amplification of T-DNA flanking region using T-DNA and adaptor based primers. To carry out genome walking and TAIL-PCR, nested primers were designed for T-DNA right and left borders. Using T-DNA right border nested primers along with adaptor primer (Genome Walking) and arbitrary degenerate primer (TAIL-PCR), VII PCR amplification was obtained. The PCR product was purified and sequenced. The location of OsPE gene on chromosome was determined using japonica rice cultivar Nipponbare sequence. Sequencing with T-RB2/AP2 ( Genome Walking) and T-RB3 (TAIL-PCR) showed significant alignment with the Oryza sativa cv. Nipponbare with only single hit at chromosome 3. The T-DNA insertion is present in the promoter region of the candidate gene Os03g0241300. The length of the candidate gene is 2.8Kb with 2 UTRs (1473bp and 193bp), 2 Exons (154bp and 935bp), and one Intron (81bp). To confirm the insertion on chromosome 3, primers were designed for rice genome flanking the T-DNA borders. PCRwas done in combination with the T-DNA (T-RB) based primers and genome-specific primers. Amplification of expected size was obtained in polyembryonic mutant but not in Basmati 370. PCR with genomespecific primers gave amplification in Basmati 370 and not in OsPE because of the insertion of approximately 10Kb fragment of the T-DNA cassette. All the 14 polyembryonic hpt positive F3 progenies also showed amplification with the T-DNA specific (T-RB1) and genome specific (PE-RB1) primers. The protein searched corresponding to the OsPE candidate gene sequence has been reported as a hypothetical protein (Os03g0241300) in Oryza sativa. OsPE gene lacked functional homologs in other species. Although using KEGG search around 401 sequences orthologous to OsPE were found through out the living kingdom but majority had low identity value (> 40%). It most closely matched with the Vitis and Arabidopsis hypothetical and unknown proteins, respectively. No OsPE paralog was found in rice. No conserved domains were found in the protein coded by OsPE. Availability ofhomologs genes ofknown function, specific tissue expression patterns and conserved domains would have supported assigning function to this novel gene, OsPE. vin RT-PCR using reverse transcribed mRNA and primer pair designed on intron exon junction, the expression of tWfgene was found in Basmati 370 shoots and no, in roots. The expression profile of the candidate gene suggested by EST counts showed maximum ESTs in seed (11/32357) followed by flower (23/136502) and panicle (23/132789). The polyembryony is associated with seed where maxtmum ESTs were found for the OsPE gene. Full length OsPE gene was cloned in Basmati 370 on the basis of the sequences obtained by genome walking and TAIL-PCR. The nucleotide sequence of the candidate gene OsPE in japonica rice was used to design overlapping PCR Primers and the OsPE gene was amplified ,„ Basmati 370 genomic DNA as the template. The sequence obtained using corresponding forward and reverse primers were aligned to determine the sequence errors and full length OsPE gene in Basmati 370 was obtained. BLAST search was done for homologous sequence in N.pponbare **riC6' Te" SNPS (Si"8le N"*otide Polymorphism) and one gap were found in the OsPE candidate gene sequence as compared to Nipponbare sequence thus suggesting atotal of 1% dissimilarity between the two sequences. The predicted protein was searched and variation was found in only two amino acids. With the minor differences in coding region the OsPE gene is expected to translate asimilar protein in Basmati 370. The OsPE gene BLAST alignment against rice FST database (Rice Functional Genomics Dataabase RiceGE) showed two hits. The OsPE gene has insertion in the promoter region whereas FST searched insertions are found to be in the coding region. No phenotypic description or associated annotations related to these insertions were reported. IX Identification for flanking sequences for two more independent T-DNA insertional mutants, B-2-2 and B-8-7 was done using TAIL-PCR. The insertion in chromosome 11 of B-2-2 is present in the gene that codes for Pectin methylesterase (PME). The combined use of Southern blot, BSA, genome walking, TAIL-PCR, RTPCR techniques and bioinformatics led to the identification of a candidate gene controlling the multiple embryo formation in Basmati rice. A detailed molecular characterization and mapping of the OsPE insertional mutant is available through this study. It can thus be speculated that OsPE gene controls the number of embryos or embryological divisions as the phenotype of the OsPE insertional mutant shows polyembryony with no sterility. The validation of the OsPE and other candidate genes can be done using two elementAc/Ds transposon system and RNAi approach.