Wild species of Oryza are an important source of useful genes. Though their phylogenetic relationships have been studied using various approaches there is a discrepancy regarding the species relationships of E, F, and HK genomes. The species O. brachyantha (F) and O. schlecteri (HK) in section ridleyanae of Vaughan's classification were considered as a separate section and series respectively by Lu. Also the grouping of O. australiensis (E) in O. officinalis complex is disputed. In this study the species relationships were analysed based on selected informative ISSR and SSR markers.

Thirty two accessions representing 17 species from the genus Oryza and Porteresia were examined for Inter simple sequence repeat (ISSR) polymorphism using 8 informative primers (Table 1). Five simple sequence repeat (SSR) loci in centromeric region were also included (Table 2). Standard procedures were followed for DNA extraction, PCR analysis, agarose gel

electrophoresis, band scoring and cluster analysis as reported earlier (Sarla et al. 2003).

ISSR analysis using 8 primers amplified 138 band positions. All the primers showed 100% polymorphism. UBC 841 showed the highest polymorphism information content (0.969) and the least mean genetic similarity (0.19) followed by UBC 815 and UBC 842. Thus these three primers were the most informative. Data based on all the ISSR primers showed a mean genetic similarity of 0.32 indicating a large variation among the accessions of different genomes. Two primers UBC-811 and UBC-842 amplified 3 species specific bands in O. punctata and O. alta. Seven primers amplified 22 accession specific markers.

Clustering based on ISSR data showed two clusters one consisting of the diploid species of A, B and C genomes and the second consisting of the tetraploid species of BC, CD and HJ genomes. However the C genome species O. eichengeri and the E genome species O. australiensis grouped in cluster II. O. brachyantha(F) and Porteresia(HK) did not cluster in the two groups but remained separate. The A genome species and the B and C genome species grouped in separate subclusters within the cluster I. In cluster II, the BC and CD genome species grouped in one subcluster and the E and HJ genome species grouped in the second cluster.

SSSR analysis using 5 primers mapped to centromeric or proximal regions amplified 21 alleles and a total of 183 bands. RM3 amplified 4 alleles in the A genome species but none in the others. The number of alleles amplified by each primer pair ranged from 2 (RM155) to 6 (RM156, RM219) with an average value of 4.2. The polymorphic information content (PIC) ranged from 0.86 (RM 464) to 0.99 (RM 3). RM 219 showed the maximum resolving power (1.8) and RM3 showed the minimum (0.77). RM 156 amplified an allele specific to O. longiglumis and 2 alleles specific to O. meridionalis. RM 219 amplified one allele specific to O. brachyantha and RM 3 amplified an allele specific to O. glumaepatula. SSR markers helped differentiate the A, E, F, HJ and HK genome species while the B, C, BC and CD genome species were highly similar. Three of the 5 primers used showed null alleles in various species.

Clustering based on 159 pooled ISSR and SSR markers grouped the species into two clusters, one consisting of the A genome species and the other consisting of B, C, BC, CD and HJ genome species. O. australiensis (E), O. brachyantha (F) and Porteresia (HK) remained separate. Cluster I and cluster II correspond to the O.sativa complex and O. officinalis complex proposed by Vaughan (1994). However there is discrepancy with respect to the grouping of the species O. australiensis (E) and O. longiglumis (HJ). The species O. australiensis (E) and O. longiglumis (HJ) were placed in the O. officinalis and O. ridleyi complex respectively (Vaughan 1994).

In the A genome cluster Jaya the indica cultivar of O. sativa grouped with one accession of O. nivara while Moroberekan the japonica cultivar was closer to O. rufipogon supporting the diphyletic origin of rice (Yamanaka et al. 2003). One of the accessions of O. glumaepatula grouped with O. rufipogon. Wang et al. (1992) considered O. glumaepatula as a subtype of O. rufipogon growing in South America. O. longistaminata grouped as the most distant species. This is in confirmation with earlier reports (Aggarwal et al. 1999, Bautista et al. 2001, Ren et al. 2003).

In the second cluster the B, C, BC, CD genome species grouped genome-wise except O. eichengeri (C) which was with O. minuta (BC). Close affinity of O. minuta and O. eichengeri has been reported earlier (Federici et al. 2002, Mullins et al. 2002). O. australiensis (E) separated out and grouped along with O. brachyantha and Porteresia. Vaughan (1994) grouped O. australiensis in the O. officinalis complex. However, studies based on molecular markers and sequence variation in genes coding prolamine protein support its exclusion from the O. officinalis complex (Xie and Zhou 1998, Joshi et al. 2000, Mullins et al. 2002) as proposed by Tateoka et al. (1962a).

O. brachyantha grouped as the most distant species as reported earlier (Aggarwal et al. 1999, Ge et al. 1999, Joshi et al. 2000). However Wang et al. (1992) reported closer affinity of this species with the A genome species based on RFLP markers. Porteresia grouped along with O. branchyantha as shown in earlier studies also (Xie and Zhou 1998, Aggarwal et al. 1999 and Joshi et al. 2000).

References

Aggarwal, R.K., D.S. Brar, S. Nandi, N. Huang and G.S. Khush, 1999. Phylogenetic relationships among Oryza species revealed by AFLP markers. Theor. Appl. Genet. 98: 1320-1328.

Bautista, N.S., R. Solis, O. Kamijima and T. Ishii, 2001. RAPD, RFLP and SSLP analysis of phylogenetic relationships between cultivated and wild species of rice. Genes Genet. Syst. 76: 71-79.

Federici, M.T., A.B. Shcherban, F. Capdevielle, M. Francis and D. Vaughan, 2002. Analysis of genetic diversity in the Oryza officinalis complex. Electronic journal of biotechnology. 5(2): 174-181.

Ge, S., T. Sang, B.R. Lu and D.Y. Hong, 1999. Phylogeny of rice genomes with emphasis on origins of allotetraploid species. Proc. Natl. Acad. Sci USA. 96: 14400-14405.

Joshi, S.P., V.S. Gupta, R.K. Aggarwal, P.K. Ranjekar and D.S. Brar, 2000. Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus oryza. Theor. Appl. Genet. 100: 1311-1320.

Ren, F., B. Lu, S. Li, J. Huang and Y. Zhu, 2003. A comparative study of genetic relationships among the AA-genome oryza species using RAPD and SSR markers. Theor. Appl. Genet. 108: 113-120.

Sarla, N., B. Sunil and E.A. Siddiq, 2003. ISSR and SSR markers based on AG and GA repeats delineate geographically diverse Oryza nivara accessions and reveal rare alleles. Curr. Sci. 84(5): 683-690.

Tateoka, T., 1962a. Taxonomic studies of Oryza I, O. latifolia complex. Bot Mag Tokyo., 75: 418-427.

Vaughan, D.A., 1994. The wild relatives of rice:a genetic resources guide book. International Rice Research Institute, Los Banos, Phillipines.

Wang, Z.M., G. Second and S.D. Tanksley, 1992. Polymorphism and phylogenetic relationships among species in the genus oryza as determined by analysis of nuclear RFLPs. Theor. Appl. Genet. 83: 565-581.

Xie, Z.W. and Y. Zhou, 1998. Phylogenetic relationship of the genus Oryza as revealed by RAPD analysis. Int. Rice Res. Notes. 23(3): 6-8.

Yamanaka, S., I. Nakamura, H. Nakai and Y. Sato, 2003. Dual origin of the cultivated rice based on molecular markers of newly collected annual and perennial strains of wild rice species, Oryza nivara and O. rufipogon. Genet. Res. and Crop Evol. 50: 529-538.