8. Differentiation within CCDD genome species in the genus Oryza as revealed by

total genomic hybridization and RFLP analysis

R.K. aGGarwal, D.S. brar, N. huang and G.S. khush Division of Plant Breeding, Genetics and Biochemistry IRRI, P.O. Box 933, 1099 Manila. Philippines

 The genus Oryza has been divided into 4 species complexes largest of which is the 0. officinalis complex, also called the O. latifolia complex (Tateoka 1964). This complex has three allotetraploid species, O. latifolia Desv., 0. alta Swallen and 0. grandiglumis (Doell) Prod., having CCDD genomes (2n = 48). Comparative morphology and cytological observations of the interspecific hybrids reveal these as a closely

Research Notes

related group with continuous variation in several of the morphological characteristics and relatively weak reproductive isolation mechanism. Based on these, Tateoka (1964) considered them to be independent species. Sampath (1962), Gopalakrishana and Sampath (1967) and Jena and Khush (1988) on the other hand, concluded that the divergence between the three species is not sufficient to justify their species status and hence should be merged together and considered as subspecies under 0. latifolia. We, therefore reexamined the differentiation in the three CCDD species at the molecular level.

A Southern blot having DraI digested genomic DNA of four accessions each of the three CCDD species and representative accessions of all other Oryza species, was used for hybridization with 32^P-labeled total genomic DNA of 0. alta, 0. latifolia, 0. grandiglumis and other species as probe at different stringencies of hybridization and washings. Use of each of the CCDD species DNA as a genomic probe resulted in considerable cross-hybridization to lanes representing the three species (12 accessions), and low (to lanes having BBCC species/accessions) to negligible hybridization to the rest of the species tested. The hybridization profiles of the three species revealed distinctive differences, separating clearly the 0. latifolia from the other two species 0. alta and 0. grandiglumis which had almost identical pattern and signal intensity of hybridization (Fig. 1). These differences in hybridization signal/pattern for CCDD species were more striking than observed for other species having similar genome(s) i.e., species of AA genome on probing with 0. sativa DNA or CC genome species when probed with 0. eichengeri/O. officinalis genomic DNA. The results of total genomic DNA hybridization thus show that the genome(s) in the three CCDD species, although closely related yet are significantly differentiated from each other.

RFLP analysis involving 46 single locus probes spread over all the 12 chromosomes on the molecular map of rice and one conserved probe of rDNA, also showed distinct divergence between the three species. The dendrogram constructed from the pooled RFLP data revealed 0. latifolia to be the most diverged among the three CCDD species and 0. alta and 0. grandiglumis to be more similar to each other (Fig. 2). More importantly, the distance matrix (not shown) calculated from the RFLP data following Nei and Li (1979) shows that the level of divergence between the three species is significantly more than that observed for within species (between accessions of the same species) for all the Oryza species, and is comparable to the other species carrying similar genome(s), thus further supporting the conclusion that these are independent species rather than subspecies/ecotypes of one species. Furthermore, 0. latifolia is widely distributed in its habitat whereas (9. alta and 0. grandiglumis grow sympatrically in comparatively limited areas (Vaughan 1989). Accordingly, the relative divergence between accessions of 0. latifolia is found to be higher than for accessions of 0. alta and 0. grandiglumis, corresponding well with their distribution. It is possible that 0. latifolia genomes have undergone selective differentiation during evolution in response to its diverse adaptive needs.

Our results demonstrate that the three CCDD genome species, although closely related are considerably differentiated at the molecular level comparable to other species

56 Rice Genetics Newsletter Vol. 13 Fig. 1. Dra-I-Southern profiles of some of the representative Oryza species obtained after hybridization with 32^P-labeled total genomic DNA (4 h exposure after low-stringency wash of 2 x SSC at 60°C) from 0. alta (upper half) and 0. latifolia (lower half). Note the striking differences in hybridization signal and pattern across tracks 10-21 and also with rest of the lanes. Tracks: 1-5: O. eichingeri : 6-9. O. minuta: 10-13: O. alta Accessions 10888. 10952, 10967. 105143: 14-17: 0. latifolia Accessions 1100168, 100914. 100955. 103787: 18-21: 0. grandiglumis Accessions 105155. 105157. 105560. 105669: 22-23: 0. malamphuzhaensis: 24-27: 0. ridleyi: and 28-30: 0. longiglumis. Research Notes in the genus 0ryza. The differentiation in their genome(s) encompasses evenly across the single locus as well as repeated DNA sequences. These results considered along with earlier observations (differences in morphological traits, crossability barriers, high F1 sterility) thus suggest that the three CCDD Latin American tetraploid species are independent species. The study also demonstrates that molecular analysis can serve as a complementary tool in the analysis of species relationships and biosystematics. Fig. 2. An UPGMA dendrogram based on mean genetic distances (according to Nei and Li, 1979) for nuclear RFLPs between species. The scale shows the genetic similarity values. References 

Gopalakrishana, R. and S. Sampath, 1967. Taxonomic status and origin of American tetraploid species of the-

series Latifoliae Tateoka in the genus 0ryza Indian J. Agr. Sci. 37: 465-475. Jena. K.K. and G.S. Khush, 1988. Cytogenetic relationships among the three species of Oryza latifolia complex. RGN 5:74-75. Nei. M. and W.H. Li, 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci., USA 76: 5269-5273. Sampath, S.. 1962. The genus 0ryza. its taxonomy and species relationships. Oryza l( I ): 1-29.

Tateoka. T., 1964. Taxonomic studies of the genus Oryza. In IRRI (ed.). Rice Genetics and Cytogenetics.

pp. 15.21. Elsevier. Amsterdam. Vaughan, D.A., 1989. Tile genus Oryza L.. Current status of taxonomy. Intl. Rice Res. lnst. Research Paper Series No. 138, pp.21.