Lactate dehydrogenase (Ldh, E.C. 1.1.1.27) locus was assayed by starch-gel (system H, Tris-citric pH 7.6) methods (Glaszmann et al. 1988) for genetic characterization of Oryza sativa complex.

A total of 98 rice strains consisting of Oryza sativa, Oryza rufipogon, and Oryza barthii from worldwide distribution were studied. Enzyme extracts were prepared from 1-5 shoots, 2-4 days after germination at 32^oC in incubator. The gel staining was performed using the modified procedure described by Wang (1996), in which stain formulation consisted of 50 ml 0.05 mol pH 8.0 Tris-HCl, 100 mg lactic acid (lithium salt), 1 ml MgCl₂ (1%), 1ml NAD (1%), 1 ml NBT (1%) or 1 ml MTT (1%), and 0.25 ml PMS (1%). The numerical nomenclature systems (Morishima and Glaszmann, 1986) was employed for designating the alleles of Ldh.

Three distinct bands and a null were observed, suggesting that Ldh locus has four alleles corresponding to null, slow, medium and fast-migrating bands. Thus, four alleles of Ldh here were designated as Ldh1-1, Ldh1-2, Ldh1-3, and Ldh1-0 (Fig. 1).

The stained bands of Ldh alleles appeared quite slow (over night) in contrast to Adh1 where bands appeared in about 30 min. However, allelic structure and the genetic

polymorphism patterns revealed for Ldh1 were nearly identical to those of alcohol dehydrogenase as shown elsewhere in this volume of RGN (Suh et al.). Among the strains investigated only the Pakistan variety, Basmati 385, possessed Ldh1-3 allele, and only the Chinese weedy rice strain Lu-tao possessed the null allele. Generally, polymorphism for three alleles (Ldh1-1, Ldh1-2, and Ldh1-3) was not present within group of lowland rice but was observed in upland rice (Table 1). These results suggest that Adh1 and Ldh1 play the similar function in response to environmental adaptation, especially in relation to tolerance of environmental stresses.

For detection of Ldh1, enzyme activity, extracts were found sensitive to low temperature preservation. Thus sample should be prepared just prior to loading and gel running.

References

Glaszmann, J.C., B.G. de los Reyes and G.S. Khush, 1988. Electrophoretic variation of isozymes in plumules of rice (Oryza sativa L.)- a key to the identification of 76 alleles at 24 loci. IRRI Res. Paper Series. 134: 14.

Morishima, H. and J.C. Glaszmann, 1986. Gene symbols for isozymes. RGN 3: 15-17. Wang, Z.R. (ed.), 1996. Analysis of Allozymes in Plants. Scientific Publisher. p. 196. (in Chinese)