VARIATION OF NITRATE REDUCTASE GENES IN SELECTED GRASS SPECIES

In order to study the variation of nitrate reductase (NR) genes among grass species, gene number, intron size and number, and the heme-hinge fragment sequence of 25 grass species were compared. Genomic DNA cut with six restriction enzymes and hybridized with the barley NAD(P)H an d NADH NR gene probes revealed a single NAD(P)H NR gene copy and two o r more NADH NR gene copies per haploid genome in most of the species e xamined. Major exceptions were Hordeum vulgare, H. vulgare ssp. sponta neum, and Avena strigosa, which appeared to have a single NADH NR gene copy. The NADH NR gene intron number and lengths were examined by pol ymerase chain reaction amplification. Introns I and III appeared to be absent in at least one of the NADH NR genes in the grass species, whi le intron II varied from 0.8 to 2.4 kilobases in length. The NADH NR g ene heme-hinge regions were amplified and sequenced. The estimated ave rage overall nucleotide substitution rate in the sequenced region was 7.8 x 10(-10) substitutions/site per year. The synonymous substitution rate was 2.11 x 10(-9) substitutions/synonymous site per year and the nonsynonymous substitution rate was 4.10 x 10(-10) substitutions/nons ynonymous site per year. Phylogenetic analyses showed that all of the wild Hordeum species examined clustered in a group separate from H. vu lgare and H. vulgare ssp. spontaneum.