THE NIR1 LOCUS IN BARLEY IS TIGHTLY LINKED TO THE NITRITE REDUCTASE APOPROTEIN GENE NII

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apo protein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pC IB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 bp in length. The nucleotide coding sequence could be aligned with the 3' en d of other higher plant nitrite reductase apoprotein cDNA sequences bu t diverges in the 3' untranslated region. The whole-plant barley mutan t STA3999, previously isolated from the cultivar Tweed, accumulates ni trite after nitrate treatment in the light, has very much lowered leve ls of nitrite reductase activity and lacks detectable nitrite reductas e cross-reacting material due to a recessive mutation in a single nucl ear gene which we have designated Nir1. STA3999 has the characteristic s expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carri ed by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tw eed (major hybridising band of 11.5 kb) and Golden Promise (major hybr idising band of 7.5 kb) when DraI-digested DNA was probed with the ins ert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI -digested DNA from the mutant STA3999 also exhibited a major hybridisi ng band of 11.5 kb after hybridisation with the insert from pBNiR1. F- 1 progeny derived from the cross between the cultivar Golden Promise a nd the homozygous nir1 mutant STA3999 were heterozygous for these band s as anticipated. Go-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatme nt/loss of detectable nitrite reductase cross-reacting material at M(r ) 63000) was scored in an F-2 population of 312 plants derived from th e cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showe d strict co-segregation (in approximately one quarter (84) of the 312 F-2 plants examined). Only those F-2 individuals heterozygous for the RFLP pattern gave rise to F-3 progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase ap oprotein gene Nii are very tightly linked.