ISOLATION OF THE BIFUNCTIONAL NAD(P)H NITRATE REDUCTASE GENE IN WHEAT

Nitrogen uptake is an essential element in crop improvement and for breeding cereal cultivars that absorb and metabolize nitrogen most efficiently for grain or silage production. Varieties which have both high grain yield and good end-use quality at a low nitrogen level are therefore needed. This aim requires a better understanding of nitrogen metabolism and its regulation, and identification of target genes to monitor N uptake by either direct gene transfer or marker-assisted breeding. The early steps of nitrate and ammonia assimilation or reassimilation involve three gene families, coding for nitrate reductase (NAR), nitrite reductase (NIR), and glutamate synthase (GOGAT). Eukaryotic nitrate reductase catalyzes the reduction of nitrate to nitrite and are classified into three groups based on reduced pyridine nucleotide dependency: NADH-specific, NAD(P)H- bispecific and NADPH-specific. Both NADH and NAD(P)H- bi-specific NRs have been reported in plants. Genes encoding NADH-NAR have been sequenced in several plants, including Arabidopsis, maize and barley. A gene encoding for a bi-specific NAD(P)H-NAR was characterized in maize and barley. In wheat, although no sequence data are available in the databases, two NADH-NAR genes located on linkage groups (LGs) 6 and 7 and one bispecific NAR gene on LG 6 have been reported. We focused on bi-specific NAD(P)H-NAR gene as one of the potential candidate genes for determining grain protein content (GPC). Using a barley bi-specific NAD(P)H-NAR sequence as an initial query, we extracted and assembled 454-reads of cv. Chinese Spring (http://www.cerealsdb.uk.net/search_reads.htm) and of a Triticum tauschii accession (http://avena.pw.usda.gov/RHmapping/blast/). Using the 454 reads we were able to assemble the three orthologous genes from the hexaploid genomes and from the D-genome ancestor Triticum tauschii. PCR primer pairs were designed for the three NAD(P)H-NAR hexaploid sequences and used to identify genome assignments on group chromosome 6 by using the Chinese Spring nullitetrasomic lines. We also obtained the NAD(P)H-NAR genomic sequence in the two Italian durum wheat cultivars Svevo and Ciccio, characterized by different grain protein content.