Partial sequences of nitrogen metabolism genes in hexaploid wheat

Our objective was to partially sequence genes controlling nitrogen metabolism in wheat species in order to find sequence polymorphism that would enable their mapping. Primers were designed for nitrate reductase, nitrite reductase, glutamate dehydrogenase and glutamate synthase (GOGAT), and gene fragments were amplified on Triticum aestivum, T. durum, T. monococcum, T. speltoides and T. tauschii. We obtained more than 8 kb of gene sequences, mainly as coding regions (60%). Polymorphism was quantified by comparing two-by-two the three genomes of the hexaploid cultivar Arche and genomes of diploid wheat species. On average, the polymorphism rate was higher for non-coding regions, where it ranged from 1/60 to 1/23, than for coding regions (range: 1/110-1/40) except when the hexaploid D genome was compared to that of T. tauschii (1/800 and 1/816, respectively). Genome-specific primers were devised for the ferredoxin-dependent (Fd)-GOGAT gene, and they enabled the mapping of this gene on homoeologous chromosomes of group 2 using Chinese Spring deletion lines. A single nucleotide polymorphism (SNP) detected between the two hexaploid wheat cultivars Arche and Recital was used to genetically map Fd-GOGAT on chromosome 2D using a population of dihaploid lines. Fd-GOGAT-specific primers were used to estimate the SNP rate on a set of 11 hexaploid and nine Durum wheat genotypes leading to the estimate of 1 SNP/515 bp. We demonstrate that polymorphism detection enables heterologous, homeologous and even paralogous copies to be assigned, even if the elaboration of specific primer pairs is time-consuming and expensive because of the sequencing.