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Background  The 6AL/6VS translocation lines, carrying the wheat powdery mildew resistance gene  Pm21 , are planted on more than 3.4 million hectares. The  NAM - A1  gene, located on chromosome 6AS of hexaploid wheat, has been implicated with increased wheat grain protein content (GPC). However, the  NAM - A1  gene was removed from the 6AL/6VS translocation lines after the original chromosome 6AS was replaced by chromosome 6VS of  Haynaldia villosa . The present study aimed to clone the  NAM  homologous gene from chromosome 6VS, to analyze the changes of GPC in the 6AL/6VS translocation lines, and to develop related molecular markers for wheat molecular breeding.    Results  A new  NAM  family gene,  NAM - V1 , was cloned from 6VS of  H. villosa  (GenBank ACC. no. KR873101).  NAM - V1  contained an intact open reading frame (ORF) and putatively encodes a protein of 407 amino acids. Phylogenetic analysis indicated that  NAM - V1  was an orthologous gene of  NAM - A1 ,  B1 , and  D1 . The determination of GPC in four  Pm21  F2 segregation populations demonstrated that the replacement of  NAM - A1  by  NAM - V1  confers increased GPC in hexaploid wheat. Multiple sequence alignment of  NAM - A1 ,  B1 ,  B2 ,  D1 ,  D2 , and  V1  showed the single nucleotide polymorphism (SNP) sites for each of the  NAM  genes, allowing us to develop a molecular marker,  CauNAM - V1 , for the specific detection of  NAM - V1  gene. Our results indicate that  CauNAM - V1  can be used as a novel DNA marker for  NAM - V1 , and can also be used for selecting  Pm21  in wheat breeding programs. Further, we developed a marker,  CauNAM - ABD , for the amplification and simultaneously distinguish among the  NAM - A1 ,  NAM - B1 ,  NAM - B2 ,  NAM - D1 , and  NAM - D2  genes in a single step.  CauNAM - ABD  enabled us to develop an efficient “one-marker-for-five-genes” procedure for identifying genes and its copy numbers related with grain protein content.    Conclusion  Here, we report the isolation of the  NAM - V1  gene of  H. villosa . This gene contributes to increasing GPC in 6AL/6VS translocation wheat lines. We developed a molecular marker for the specific detection of  NAM - V1  and a molecular marker that can be used to simultaneously distinguished among the  NAM - A1 ,  NAM - B1 ,  NAM - B2 ,  NAM - D1 , and  NAM - D2  genes in a single step.    Electronic supplementary material  The online version of this article (doi:10.1186/s12863-016-0391-4) contains supplementary material, which is available to authorized users.

Haynaldia villosa NAM-V1 is linked with the powdery mildew resistance gene Pm21 and contributes to increasing grain protein content in wheat