Abstract
BackgroundThe 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.ResultsA 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.ConclusionHere, 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 materialThe online version of this article (doi:10.1186/s12863-016-0391-4) contains supplementary material, which is available to authorized users.
Highlights
The 6AL/6VS translocation lines, carrying the wheat powdery mildew resistance gene Pm21, are planted on more than 3.4 million hectares
Cloning of the no apical meristem (NAM)-V1 gene The genomic DNA and cDNA of the NAM-V1 gene were amplified from H. villosa using two pairs of primers (Fig. 1a)
Our results demonstrate that the replacement of NAM-A1 by NAM-V1 confers increased grain protein content, implying that NAM-V1 is more efficient than NAM-A1 in increasing the GPC
Summary
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). 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. Grain protein concentration (GPC) is an important agronomic trait in wheat. In 1991, a complete set of disomic substitution lines were developed by the introgression of each of the chromosomes of wild emmer wheat with high GPC (DIC) into the durum cultivar ‘Langdon’ (LDN). A quantitative trait locus (QTL) for wheat GPC was mapped onto the short arm of chromosome (6BS) using the recombinant inbred lines DIC and LDN [5], and later mapped within a 2.7 cM region as a single Mendelian locus, Gpc-B1 [6]. Uauy et al (2006) positionally cloned Gpc-B1 and established that it is a member of the NAC transcription factor; they renamed it NAM-B1 [7]
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