Wheat grain protein, zinc (Zn), and iron (Fe) content are important wheat qualities crucial for human nutrition and health worldwide. Increasing these three components simultaneously in wheat grains by a single gene came into the picture through NAM-B1 cloning. NAM-B1 gene and its association with the mentioned grain quality traits have been primarily studied in common and durum wheat and their progenitors T. dicoccum and T. dicoccoides. In the present study, for the first time, 38 wheat accessions comprising ten hexaploids from five species and 28 tetraploids from nine species were evaluated in the field for two consecutive years. Additionally, the 582 first nucleotides of the NAM-B1 gene were examined. The NAM-B1 gene was present in 21 tetraploids and five hexaploid accessions. Seven tetraploid accessions contained the wild-type allele (five T. dicoccum, one T. dicoccoides, and one T. ispahanicum) and fourteen the mutated allele with a 'T' insertion at position 11 in the open reading frame, causing a frameshift. In hexaploid wheat comprising the gene, only one accession of T. spelta contained the wild-type allele, and the rest resembled the insertion mutated type. In the two-year field experiment, eight accessions with the wild-type NAM-B1 allele had significantly higher protein, Zn and Fe grain content when compared to indel-type accessions. Additionally, these accessions exhibited a lower mean for seed-filling duration than all other accessions containing indel-type alleles. In terms of grain yield, 1,000-kernel weight, kernel diameter, and kernel length, T. dicoccum accessions having wild-type alleles were similar to the indel-type accessions over two years of evaluation. These findings further support the possibility of simultaneous improvement of wheat grain protein, Zn, and Fe content by a single gene crucial for human nutrition and health worldwide.
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