Development and characterization of NB-ARC gene-derived SSRs in wheat (Triticum aestivum L.)

Bread wheat (Triticum aestivum L.), among the earliest cultivated crops, provides approximately 20% of the overall protein and calorie intake in human diets. Considering its significance for global food and nutritional security, enhancing gene-based resources is essential for maintaining advancements in yield and quality in the future. Microsatellites or simple sequence repeats (SSRs) located within genes involved in important biological functions offer useful resources for developing gene-based markers. NB-ARC (nucleotide-binding adaptor shared by Apaf-1, R proteins, and CED-4) domain-containing genes, owing to their role in plant defense and stress responses, represent important targets for developing gene-based SSR markers.

In this study, NB-ARC gene-based SSRs were systematically identified, characterized, and validated to generate novel gene-based functional markers for crop improvement. A total of 972 perfect SSRs were identified in 655 genes from 2,105 NB-ARC sequences, with even distribution between coding and untranslated regions. Tri-nucleotide repeats were predominant, and SSRs showed uneven chromosomal distribution, greater representation in the B sub-genome, and the highest density on chromosome 7 A. For validation, 65 SSRs were randomly selected, of which 48 (~ 74%) produced clear amplicons and showed moderate transferability (68.40%) across wild and related wheat species. Genotyping of 42 Indian wheat varieties using 21 polymorphic SSRs identified 64 alleles, with observed (Ho) and expected heterozygosity (He) values of 0.536 and 0.568, respectively, indicating a moderate level of genetic diversity with PIC of 0.487. The NB-ARC SSRs successfully distinguished wild/related wheat genotypes as well as cultivated wheat varieties through phylogenetic, PCoA, and STRUCTURE analysis. Segregation analysis of the selected NB-ARC SSRs in two bi-parental mapping populations indicated Mendelian inheritance with a certain population distortion.

Overall, NB-ARC gene-based SSRs represent reliable gene-based markers for diversity analysis, population structure studies, and molecular breeding in wheat.