Grain protein content (GPC) is a key aspect of grain quality, a major determinant of the flour functional properties and grain nutritional value of bread wheat. Exploiting diverse germplasms to identify genes for improving crop performance and grain nutritional quality is needed to enhance food security. Here, we evaluated GPC in a panel of 255 Triticum aestivum L. accessions from 27 countries. GPC determined in seeds from three consecutive crop seasons varied from 8.6 to 16.4% (11.3% on average). Significant natural phenotypic variation in GPC among genotypes and seasons was detected. The population was evaluated for the presence of the trait-linked single nucleotide polymorphism (SNP) markers via a genome-wide association study (GWAS). GWAS analysis conducted with calculated best linear unbiased estimates (BLUEs) of phenotypic data and 90 K SNP array using the fixed and random model circulating probability unification (FarmCPU) model identified seven significant genomic regions harboring GPC-associated markers on chromosomes 1D, 3A, 3B, 3D, 4B and 5A, of which those on 3A and 3B shared associated SNPs with at least one crop season. The verified SNP–GPC associations provide new promising genomic signals on 3A (SNPs: Excalibur_c13709_2568 and wsnp_Ku_c7811_13387117) and 3B (SNP: BS00062734_51) underlying protein improvement in wheat. Based on the linkage disequilibrium for significant SNPs, the most relevant candidate genes within a 4 Mbp-window included genes encoding a subtilisin-like serine protease; amino acid transporters; transcription factors; proteins with post-translational regulatory functions; metabolic proteins involved in the starch, cellulose and fatty acid biosynthesis; protective and structural proteins, and proteins associated with metal ions transport or homeostasis. The availability of molecular markers within or adjacent to the sequences of the detected candidate genes might assist a breeding strategy based on functional markers to improve genetic gains for GPC and nutritional quality in wheat.

Genetic diversity and population structure are key resources for breeding purposes and genetic studies of important agronomic traits in crops. In this study, we described SNP-based genetic diversity, linkage disequilibrium and population structure in a panel of 179 bread wheat advanced cultivars and old accessions from Bulgaria, using an optimized wheat 25K Infinium iSelect array. Out of 19,019 polymorphic SNPs, 17,968 had a known chromosome position on the A (41%), B (42%) and D (11%) genome, and 6% were not assigned to any chromosome. Homoeologous group 4, in particular chromosome 4D, was the least polymorphic. In the total population, the Nei’s gene diversity was within the range 0.1–0.5, and the polymorphism information content ranged from 0.1 to 0.4. Significant differences between the old and modern collections were revealed with respect to the linkage disequilibrium (LD): the average values for LD (r²), the percentage of the locus pairs in LD and the LD decay were 0.64, 16% and 3.3 for the old germplasm, and 0.43, 30% and 4.1 for the modern releases, respectively. Structure and k-means clustering algorithm divided the panel into three groups. The old accessions formed a distinct subpopulation. The cluster analysis further distinguished the modern releases according to the geographic region and genealogy. Gene exchange was evidenced mainly between the subpopulations of contemporary cultivars. The achieved understanding of the genetic diversity and structure of the Bulgarian wheat population and distinctiveness of the old germplasm could be of interest for breeders developing cultivars with improved characteristics. The obtained knowledge about SNP informativeness and the LD estimation are worthwhile for selecting markers and for considering the composition of a population in association mapping studies of traits of interest.

Strategies and coping mechanisms for stress tolerance under sub-optimal nutrition conditions could provide important guidelines for developing selection criteria in sustainable agriculture. Nitrogen (N) is one of the major nutrients limiting the growth and yield of crop plants, among which wheat is probably the most substantial to human diet worldwide. Physiological status and photosynthetic capacity of two contrasting wheat genotypes (old Slomer and modern semi-dwarf Enola) were evaluated at the seedling stage to assess how N supply affected osmotic stress tolerance and capacity of plants to survive drought periods. It was evident that higher N input in both varieties contributed to better performance under dehydration. The combination of lower N supply and water deprivation (osmotic stress induced by polyethylene glycol treatment) led to greater damage of the photosynthetic efficiency and a higher degree of oxidative stress than the individually applied stresses. The old wheat variety had better N assimilation efficiency, and it was also the one with better performance under N deficiency. However, when both N and water were deficient, the modern variety demonstrated better photosynthetic performance. It was concluded that different strategies for overcoming osmotic stress alone or in combination with low N could be attributed to differences in the genetic background. Better performance of the modern variety conceivably indicated that semi-dwarfing (Rht) alleles might have a beneficial effect in arid regions and N deficiency conditions.

Genetic enhancement of grain production and quality is a priority in wheat breeding projects. In this study, we assessed two key agronomic traits—grain protein content (GPC) and thousand kernel weight (TKW)—across 179 Bulgarian contemporary and historic varieties and landraces across three growing seasons. Significant phenotypic variation existed for both traits among genotypes and seasons, and no discernible difference was evident between the old and modern accessions. To understand the genetic basis of the traits, we conducted a genome-wide association study with MLM using phenotypic data from the crop seasons, best linear unbiased estimators, and genotypic data from the 25K Infinium iSelect array. As a result, we detected 16 quantitative trait nucleotides (QTNs) associated with GPC and 15 associated with TKW, all of which passed the false discovery rate threshold. Seven loci favorably influenced GPC, resulting in an increase of 1.4% to 8.1%, while four loci had a positive impact on TKW with increases ranging from 1.9% to 8.4%. While some loci confirmed previously published associations, four QTNs linked to GPC on chromosomes 2A, 7A, and 7B, as well as two QTNs related to TKW on chromosomes 1B and 6A, may represent novel associations. Annotations for proteins involved in the senescence-associated nutrient remobilization and in the following buildup of resources required for seed germination have been found for selected putative candidate genes. These include genes coding for storage proteins, cysteine proteases, cellulose-synthase, alpha-amylase, transcriptional regulators, and F-box and RWP-RK family proteins. Our findings highlight promising genomic regions for targeted breeding programs aimed at improving grain yield and protein content.

In the context of crop breeding, plant height (PH) plays a pivotal role in determining straw and grain yield. Although extensive research has explored the genetic control of PH in wheat, there remains an opportunity for further advancements by integrating genomics with growth-related phenomics. Our study utilizes the latest genome-wide association scan (GWAS) techniques to unravel the genetic basis of temporal variation in PH across 179 Bulgarian bread wheat accessions, including landraces, tall historical, and semi-dwarf modern varieties. A GWAS was performed with phenotypic data from three growing seasons, the calculated best linear unbiased estimators, and the leveraging genotypic information from the 25K Infinium iSelect array, using three statistical methods (MLM, FarmCPU, and BLINK). Twenty-five quantitative trait loci (QTL) associated with PH were identified across fourteen chromosomes, encompassing 21 environmentally stable quantitative trait nucleotides (QTNs), and four haplotype blocks. Certain loci (17) on chromosomes 1A, 1B, 1D, 2A, 2D, 3A, 3B, 4A, 5B, 5D, and 6A remain unlinked to any known Rht (Reduced height) genes, QTL, or GWAS loci associated with PH, and represent novel regions of potential breeding significance. Notably, these loci exhibit varying effects on PH, contribute significantly to natural variance, and are expressed during seedling to reproductive stages. The haplotype block on chromosome 6A contains five QTN loci associated with reduced height and two loci promoting height. This configuration suggests a substantial impact on natural variation and holds promise for accurate marker-assisted selection. The potentially novel genomic regions harbor putative candidate gene coding for glutamine synthetase, gibberellin 2-oxidase, auxin response factor, ethylene-responsive transcription factor, and nitric oxide synthase; cell cycle-related genes, encoding cyclin, regulator of chromosome condensation (RCC1) protein, katanin p60 ATPase-containing subunit, and expansins; genes implicated in stem mechanical strength and defense mechanisms, as well as gene regulators such as transcription factors and protein kinases. These findings enrich the pool of semi-dwarfing gene resources, providing the potential to further optimize PH, improve lodging resistance, and achieve higher grain yields in bread wheat.

Genetic enhancement of grain production and quality is a priority in wheat breeding projects. In this study, we assessed two key agronomic traits - grain protein content (GPC) and thousand kernel weight (TKW) - across 179 Bulgarian contemporary and historic varieties, and landraces in three growing seasons. Significant phenotypic variation existed for both traits among genotypes and seasons, and no discernible difference was evident between the old and modern accessions. To understand the genetic basis of the traits, we conducted a genome-wide association study with MLM using phenotypic data from the crop seasons, best linear unbiased estimators, genotypic data from the 25K Infinium iSelect array, and a Bonferroni-adjusted p-value. As a result, 776 significant quantitative trait nucleotides (QTNs) organized into quantitative trait loci were discovered, including 25 loci associated with GPC, and 35 loci linked to TKW. Additionally, 14 environmentally stable QTNs were identified for TKW. Certain loci on chromosomes 2A, 3A, and 6B favorably in-fluenced GPC, while the locus on chromosome 6D impacted TKW positively. These loci may reflect novel associations to the two traits. Annotations for proteins involved in the remobilization of carbon and nitrogen from vegetative tissues to developing grain during senescence, as well as in the following buildup of resources required for seed germination, have been found for selected pu-tative candidate genes. These include genes coding for 11S globulin seed storage protein, glutamine synthetase, metalloendopeptidase, components of the ubiquitin/proteasome system, sucrose transporter, alpha-amylase, NAC and bZIP transcription factors. Our findings highlight promising genomic regions for targeted breeding programs aimed at improving grain yield and protein content.