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Nitrogen is an essential element for maize growth, but excessive application can lead to various environmental and ecological issues, including water pollution, air pollution, greenhouse gas emissions, and biodiversity loss. Hence, developing maize hybrids resilient to low-N conditions is vital for sustainable agriculture, particularly in nitrogen-deficient soils. Combining ability and genetic relationships among parental lines is crucial for breeding superior hybrids under diverse nitrogen levels. This study aimed to assess the genetic diversity of maize inbred lines using simple sequence repeat (SSR) markers and evaluate their combining ability to identify superior hybrids under low-N and recommended conditions. Local and exotic inbred lines were genotyped using SSR markers, revealing substantial genetic variation with high gene diversity (He = 0.60), moderate polymorphism information content (PIC = 0.54), and an average of 3.64 alleles per locus. Twenty-one F1 hybrids were generated through a diallel mating design using these diverse lines. These hybrids and a high yielding commercial check (SC-131) were field-tested under low-N and recommended N conditions. Significant variations (p < 0.01) were observed among nitrogen levels, hybrids, and their interaction for all recorded traits. Additive genetic variances predominated over non-additive genetic variances for grain yield and most traits. Inbred IL3 emerged as an effective combiner for developing early maturing genotypes with lower ear placement. Additionally, inbreds IL1, IL2, and IL3 showed promise as superior combiners for enhancing grain yield and related traits under both low-N and recommended conditions. Notably, hybrids IL1×IL4, IL2×IL5, IL2×IL6, and IL5×IL7 exhibited specific combining abilities for increasing grain yield and associated traits under low-N stress conditions. Furthermore, strong positive associations were identified between grain yield and specific traits like plant height, ear length, number of rows per ear, and number of kernels per row. Due to their straightforward measurability, these relationships underscore the potential of using these traits as proxies for indirect selection in early breeding generations, particularly under low-N stress. This research contributes to breeding nitrogen-efficient maize hybrids and advances our understanding of the genetic foundations for tolerance to nitrogen limitations.

Calcium is one of the most limiting factors for the growth and reproduction of peanut, which ultimately affects pod and seed yields. A two-year field experiment was carried out to assess the impact of five calcium applications, including nano-calcium and conventional forms, on growth, leaf nutrient content, yield traits, and quality parameters of three diverse peanut cultivars (Ismailia-1, Giza-5, and Giza-6). The applied calcium applications were calcium sulfate, which is recommended for commercial peanut cultivation and commonly referred to as gypsum (coded as Ca-1), calcium nitrate (Ca-2), nano-calcium nitrate (Ca-3), 50% calcium nitrate + 50% nano-calcium (Ca-4), and 50% calcium sulfate + 50% nano-calcium (Ca-5). Calcium sulfate (gypsum, Ca-1) was soil-supplied during the seedbed preparation as recommended, while the other calcium applications (Ca-2, Ca-3, Ca-4, and Ca-5) were exogenously sprayed three times at 30, 45, and 60 days after sowing. The soil of the experimental site was alkaline, with a high pH of 8.6. The results revealed significant differences among cultivars, calcium applications, and their interactions. The soil-supplied gypsum Ca-1 displayed lower agronomic performance on all recorded growth, leaf nutrient content, yield traits, and quality parameters. On the other hand, the foliar-supplied calcium, particularly Ca-4 and Ca-5, displayed superior effects compared to the other simple calcium forms. Ca-4 and Ca-5 produced significantly higher seed yield (3.58 and 3.38 t/ha) than the simple recommended form (Ca-1, 2.34 t/ha). This could be due to the difficulty of calcium uptake from soil-supplied calcium under high soil pH compared to the exogenously sprayed nano-calcium form. Moreover, the superior performance of Ca-4 and Ca-5 could be caused by the mixture of fertilizers from the synergistic effect of calcium and nitrate or sulfate. Furthermore, the effect of nitrate was applied in nano form in the Ca4 and Ca-5 treatments, which contributed to improving nutrient uptake efficiency and plant growth compared to the other treatments. The peanut cultivar Giza-6 showed superiority for most measured traits over the other two cultivars. The interaction effect between the assessed cultivars and calcium applications was significant for various traits. The cultivar Giza-6 showed a significant advantage for most measured traits with the mixture of 50% calcium nitrate + 50% nano-calcium (Ca-4). Conclusively, the results pointed out the advantage of the exogenously sprayed nano-calcium form combined with calcium nitrate or calcium sulfate for promoting growth, leaf nutrient content, yield, and quality traits of peanut, particularly with high-yielding cultivars under sandy soil with high pH.

Water deficit is a decisive challenge that tremendously affects maize physiological functions and productivity. Hence, it is crucial to ameliorate its tolerance to drought stress, in particular under abrupt climate change and a growing population. The present study aimed to explore the influence of exogenously sprayed moringa seed extract (Moringa oleifera) and α-tocopherol on physio-biochemical, morphological, and yield attributes of six diverse maize hybrids under three irrigation levels in poor-fertility sandy soil. The applied irrigation regimes were based on estimated crop evapotranspiration (ET) using the FAO Penman–Monteith equation. A split–split plot arrangement with a randomized complete block design and three replicates was applied for different treatments. Irrigation levels (100% ET, 75% ET and 50% ET) were established in the main plots, while foliar applications (moringa extract and α-tocopherol) were located in subplots and the assessed hybrids (SC162, SC166, SC167, SC168, SC176, and SC178) in subsubplots. Mild (75% ET) and severe (50% ET) drought stress gradually reduced the gas exchange, photosynthetic efficiency, water relations, and yield traits compared with well-watered conditions (100% ET). However, foliar application of moringa seed extract or α-tocopherol was effective in reinforcing maize tolerance to drought stress by enhancing the accumulation of osmoprotectants, improving antioxidant enzymes, and decreasing levels of peroxidation of membrane lipids and electrolyte leakage compared to untreated control. These positive impacts were reflected in boosting yield traits and crop water productivity under water deficit conditions. The physiological and agronomic performance of the assessed maize hybrids considerably varied under water deficit conditions. The hybrids SC168, SC176, and SC178 exhibited the best performance under mild and severe drought conditions compared with the other hybrids. Consequently, the integration of exogenously applied moringa seed extract or α-tocopherol with tolerant maize hybrids such as SC168, SC176, and SC178 is an efficient approach to ameliorating drought tolerance under water-scarce conditions in arid environments.

Drought is a major abiotic stress that greatly affects canola growth, production, and quality. Moreover, water scarcity is projected to be more severe and frequent as a result of climate change, in particular in arid environments. Thereupon, developing drought-tolerant and high-yielding canola genotypes has become more critical to sustaining its production and ensuring global food security with the continuing population growth. In the present study, ten canola genotypes comprising six developed tissue-cultured canola genotypes, two exotic genotypes, and two commercial cultivars were evaluated under four irrigation regimes. The applied irrigation regimes were well-watered (100% crop evapotranspiration, ETc), mild drought (80% ETc), moderate drought (60% ETc), and severe drought (40% ETc) conditions. Drought-stress treatments (80, 60, and 40% ETc) gradually reduced the chlorophyll content, relative water content, flowering time, days to maturity, plant height, number of pods, number of branches, seed yield, and oil percentage, and increased proline, phenolic, anthocyanin, and glycine betaine contents. The evaluated genotypes exhibited varied responses to drought-stress conditions. The developed tissue-cultured genotypes T2, T3, and T1, as well as exotic genotype Torpe, possessed the highest performance in all evaluated parameters and surpassed the other tested genotypes under water-deficit conditions. Overall, our findings elicited the superiority of certain newly developed tissue-cultured genotypes and exotic ones compared with commercial cultivars, which could be exploited in canola breeding under water-deficit conditions.

The Iberian Peninsula was the place where pepper (Capsicum annuum) entered Europe and dispersed to other continents but was also an important secondary center for its diversification. The current work evaluated the phenotypic diversity existing in this region and investigated how that evolved from Capsicum native areas (Mexico and Andean Region). For that purpose, the high-throughput phenotyping tool Tomato Analyzer was employed. Descriptors related to size and shape were the most distinctive among fruit types, reflecting a broad diversity for Iberian peppers. These traits likely reflected those suffering from more intensive human selections, driving the worldwide expansion of C. annuum. Iberian peppers maintained close proximity to the American accessions in terms of fruit phenomics. The highest similarities were observed for those coming from the southeastern edge of the Peninsula, while northwestern accessions displayed more significant differences. Common fruit traits (small, conical) suggested that Portuguese and Spanish landraces may have arisen from an ancient American population that entered the south of Spain and promptly migrated to the central and northern territories, giving rise to larger, elongated, and blocky pods. Such lineages would be the result of adaptations to local soil–climate factors prevailing in different biogeographic provinces.

The main goal of this work was to investigate the relationship between the fruit morphology and biochemical composition of peppers (Capsicum spp.). For that purpose, one hundred native varieties from the Andean region, where the genus Capsicum has its origin, were analysed for different phytochemical compounds. In addition, pepper fruits were assessed with the highly precise phenomics tool Tomato Analyzer. The collection showed a broad variability which was more evident within the C. annuum group. On average, C. frutescens accessions displayed the highest levels of solid soluble content, pH, polyphenols and antioxidant activity. The Tomato Analyzer descriptors under the categories of size, shape index, and latitudinal section, mostly contributed to the variance among Capsicum groups. C. annuum hold the larger fruits, whereas C. frutescens comprised fruits of smaller sizes. The correlation analysis revealed that biochemical traits were negatively correlated with the fruit parameters related to size, suggesting that huger fruits contain lower amounts of chemical metabolites. The multivariate approximations demonstrated that Andean peppers assorted according to morphometric and colorimetric characteristics, but independently of their species or geographical origin. Groups of valuable native varieties carrying promising traits were identified.

In this work, the impact of pepper (Capsicum spp.) fruits morphology on their composition for health-promoting compounds was investigated. For that purpose, pepper accessions from Ecuador, one of the hotspots in Capsicum’s origin, were analyzed for ascorbic acid, polyphenols, capsaicinoids, and prevention of cholesterol oxidation. Plant and fruit phenomics were assessed with conventional descriptors and Tomato Analyzer digital traits. Significant differences among accessions and species revealed a large diversity within the collection. The Capsicum frutescens group displayed the highest levels of capsaicinoids, whereas the polyphenols shortly varied among the five domesticated species. Capsicum pubescens exhibited the lowest content of ascorbic acid. The conventional descriptors describing the magnitude of plants and fruits, as well as digital attributes under the categories of size, shape index, and latitudinal section, mostly explained the variance among Capsicum groups. Correlation test revealed that phytochemical components were negatively correlated with the morphometric fruit attributes, suggesting that huge fruits contained lower amounts of nutraceutical compounds. Multivariate analysis showed that parameters related to fruit size, shape, and nutraceutical composition primarily contribute to the arrangement of pepper accessions. Such results suggested that those traits have been subjected to higher selection pressures imposed by humans.

Powdery mildew causes severe yield losses in barley production worldwide. Although many resistance genes have been described, only a few have already been cloned. A strong QTL (quantitative trait locus) conferring resistance to a wide array of powdery mildew isolates was identified in a Spanish barley landrace on the long arm of chromosome 7H. Previous studies narrowed down the QTL position, but were unable to identify candidate genes or physically locate the resistance. In this study, the exome of three recombinant lines from a high-resolution mapping population was sequenced and analyzed, narrowing the position of the resistance down to a single physical contig. Closer inspection of the region revealed a cluster of closely related NBS-LRR (nucleotide-binding site–leucine-rich repeat containing protein) genes. Large differences were found between the resistant lines and the reference genome of cultivar Morex, in the form of PAV (presence-absence variation) in the composition of the NBS-LRR cluster. Finally, a template-guided assembly was performed and subsequent expression analysis revealed that one of the new assembled candidate genes is transcribed. In summary, the results suggest that NBS-LRR genes, absent from the reference and the susceptible genotypes, could be functional and responsible for the powdery mildew resistance. The procedure followed is an example of the use of NGS (next-generation sequencing) tools to tackle the challenges of gene cloning when the target gene is absent from the reference genome.

The aim of this study was to estimate the accuracy and convergence of newly developed barley ( L.) genomic resources, primarily genome zipper (GZ) and population sequencing (POPSEQ), at the genome-wide level and to assess their usefulness in applied barley breeding by analyzing seven known loci. Comparison of barley GZ and POPSEQ maps to a newly developed consensus genetic map constructed with data from 13 individual linkage maps yielded an accuracy of 97.8% (GZ) and 99.3% (POPSEQ), respectively, regarding the chromosome assignment. The percentage of agreement in marker position indicates that on average only 3.7% GZ and 0.7% POPSEQ positions are not in accordance with their centimorgan coordinates in the consensus map. The fine-scale comparison involved seven genetic regions on chromosomes 1H, 2H, 4H, 6H, and 7H, harboring major genes and quantitative trait loci (QTL) for disease resistance. In total, 179 GZ loci were analyzed and 64 polymorphic markers were developed. Entirely, 89.1% of these were allocated within the targeted intervals and 84.2% followed the predicted order. Forty-four markers showed a match to a POPSEQ-anchored contig, the percentage of collinearity being 93.2%, on average. Forty-four markers allowed the identification of twenty-five fingerprinted contigs (FPCs) and a more clear delimitation of the physical regions containing the traits of interest. Our results demonstrate that an increase in marker density of barley maps by using new genomic data significantly improves the accuracy of GZ. In addition, the combination of different barley genomic resources can be considered as a powerful tool to accelerate barley breeding.

Single Nucleotide Polymorphisms (SNPs), which constitute single base-pair variations in the DNA sequence, are the most abundant molecular markers in plant and animal genomes. They are becoming the markers of choice for genotyping in all fields of molecular biology, as they are easily prone to automation and high throughput, for example through pyrosequencing. This technology is accurate, flexible and can be easily automated. However, the need for primers labelled with biotin, promptly rise the cost of any methodology employing a pyrosequencing approach. In this protocol we described an improved, efficient, reliable and cost-effective pyrosequencing protocol, based on a universal M13 biotinylated primer, for SNP genotyping in plants.

Recent advances in next-generation sequencing techniques allow the detection of a large number of SNPs and their use in a high throughput manner. However, Cleaved Amplified Polymorphic Sequences (CAPSs) still play a significant role as complement to other high throughput methods for SNP genotyping. Therefore, new methods focusing on the acceleration of this type of markers are highly desirable. The combination of the classical CAPS technique and a M13-tailed primer multiplexing assay was used to develop an agarose gel free protocol for the analysis of SNPs via restriction enzyme digestion. PCR products were fluorescence labeled with a universal M13 primer and subsequently digested with the appropriate restriction endonuclease. After mixing differently labeled products, they were detected on a capillary electrophoresis system. This method allows the cost-effective genotyping of several SNPs in a multiplexed manner at an overall low cost in a short period of time. Additionally, this method could be efficiently combined with the simultaneous detection of SSRs at the same electrophoresis run resulting in a procedure well suited for marker-based selection procedures, genotyping of mapping populations and the assay of genetic diversity.

The successful exploitation of germplasm banks, harbouring plant genetic resources indispensable for plant breeding, will depend on our ability to characterize their genetic diversity. The Vegetable Germplasm Bank of Zaragoza (BGHZ) (Spain) holds an important Capsicum annuum collection, where most of the Spanish pepper variability is represented, as well as several accessions of other domesticated and non-domesticated Capsicum spp from all over the five continents. In the present work, a total of 51 C. annuum landraces (mainly from Spain) and 51 accessions from nine Capsicum species maintained at the BGHZ were evaluated using 39 microsatellite (SSR) markers spanning the whole genome. The 39 polymorphic markers allowed the detection of 381 alleles, with an average of 9.8 alleles per locus. A sizeable proportion of alleles (41.2%) were recorded as specific alleles and the majority of these were present at very low frequencies (rare alleles). Multivariate and model-based analyses partitioned the collection in seven clusters comprising the ten different Capsicum spp analysed: C. annuum, C. chinense, C. frutescens, C. pubescens, C. bacatum, C. chacoense and C. eximium. The data clearly showed the close relationships between C. chinense and C. frutescens. C. cardenasii and C. eximium were indistinguishable as a single, morphologically variable species. Moreover, C. chacoense was placed between C. baccatum and C. pubescens complexes. The C. annuum group was structured into three main clusters, mostly according to the pepper fruit shape, size and potential pungency. Results suggest that the diversification of C. annuum in Spain may occur from a rather limited gene pool, still represented by few landraces with ancestral traits. This ancient population would suffer from local selection at the distinct geographical regions of Spain, giving way to pungent and elongated fruited peppers in the South and Center, while sweet blocky and triangular types in Northern Spain.

Three quantitative trait loci (QTL) conferring broad spectrum resistance to powdery mildew, caused by the fungus Blumeria graminis f. sp. hordei, were previously identified on chromosomes 7HS, 7HL and 6HL in the Spanish barley landrace-derived lines SBCC097 and SBCC145. In the present work, a genome-wide putative linear gene index of barley (Genome Zipper) and the first draft of the physical, genetic and functional sequence of the barley genome were used to go one step further in the shortening and explicit demarcation on the barley genome of these regions conferring resistance to powdery mildew as well as in the identification of candidate genes. First, a comparative analysis of the target regions to the barley Genome Zippers of chromosomes 7H and 6H allowed the development of 25 new gene-based molecular markers, which slightly better delimit the QTL intervals. These new markers provided the framework for anchoring of genetic and physical maps, figuring out the outline of the barley genome at the target regions in SBCC097 and SBCC145. The outermost flanking markers of QTLs on 7HS, 7HL and 6HL defined a physical area of 4 Mb, 3.7 Mb and 3.2 Mb, respectively. In total, 21, 10 and 16 genes on 7HS, 7HL and 6HL, respectively, could be interpreted as potential candidates to explain the resistance to powdery mildew, as they encode proteins of related functions with respect to the known pathogen defense-related processes. The majority of these were annotated as belonging to the NBS-LRR class or protein kinase family.