First report of aflatoxin and ochratoxin contamination in ginger collected from different agroclimatic zones from Punjab, Pakistan.

Ginger, a fresh rhizome, an economically important spice with extensive nutraceutical activities finds itself in vegetable and therapeutic market. Aflatoxins (AFB1, AFB2, AFG1 and AFG2) along with ochratoxin A (OTA) are the most significant and the most toxic form of mycotoxins which are produced by various fungi. This study was initiated to assess the contamination of AFs and OTA in raw and dried ginger products, collected from different agro-climatic zones in Punjab, Pakistan employing the high performance liquid chromatography. We found all (raw ginger samples commercial ginger powders) samples contaminated with AFB1 (range: 29.88-1060.12 µg/kg). AFB2 contamination was much lower (range: 0-17.54 µg/kg). Variable contamination of AFG1 was also observed (range: 0-170.58 µg/kg) whereas AFG2 contamination was found in only three (range: 0-21.88 µg/kg) out of 19 raw ginger samples. OTA contamination ranged from 0.05 to 3.42 µg/kg. Ginger samples from lower altitudes (<1000 m) were more contaminated with AFB1 sub type mycotoxin. Keeping in view that the toxicity of AFs is in the order of B1>G1> B2>G2, it was alarming to find that 100% of the samples were contaminated with AFB1 way beyond the permissible limits. Our very first report about the contamination of ginger with AFs presents a grave health issue because of wide use of ginger. We conclude that ginger production in Pakistan needs to be carefully crafted and due diligence is needed during ginger cultivation, harvest and post-harvest operations because the amount of aflatoxins detected in this study are very much above the permissible limits. In this regard, ginger storage in cooler environments such as refrigerator should be encouraged to contain the AFs proliferation.

GWAS in a Collection of Bulgarian Old and Modern Bread Wheat Accessions Uncovers Novel Genomic Loci for Grain Protein Content and Thousand Kernel Weight.

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.

Nutritional Genomic Approach for Improving Grain Protein Content in Wheat.

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 Analyses of Seed Longevity in Capsicum annuum L. in Cold Storage Conditions.

Seed longevity is the most important trait in the genebank management system. No seed can remain infinitely viable. There are 1241 accessions of Capsicum annuum L. available at the German Federal ex situ genebank at IPK Gatersleben. C. annuum (Capsicum) is the most economically important species of the genus Capsicum. So far, there is no report that has addressed the genetic basis of seed longevity in Capsicum. Here, we convened a total of 1152 Capsicum accessions that were deposited in Gatersleben over forty years (from 1976 to 2017) and assessed their longevity by analyzing the standard germination percentage after 5-40 years of storage at -15/-18 °C. These data were used to determine the genetic causes of seed longevity, along with 23,462 single nucleotide polymorphism (SNP) markers covering all of the 12 Capsicum chromosomes. Using the association-mapping approach, we identified a total of 224 marker trait associations (MTAs) (34, 25, 31, 35, 39, 7, 21 and 32 MTAs after 5-, 10-, 15-, 20-, 25-, 30-, 35- and 40-year storage intervals) on all the Capsicum chromosomes. Several candidate genes were identified using the blast analysis of SNPs, and these candidate genes are discussed.

Estimation of genetic diversity using seed storage protein (SSP) profiling in wild and cultivated species of Cicer L.

BACKGROUND: The narrow genetic diversity of chickpea is a serious impediment to modern cultivar creation. Seed storage proteins (SSPs) are stable and have minimal or no degradation when subjected to isolation and SDS-PAGE. METHODS AND RESULTS: We have characterized SSPs of 436 chickpea genotypes, belonging to nine annual Cicer species, originated from 47 countries by SDS-PAGE and determined the extent of genetic diversity in chickpea through clustering. Based on scoring, a total of 44 bands (10 to 170 kDa) were identified, which were all polymorphic. The least appeared protein bands were 11, 160 and 170 kDa where band of 11 and 160 kDa was present exclusively in wild type. Five bands were present in < 10% of genotypes. Bands appeared in 200-300 genotypes were suggested less polymorphic, on contrary bands present in 10-150 genotypes were suggested more polymorphic. Polymorphism of protein bands in context to their potential functions reported in literature were explored and suggested that the glubulins were most and glutelins were least abundant, whereas albumins with their known role in stress tolerance can be used as marker in chickpea breeding. Cluster analysis produced 14 clusters, interestingly three clusters contained only Pakistani genotypes and thus Pakistani genotypes appeared as a separate entity from the rest of the genotypes. CONCLUSION: Our results indicate that SDS-PAGE of SSPs is a powerful technique in determining the genetic diversity plus it is easily adaptable, due to its cost effectiveness in comparison to other genomics tools.

A GBS-based genome-wide association study reveals the genetic basis of salinity tolerance at the seedling stage in bread wheat (Triticum aestivum L.).

High salinity levels affect 20% of the cultivated area and 9%-34% of the irrigated agricultural land worldwide, ultimately leading to yield losses of crops. The current study evaluated seven salt tolerance-related traits at the seedling stage in a set of 138 pre-breeding lines (PBLs) and identified 63 highly significant marker-trait associations (MTAs) linked to salt tolerance. Different candidate genes were identified in in silico analysis, many of which were involved in various stress conditions in plants, including glycine-rich cell wall structural protein 1-like, metacaspase-1, glyceraldehyde-3-phosphate dehydrogenase GAPA1, and plastidial GAPA1. Some of these genes coded for structural protein and participated in cell wall structure, some were linked to programmed cell death, and others were reported to show abiotic stress response roles in wheat and other plants. In addition, using the Multi-Trait Genotype-Ideotype Distance Index (MGIDI) protocol, the best-performing lines under salt stress were identified. The SNPs identified in this study and the genotypes with favorable alleles provide an excellent source to impart salt tolerance in wheat.

A GBS-Based GWAS Analysis of Leaf and Stripe Rust Resistance in Diverse Pre-Breeding Germplasm of Bread Wheat (Triticum aestivum L.).

Yellow (YR) and leaf (LR) rusts caused by Puccinia striiformis f. sp. tritici (Pst) and Puccinia triticina, respectively, are of utmost importance to wheat producers because of their qualitative and quantitative effect on yield. The search for new loci resistant to both rusts is an ongoing challenge faced by plant breeders and pathologists. Our investigation was conducted on a subset of 168 pre-breeding lines (PBLs) to identify the resistant germplasm against the prevalent local races of LR and YR under field conditions followed by its genetic mapping. Our analysis revealed a range of phenotypic responses towards both rusts. We identified 28 wheat lines with immune response and 85 resistant wheat genotypes against LR, whereas there were only eight immune and 52 resistant genotypes against YR. A GWAS (genome-wide association study) identified 190 marker-trait associations (MTAs), where 120 were specific to LR and 70 were specific to YR. These MTAs were confined to 86 quantitative trait loci (QTLs), where 50 QTLs carried MTAs associated with only LR, 29 QTLs carried MTAs associated with YR, and seven QTLs carried MTAs associated with both LR and YR. Possible candidate genes at the site of these QTLs are discussed. Overall, 70 PBLs carried all seven LR/YR QTLs. Furthermore, there were five PBLs with less than five scores for both LR and YR carrying positive alleles of all seven YR/LR QTLs, which are fit to be included in a breeding program for rust resistance induction.

QTL Analysis for Bread Wheat Seed Size, Shape and Color Characteristics Estimated by Digital Image Processing.

The size, shape, and color of wheat seeds are important traits that are associated with yield and flour quality (size, shape), nutritional value, and pre-harvest sprouting (coat color). These traits are under multigenic control, and to dissect their molecular and genetic basis, quantitative trait loci (QTL) analysis is used. We evaluated 114 recombinant inbred lines (RILs) in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative, ITMI/MP) grown in 2014 season. We used digital image analysis for seed phenotyping and obtained data for seven traits describing seed size and shape and 48 traits of seed coat color. We identified 212 additive and 34 pairs of epistatic QTLs on all the chromosomes of wheat genome except chromosomes 1A and 5D. Many QTLs were overlapping. We demonstrated that the overlap between QTL regions was low for seed size/shape traits and high for coat color traits. Using the literature and KEGG data, we identified sets of genes in Arabidopsis and rice from the networks controlling seed size and color. Further, we identified 29 and 14 candidate genes for seed size-related loci and for loci associated with seed coat color, respectively.

Genetic Aspects and Molecular Causes of Seed Longevity in Plants-A Review.

Seed longevity is the most important trait related to the management of gene banks because it governs the regeneration cycle of seeds. Thus, seed longevity is a quantitative trait. Prior to the discovery of molecular markers, classical genetic studies have been performed to identify the genetic determinants of this trait. Post-2000 saw the use of DNA-based molecular markers and modern biotechnological tools, including RNA sequence (RNA-seq) analysis, to understand the genetic factors determining seed longevity. This review summarizes the most important and relevant genetic studies performed in Arabidopsis (24 reports), rice (25 reports), barley (4 reports), wheat (9 reports), maize (8 reports), soybean (10 reports), tobacco (2 reports), lettuce (1 report) and tomato (3 reports), in chronological order, after discussing some classical studies. The major genes identified and their probable roles, where available, are debated in each case. We conclude by providing information about many different collections of various crops available worldwide for advanced research on seed longevity. Finally, the use of new emerging technologies, including RNA-seq, in seed longevity research is emphasized by providing relevant examples.

Genetic Insight Into the Insect Resistance in Bread Wheat Exploiting the Untapped Natural Diversity.

Climate change is an undeniable threat to sustainable wheat production in the future as an increased temperature will significantly increase grain loss due to the increased number of generations per season of multivoltine species that are detrimental to plants. Among insects, orange wheat blossom midge (OWBM), yellow wheat blossom midge (YWBM), saddle gall midge (SGM), thrips, and frit fly (FF) are important wheat pests in the European environments, which can be managed by the development of resistant cultivars. This involves the identification, confirmation, and incorporation of insect resistance sources into new high-yielding cultivars. We used two diverse and unrelated wheat [winter wheat (WW) and spring wheat (SW)] panels to associate single-nucleotide polymorphism (SNP) markers with the mentioned pests using the tools of association mapping. All in all, a total of 645 and 123 significant associations were detected in WW and SW, respectively, which were confined to 246 quantitative trait loci. Many candidate genes were identified using the BLAST analysis of the sequences of associated SNPs. Some of them are involved in controlling the physical structures of plants such as stomatal immunity and closure, cuticular wax in leaf blade, whereas others are involved in the production of certain enzymes in response to biotic and abiotic stresses. To our knowledge, this is the first detailed investigation that deals with YWBM, SGM, thrips, and FF resistance genetics using the natural variation in wheat. The reported germplasm is also readily available to breeders across the world that can make rational decisions to breed for the pest resilience of their interest by including the resistant genotypes being reported.

Mapping Resistance to Argentinean Fusarium (Graminearum) Head Blight Isolates in Wheat.

Fusarium head blight (FHB) of wheat, caused by Fusarium graminearum (Schwabe), is a destructive disease worldwide, reducing wheat yield and quality. To accelerate the improvement of scab tolerance in wheat, we assessed the International Triticeae Mapping Initiative mapping population (ITMI/MP) for Type I and II resistance against a wide population of Argentinean isolates of F. graminearum. We discovered a total of 27 additive QTLs on ten different (2A, 2D, 3B, 3D, 4B, 4D, 5A, 5B, 5D and 6D) wheat chromosomes for Type I and Type II resistances explaining a maximum of 15.99% variation. Another four and two QTLs for thousand kernel weight in control and for Type II resistance, respectively, involved five different chromosomes (1B, 2D, 6A, 6D and 7D). Furthermore, three, three and five QTLs for kernel weight per spike in control, for Type I resistance and for Type II resistance, correspondingly, involved ten chromosomes (2A, 2D, 3B, 4A, 5A, 5B, 6B, 7A, 7B, 7D). We were also able to detect five and two epistasis pairs of QTLs for Type I and Type II resistance, respectively, in addition to additive QTLs that evidenced that FHB resistance in wheat is controlled by a complex network of additive and epistasis QTLs.

A SNP-based genetic dissection of versatile traits in bread wheat (Triticum aestivum L.).

The continuous increase in global population prompts increased wheat production. Future wheat (Triticum aestivum L.) breeding will heavily rely on dissecting molecular and genetic bases of wheat yield and related traits which is possible through the discovery of quantitative trait loci (QTLs) in constructed populations, such as recombinant inbred lines (RILs). Here, we present an evaluation of 92 RILs in a bi-parental RIL mapping population (the International Triticeae Mapping Initiative Mapping Population [ITMI/MP]) using newly generated phenotypic data in 3-year experiments (2015), older phenotypic data (1997-2009), and newly created single nucleotide polymorphism (SNP) marker data based on 92 of the original RILs to search for novel and stable QTLs. Our analyses of more than 15 unique traits observed in multiple experiments included analyses of 46 traits in three environments in the USA, 69 traits in eight environments in Germany, 149 traits in 10 environments in Russia, and 28 traits in four environments in India (292 traits in 25 environments) with 7584 SNPs (292 × 7584 = 2 214 528 data points). A total of 874 QTLs were detected with limit of detection (LOD) scores of 2.01-3.0 and 432 QTLs were detected with LOD > 3.0. Moreover, 769 QTLs could be assigned to 183 clusters based on the common markers and relative proximity of related QTLs, indicating gene-rich regions throughout the A, B, and D genomes of common wheat. This upgraded genotype-phenotype information of ITMI/MP can assist breeders and geneticists who can make crosses with suitable RILs to improve or investigate traits of interest.

Genetic basis of some physiological traits and yield in early and late sowing conditions in bread wheat (Triticum aestivum L.).

The rise in human population necessitates the use of all available tools to enhance wheat productivity. In this regard, pre-breeding has mobilized novel underutilized genetic variation into breeding programs. However, this germplasm needs to be characterized for its efficient utilization. This investigation was initiated to evaluate the early and late sown wheat pre-breeding germplasm for physiology- and yield-related traits and to associate the mapped SNPs using association mapping approach. Our results indicate that the germplasm performed better in early sowing in comparison to late planting where grain yield (Yd) was found positively correlated with water use efficiency (WUE), heading time, and chlorophyll contents (Chl). We discovered a total of 210 associations involving 155 SNPs. Taking into consideration either early or late sowing and the mean values, only 12 marker traits were associated with trait germination, plant height, stomatal conductance, transpiration rate, Chl, carotenoids, and Yd. Our correlations and mapping results indicate that higher WUE along with Chl can be targeted as indirect physiological markers to enhance wheat yield.

A GWAS to identify the cereal cyst nematode (Heterodera filipjevi) resistance loci in diverse wheat prebreeding lines.

Yield losses because of cereal cyst nematodes could be as high as 92%, causing a bottleneck for wheat production. An integrated approach (application of pesticides, crop rotation, and use of host resistance) is needed to manage this devastating pathogen where resistant cultivars are considered most effective. This necessitates the identification of nematode-resistant sources in the available germplasm. Here, we report on the genetic mapping of nematode resistance in 255 diverse prebreeding lines (PBLs) employing an association mapping strategy. Altogether, seven additive quantitative trait loci (QTL) were identified on chromosomes 1A, 2A, 2B, 2D, 3A, 6B, and 6D explaining a maximum of 9.42% phenotypic variation where at least five QTL (on chromosomes 2A, 2B, 2D, 6B, and 6D) are located on the same chromosomes that harbor the already known nematode resistance genes. Resistant PBLs carried Aegilops squarrosa (436) in their pedigree which could be the possible source of positive alleles. To add to it, better yield performance of the identified nematode-resistant lines under stress conditions indicates that the germplasm can provide both nematode resistance and high-yielding cultivars.

A GBS-based GWAS analysis of adaptability and yield traits in bread wheat (Triticum aestivum L.).

Wheat is a foremost food grain of Pakistan and occupies a vital position in agricultural policies of the country. Wheat demand will be increased by 60% by 2050 which is a serious concern to meet this demand. Conventional breeding approaches are not enough to meet the demand of growing human population. It is paramount to integrate underutilized genetic diversity into wheat gene pool through efficient and accurate breeding tools and technology. In this study, we present the genetic analysis of a 312 diverse pre-breeding lines using DArT-seq SNPs seeking to elucidate the genetic components of emergence percentage, heading time, plant height, lodging, thousand kernel weight, and yield (Yd) which resulted in detection of 201 significant (p value < 10-3) and 61 highly significant associations (p value < 1.45 × 10-4). More importantly, chromosomes 1B and 2A carried loci linked to Yd in two different seasons, and an increase of up to 8.20% is possible in Yd by positive allele mining. We identified seven lines with > 4 positive alleles for Yd whose pedigree carried Aegilops squarrosa as one of the parents providing evidence that Aegilops species, apart from imparting resistance against biotic stresses, may also provide alleles for yield enhancement.

Mapping of QTL associated with seed longevity in durum wheat (Triticum durum Desf.).

Regeneration cycle of seeds kept at genebanks is determined by seed longevity. Information about longevity of species is important for storage periods, germination test intervals, and reproduction cycles. Seed longevity is different between species and depends on the storage conditions. It also differs between genotypes of a species providing the basis of genetic analyses of seed longevity. Studies in hexaploid wheat and barley have identified numerous quantitative trait locus (QTL) linked to the trait. Seed longevity in durum wheat, however, has not been attempted so far. Here, we present the first report of genetic analysis of grain longevity in durum wheat using a bi-parental mapping population composed of 114 recombinant inbred lines. QTL analysis identified three highly significant and one significant QTL for initial germination (on chromosomes 4B, 5A (2 QTL), and 6B), three significant QTL for germination after accelerated aging treatment (on chromosomes 5A and 7B (2 QTL)), and five significant QTL determining relative germination and distributed on chromosomes 3A, 3B, 5A, 6B, and 7B. This study confirms the results of previous investigations in bread wheat and provides a baseline for further research in durum wheat.

Genetic architecture of seed longevity in bread wheat (Triticum aestivum L.).

The deterioration in the quality of ex situ conserved seed over time reflects a combination of both physical and chemical changes. Intraspecific variation for longevity is, at least in part, under genetic control. Here, the grain of 183 bread wheat accessions maintained under low-temperature storage at the IPK-Gatersleben genebank over some decades have been tested for their viability, along with that of fresh grain subjected to two standard artificial ageing procedures. A phenotype-genotype association analysis, conducted to reveal the genetic basis of the observed variation between accessions, implicated many regions of the genome, underling the genetic complexity of the trait. Some, but not all, of these regions were associated with variation for both natural and experimental ageing, implying some non-congruency obtains between these two forms of testing for longevity. The genes underlying longevity appear to be independent of known genes determining dormancy and pre-harvest sprouting.

The inheritance of wheat grain longevity: a comparison between induced and natural ageing.

Seed longevity is an important trait for both ex situ genebanks and the seed industry. It is partially determined by genetic factors, but is also dependent on the environmental conditions experienced by the mother plant during seed maturation, as well as those imposed during the post-harvest and storage periods. For practical reasons, the variation in longevity has repeatedly been analysed by treating fresh seed to various induced ageing protocols, but the extent to which these procedures mimic the natural ageing process remains debatable. Here, a comparison was attempted between the wheat genomic regions identified by biparental mapping as harbouring determinants of viability loss identified in grain which had been either aged artificially or had been stored long term. Only one locus proved to be shared, but even here, the parental origin of the positive allele differed. Correlation analysis revealed no relationship between various induced ageing treatments and long-term storage.

Molecular markers in management of ex situ PGR-a case study.

Worldwide germplasm collections contain about 7.4 million accessions of plant genetic resources for food and agriculture. One of the 10 largest ex situ genebanks of our globe is located at the Leibniz Institute of Plant Genetics and Crop Plant Research in Gatersleben, Germany. Molecular tools have been used for various gene bank management practices including characterization and utilization of the germplasm. The results on genetic integrity of longterm- stored gene bank accessions of wheat (self-pollinating) and rye (open-pollinating) cereal crops revealed a high degree of identity for wheat. In contrast, the out-pollinating accessions of rye exhibited shifts in allele frequencies. The genetic diversity of wheat and barley germplasm collected at intervals of 40 to 50 years in comparable geographical regions showed qualitative rather than a quantitative change in diversity. The inter- and intraspecific variation of seed longevity was analysed and differences were detected. Genetic studies in barley, wheat and oilseed rape revealed numerous QTL, indicating the complex and quantitative nature of seed longevity. Some of the loci identified were in genomic regions that co-localize with genes determining agronomic traits such as spike architecture or biotic and abiotic stress response. Finally, a genome-wide association mapping analysis of a core collection of wheat for flowering time was performed using diversity array technology (DArT) markers. Maker trait associations were detected in genomic regions where major genes or QTL have been described earlier. In addition, new loci were also detected, providing opportunities to monitor genetic variation for crop improvement.