Multi-environment association study highlights candidate genes for robust agronomic quantitative trait loci in a novel worldwide Capsicum core collection.

Investigating crop diversity through genome-wide association studies (GWAS) on core collections helps in deciphering the genetic determinants of complex quantitative traits. Using the G2P-SOL project world collection of 10 038 wild and cultivated Capsicum accessions from 10 major genebanks, we assembled a core collection of 423 accessions representing the known genetic diversity. Since complex traits are often highly dependent upon environmental variables and genotype-by-environment (G × E) interactions, multi-environment GWAS with a 10 195-marker genotypic matrix were conducted on a highly diverse subset of 350 Capsicum annuum accessions, extensively phenotyped in up to six independent trials from five climatically differing countries. Environment-specific and multi-environment quantitative trait loci (QTLs) were detected for 23 diverse agronomic traits. We identified 97 candidate genes potentially implicated in 53 of the most robust and high-confidence QTLs for fruit flavor, color, size, and shape traits, and for plant productivity, vigor, and earliness traits. Investigating the genetic architecture of agronomic traits in this way will assist the development of genetic markers and pave the way for marker-assisted selection. The G2P-SOL pepper core collection will be available upon request as a unique and universal resource for further exploitation in future gene discovery and marker-assisted breeding efforts by the pepper community.

Global range expansion history of pepper (Capsicum spp.) revealed by over 10,000 genebank accessions.

Genebanks collect and preserve vast collections of plants and detailed passport information, with the aim of preserving genetic diversity for conservation and breeding. Genetic characterization of such collections has the potential to elucidate the genetic histories of important crops, use marker-trait associations to identify loci controlling traits of interest, search for loci undergoing selection, and contribute to genebank management by identifying taxonomic misassignments and duplicates. We conducted a genomic scan with genotyping by sequencing (GBS) derived single nucleotide polymorphisms (SNPs) of 10,038 pepper (Capsicum spp.) accessions from worldwide genebanks and investigated the recent history of this iconic staple. Genomic data detected up to 1,618 duplicate accessions within and between genebanks and showed that taxonomic ambiguity and misclassification often involve interspecific hybrids that are difficult to classify morphologically. We deeply interrogated the genetic diversity of the commonly consumed Capsicum annuum to investigate its history, finding that the kinds of peppers collected in broad regions across the globe overlap considerably. The method ReMIXTURE-using genetic data to quantify the similarity between the complement of peppers from a focal region and those from other regions-was developed to supplement traditional population genetic analyses. The results reflect a vision of pepper as a highly desirable and tradable cultural commodity, spreading rapidly throughout the globe along major maritime and terrestrial trade routes. Marker associations and possible selective sweeps affecting traits such as pungency were observed, and these traits were shown to be distributed nonuniformly across the globe, suggesting that human preferences exerted a primary influence over domesticated pepper genetic structure.

Genome wide association mapping for agronomic, fruit quality, and root architectural traits in tomato under organic farming conditions.

BACKGROUND: Opportunity and challenges of the agriculture scenario of the next decades will face increasing demand for secure food through approaches able to minimize the input to cultivations. Large panels of tomato varieties represent a valuable resource of traits of interest under sustainable cultivation systems and for genome-wide association studies (GWAS). For mapping loci controlling the variation of agronomic, fruit quality, and root architecture traits, we used a heterogeneous set of 244 traditional and improved tomato accessions grown under organic field trials. Here we report comprehensive phenotyping and GWAS using over 37,300 SNPs obtained through double digest restriction-site associated DNA (dd-RADseq). RESULTS: A wide range of phenotypic diversity was observed in the studied collection, with highly significant differences encountered for most traits. A variable level of heritability was observed with values up to 69% for morphological traits while, among agronomic ones, fruit weight showed values above 80%. Genotype by environment analysis highlighted the strongest genotypic effect for aboveground traits compared to root architecture, suggesting that the hypogeal part of tomato plants has been a minor objective for breeding activities. GWAS was performed by a compressed mixed linear model leading to 59 significantly associated loci, allowing the identification of novel genes related to flower and fruit characteristics. Most genomic associations fell into the region surrounding SUN, OVATE, and MYB gene families. Six flower and fruit traits were associated with a single member of the SUN family (SLSUN31) on chromosome 11, in a region involved in the increase of fruit weight, locules number, and fruit fasciation. Furthermore, additional candidate genes for soluble solids content, fruit colour and shape were found near previously reported chromosomal regions, indicating the presence of synergic and multiple linked genes underlying the variation of these traits. CONCLUSIONS: Results of this study give new hints on the genetic basis of traits in underexplored germplasm grown under organic conditions, providing a framework for the development of markers linked to candidate genes of interest to be used in genomics-assisted breeding in tomato, in particular under low-input and organic cultivation conditions.

Overview of Biotic Stresses in Pepper (Capsicum spp.): Sources of Genetic Resistance, Molecular Breeding and Genomics.

Pepper (Capsicum spp.) is one of the major vegetable crops grown worldwide largely appreciated for its economic importance and nutritional value. This crop belongs to the large Solanaceae family, which, among more than 90 genera and 2500 species of flowering plants, includes commercially important vegetables such as tomato and eggplant. The genus includes over 30 species, five of which (C. annuum, C. frutescens, C. chinense, C. baccatum, and C. pubescens) are domesticated and mainly grown for consumption as food and for non-food purposes (e.g., cosmetics). The main challenges for vegetable crop improvement are linked to the sustainable development of agriculture, food security, the growing consumers' demand for food. Furthermore, demographic trends and changes to climate require more efficient use of plant genetic resources in breeding programs. Increases in pepper consumption have been observed in the past 20 years, and for maintaining this trend, the development of new resistant and high yielding varieties is demanded. The range of pathogens afflicting peppers is very broad and includes fungi, viruses, bacteria, and insects. In this context, the large number of accessions of domesticated and wild species stored in the world seed banks represents a valuable resource for breeding in order to transfer traits related to resistance mechanisms to various biotic stresses. In the present review, we report comprehensive information on sources of resistance to a broad range of pathogens in pepper, revisiting the classical genetic studies and showing the contribution of genomics for the understanding of the molecular basis of resistance.

Occurrence of variable levels of health-promoting fruit compounds in horn-shaped Italian sweet pepper varieties assessed by a comprehensive approach.

BACKGROUND: Cultivated pepper is a rich source of diverse bioactive compounds with potential properties related to the prevention of major degenerative and proliferative diseases. In the present study, 15 sweet pepper varieties, highly appreciated on the Italian market, were assessed for variation in the content of ascorbic acid, isoprenoids and flavonoids, as well as morpho-agronomic performances and molecular diversity. RESULTS: The collection under study showed a wide variability for all traits considered. Traditional cultivars, although less productive, were characterized by a high content of ß-carotene and ascorbic acid, reaching maximum concentrations of 230.5 mg kg-1 fresh weight (fw) and 2750 mg kg-1 fw, respectively. Strong correlations were demonstrated between neoxanthin and luteolins. Fruit weight was positively correlated with α-tocopherol content and negatively correlated with luteolins content. The genotyping by sequencing platform allowed the identification of 1833 single nucleotide polymorphism, which better defined the relationships among cultivars, based on provenance and improvement rate. CONCLUSIONS: The present study provides an overview of the variability in the expression of fruit nutritional traits in a collection of horn-shaped pepper cultivars, integrating agronomic and molecular data. The impact for breeding and consumers is discussed. © 2017 Society of Chemical Industry.

Polyphenol Oxidases in Crops: Biochemical, Physiological and Genetic Aspects.

Enzymatic browning is a colour reaction occurring in plants, including cereals, fruit and horticultural crops, due to oxidation during postharvest processing and storage. This has a negative impact on the colour, flavour, nutritional properties and shelf life of food products. Browning is usually caused by polyphenol oxidases (PPOs), following cell damage caused by senescence, wounding and the attack of pests and pathogens. Several studies indicated that PPOs play a role in plant immunity, and emerging evidence suggested that PPOs might also be involved in other physiological processes. Genomic investigations ultimately led to the isolation of PPO homologs in several crops, which will be possibly characterized at the functional level in the near future. Here, focusing on the botanic families of Poaceae and Solanaceae, we provide an overview on available scientific literature on PPOs, resulting in useful information on biochemical, physiological and genetic aspects.

Application of High-Resolution Melting and DNA Barcoding for Discrimination and Taxonomy Definition of Rocket Salad (Diplotaxis spp.) Species.

Nuclear and cytoplasmic DNA barcoding regions are useful for plant identification, breeding, and phylogenesis. In this study, the genetic diversity of 17 Diplotaxis species, was investigated with 5 barcode markers. The allelic variation was based on the sequences of chloroplast DNA markers including the spacer between trnL and trnF and tRNA-Phe gene (trnL-F), the rubisco (rbcl), the maturase K (matk), as well as the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA. A highly polymorphic marker (HRM500) derived from a comparison of cytoplasmic genome sequences in Brassicaceae, was also included. Subsequently, a real-time PCR method coupled with HRM analysis was implemented to better resolve taxonomic relationships and identify assays suitable for species identification. Integration of the five barcode regions revealed a grouping of the species according to the common chromosomal set number. Clusters including species with n = 11 (D. duveryrieriana or cretacea, D. tenuifolia, D. simplex and D. acris), n = 8 (D. ibicensis, D. brevisiliqua and D. ilorcitana), and n = 9 (D. brachycarpa, D. virgata, D. assurgens, and D. berthautii) chromosomes were identified. Both phylogenetic analysis and the genetic structure of the collection identified D. siifolia as the most distant species. Previous studies emphasized this species' extremely high glucosinolate content, particularly for glucobrassicin. High-resolution melting analysis showed specific curve patterns useful for the discrimination of the species, thus determining ITS1 as the best barcode for fingerprinting. Findings demonstrate that the approach used in this study is effective for taxa investigations and genetic diversity studies.

Harnessing Crop Diversity through Genetics, Genomics and Phenomics Approaches.

Developing resilient cultivars ensuring adequate productions will be the agriculture industry's primary challenge in the coming decades to ensure food security, especially with climate change and a growing global population [...].

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.

Development and application of Single Primer Enrichment Technology (SPET) SNP assay for population genomics analysis and candidate gene discovery in lettuce.

Single primer enrichment technology (SPET) is a novel high-throughput genotyping method based on short-read sequencing of specific genomic regions harboring polymorphisms. SPET provides an efficient and reproducible method for genotyping target loci, overcoming the limits associated with other reduced representation library sequencing methods that are based on a random sampling of genomic loci. The possibility to sequence regions surrounding a target SNP allows the discovery of thousands of closely linked, novel SNPs. In this work, we report the design and application of the first SPET panel in lettuce, consisting of 41,547 probes spanning the whole genome and designed to target both coding (~96%) and intergenic (~4%) regions. A total of 81,531 SNPs were surveyed in 160 lettuce accessions originating from a total of 10 countries in Europe, America, and Asia and representing 10 horticultural types. Model ancestry population structure clearly separated the cultivated accessions (Lactuca sativa) from accessions of its presumed wild progenitor (L. serriola), revealing a total of six genetic subgroups that reflected a differentiation based on cultivar typology. Phylogenetic relationships and principal component analysis revealed a clustering of butterhead types and a general differentiation between germplasm originating from Western and Eastern Europe. To determine the potentiality of SPET for gene discovery, we performed genome-wide association analysis for main agricultural traits in L. sativa using six models (GLM naive, MLM, MLMM, CMLM, FarmCPU, and BLINK) to compare their strength and power for association detection. Robust associations were detected for seed color on chromosome 7 at 50 Mbp. Colocalization of association signals was found for outer leaf color and leaf anthocyanin content on chromosome 9 at 152 Mbp and on chromosome 5 at 86 Mbp. The association for bolting time was detected with the GLM, BLINK, and FarmCPU models on chromosome 7 at 164 Mbp. Associations were detected in chromosomal regions previously reported to harbor candidate genes for these traits, thus confirming the effectiveness of SPET for GWAS. Our findings illustrated the strength of SPET for discovering thousands of variable sites toward the dissection of the genomic diversity of germplasm collections, thus allowing a better characterization of lettuce collections.

Development, Preparation, and Curation of High-Throughput Phenotypic Data for Genome-Wide Association Studies: A Sample Pipeline in R.

Genome-wide association studies (GWAS) have benefited from the advances of sequencing methods for the generation of high-density genomic data. By bridging genotype to phenotype, several genes have been associated with traits of agricultural interest. Despite this, there is still a gap between genotyping and phenotyping due to the large difference in throughput between the two disciplines. Although cutting-edge phenomics technologies are available to the community, their costs are still prohibitive at the small lab level. Semiautomated methods of investigation provide a valid alternative to generate large-scale phenotyping data able to deeply investigate the characteristics of different plant organs. Beyond automation, phenomics data management is another major constraint to consider; while bioinformatics pipelines are well-trained for releasing high-quality genomic data, fewer efforts have been done for phenotyping information. This chapter provides a guide for generating large-scale data related to the size and shape of fruits, leaves, seeds, and roots and for downstream analysis for curation and preparation of clean datasets, through removal of outliers and performing primary statistical analysis. Different steps to be carried out in the R environment will be shown for gathering the appropriate input information to use in GWAS avoiding any possible bias.

Next generation sequencing technologies to explore the diversity of germplasm resources: Achievements and trends in tomato.

Tomato is one of the major vegetable crops grown worldwide and a model species for genetic and biological research. Progress in genomic technologies made possible the development of forefront methods for high-scale sequencing, providing comprehensive insight into the genetic architecture of germplasm resources. This review revisits next-generation sequencing strategies and applications to investigate the diversity of tomato, describing the common platforms used for SNP genotyping of large collections, de novo sequencing, and whole genome resequencing. Significant findings in evolutionary history are outlined, thus discussing how genomics has provided new hints about the processes behind domestication. Finally, achievement and perspectives on pan-genome construction and graphical pan-genome development toward precise mining of the natural variation to be exploited for breeding purposes are presented.

Whole-genome resequencing reveals genomic footprints of Italian sweet and hot pepper heirlooms giving insight into genes underlying key agronomic and qualitative traits.

BACKGROUND: Pepper is a major crop species of the Solanaceae family, largely appreciated for its high nutritional and healthy contribution to human diets. In the Mediterranean basin, the favorable pedoclimatic conditions enhanced the selection of several diversified landraces cultivated pepper (Capsicum annuum), for whom Italy can be considered a main pole of diversification. Hence, a survey of traditional C. annuum genetic resources is essential for deep understanding of such diversity and for applications in genomics assisted breeding. Here, we report whole-genome resequencing analyses of two sweet and two pungent genotypes highly diffused in South Italy and representative of the variability for shape, colour and nutritional properties. RESULTS: The four genomes were reconstructed at a chromosomal scale using a reference-guided approach, based on a dataset of 2.6 billion paired-end reads, corresponding to 20× genome coverage and a mapping rate above 99% for a final genomes size of approximately 3 Gb. After five iterations of variant calling, a total of 29,258,818 single nucleotide polymorphisms (SNPs) and 1,879,112 InDels, were identified. Substantial differences were observed among the four genomes based on geographical origin, with chromosomes 9 and 11 showing more polymorphisms in the accessions with higher fruit weight and absence of pungency. Among the identified variants, a small private indel (T - > TA) shared between sweet and big fruits accessions induces a frameshift with the generation of a new stop codon in a gene annotated as extensin, whereas two private SNPs within hot types were identified in 1-aminocyclopropane-1-carboxylate oxidase (ACO), a key gene involved in fruit ripening. The estimation of repetitive elements highlights a preponderant presence of Long Terminal Repeats (LTRs), the majority of which belonged to Gypsy superfamily. By comparing the four genomes with publicly available references including 'CM334' and Zunla-1 highlight the presence of 49,475 shared gene families. CONCLUSIONS: The new genomic sequences aim to enrich the whole genome information of pepper local varieties, providing a valuable tool for precision gene mapping, marker discovery, comparative studies. Such knowledge widens the frontiers to understand the selection history of Italian pepper landraces toward the recognition of specificity local agri-food products marks.

Occurrence of Macrophomina phaseolina on Chickpea in Italy: Pathogen Identification and Characterization.

Climate change has led to the spread of plant pathogens in novel environments, causing dramatic crop losses and economic damage. Botryosphaeriaceae represents a massive fungal family, containing a huge number of plant pathogens, which are able to infect several hosts. Among them, Macrophomina phaseolina is a necrotrophic fungus, responsible for several plant diseases, including the soft stem rot of common bean, crown rot on strawberry and charcoal rot of several legumes. Here, Macrophomina, causing crown charcoal rot in chickpeas, was isolated from symptomatic plants in Cicerale (SA), Campania, South Italy. Morphological and molecular characterization was carried out and pathogenicity tests were performed. Phylogenetic analyses were performed comparing Macrophomina strains coming from different geographic areas and hosts. The experiments confirmed the pathogenicity of the isolate CREA OF 189.2 on chickpea, while host range highlighted the polyphagous nature of this strain; thus, symptoms were reported on lentils, common bean and cantaloupe. The multidisciplinary approach allows us to increase the knowledge about this emerging pathogen. To the best of our knowledge, this is the first report on Macrophomina phaseolina from chickpeas in Italy.

An integrated genomic and biochemical approach to investigate the potentiality of heirloom tomatoes: Breeding resources for food quality and sustainable agriculture.

A revival of interest in traditional varieties reflects the change in consumer preferences and the greater awareness of the quality of locally grown products. As ancient cultivars, heirlooms have been selected for decades in specific habitats and represent nowadays potential germplasm sources to consider for breeding high-quality products and cultivation in sustainable agriculture. In this study, 60 heirloom tomato (Solanum lycopersicum L.) accessions, including diverse varietal types (beefsteak, globe, oxheart, plum, and cherry), were profiled over two seasons for the main chemical and biochemical fruit traits. A medium-high level of heritability was found for all traits ranging from 0.52 for soluble solids to 0.99 for fruit weight. The average content of ascorbic acid was ~31 mg 100 g-1 of fw in both seasons, while the greatest variability was found for carotenoids with peaks of 245.65 µg g-1 of fw for total lycopene and 32.29 µg g-1 of fw for ß-carotene. Dissection of genotypic (G) and seasonal (Y) factors highlighted genotype as the main source of variation for all traits. No significant effect of Y and G × Y was found for ascorbic acid and fruit weight, respectively, whereas a high influence of Y was found on the variation of lycopene. Molecular fingerprinting was performed using the 10K SolCAP array, yielding a total of 7,591 SNPs. Population structure, phylogenetic relationships, and principal components analysis highlighted a differentiation of plum and cherry genotypes with respect to the beefsteak and globe types. These results were confirmed by multivariate analysis of phenotypic traits, shedding light on how breeding and selection focused on fruit characteristics have influenced the genetic and phenotypic makeup of heirlooms. Marker-trait association showed 11 significantly associated loci for ß-carotene and fruit weight. For ß-carotene, a single variant on chromosome 8 was found at 12 kb to CCD8, a cleavage dioxygenase playing a key role in the biosynthesis of apocarotenoids. For fruit weight, a single association was located at less than 3 Mbp from SLSUN31 and fw11.3, two candidates involved in the increasing of fruit mass. These results highlight the potentiality of heirlooms for genetic improvement and candidate gene identification.

Genotypic and Environmental Effects on Morpho-Physiological and Agronomic Performances of a Tomato Diversity Panel in Relation to Nitrogen and Water Stress Under Organic Farming.

The agricultural scenario of the upcoming decades will face major challenges for the increased and sustainable agricultural production and the optimization of the efficiency of water and fertilizer inputs. Considering the current and foreseen water scarcity in several marginal and arid areas and the need for a more sustainable farming production, the selection and development of cultivars suitable to grow under low-input conditions is an urgent need. In this study, we assayed 42 tomato genotypes for thirty-two morpho-physiological and agronomic traits related to plant, fruit, and root characteristics under standard (control) and no-nitrogen fertilization or water deficit (30% of the amount given to non-stressed trials) treatments in two sites (environments), which corresponded to organic farms located in Italy and Spain. A broad range of variation was found for all traits, with significant differences between the applied treatments and the cultivation sites. Dissection of genotypic (G), environmental (E), and treatment (T) factors revealed that the three main factors were highly significant for many traits, although G was the main source of variation in most cases. G × E interactions were also important, while G × T and E × T were less relevant. Only fruit weight and blossom end rot were highly significant for the triple interaction (G × E × T). Reduction of water supply significantly increased the soluble solid content in both locations, whereas both nitrogen and water stress led to a general decrease in fruit weight and total yield. Despite so, several accessions exhibited better performances than the control when cultivated under stress. Among the accessions evaluated, hybrids were promising in terms of yield performance, while overall landraces and heirlooms exhibited a better quality. This suggests the possibility of exploiting both the variation within ancient varieties and the heterosis for yield of hybrids to select and breed new varieties with better adaptation to organic farming conditions, both under optimal and suboptimal conditions. The results shed light on the strategies to develop novel varieties for organic farming, giving hints into the management of inputs to adopt for a more sustainable tomato cultivation.

Biochemical and molecular analysis of pink tomatoes: deregulated expression of the gene encoding transcription factor SlMYB12 leads to pink tomato fruit color.

The color of tomato fruit is mainly determined by carotenoids and flavonoids. Phenotypic analysis of an introgression line (IL) population derived from a cross between Solanum lycopersicum 'Moneyberg' and the wild species Solanum chmielewskii revealed three ILs with a pink fruit color. These lines had a homozygous S. chmielewskii introgression on the short arm of chromosome 1, consistent with the position of the y (yellow) mutation known to result in colorless epidermis, and hence pink-colored fruit, when combined with a red flesh. Metabolic analysis showed that pink fruit lack the ripening-dependent accumulation of the yellow-colored flavonoid naringenin chalcone in the fruit peel, while carotenoid levels are not affected. The expression of all genes encoding biosynthetic enzymes involved in the production of the flavonol rutin from naringenin chalcone was down-regulated in pink fruit, suggesting that the candidate gene underlying the pink phenotype encodes a regulatory protein such as a transcription factor rather than a biosynthetic enzyme. Of 26 MYB and basic helix-loop-helix transcription factors putatively involved in regulating transcription of genes in the phenylpropanoid and/or flavonoid pathway, only the expression level of the MYB12 gene correlated well with the decrease in the expression of structural flavonoid genes in peel samples of pink- and red-fruited genotypes during ripening. Genetic mapping and segregation analysis showed that MYB12 is located on chromosome 1 and segregates perfectly with the characteristic pink fruit color. Virus-induced gene silencing of SlMYB12 resulted in a decrease in the accumulation of naringenin chalcone, a phenotype consistent with the pink-colored tomato fruit of IL1b. In conclusion, biochemical and molecular data, gene mapping, segregation analysis, and virus-induced gene silencing experiments demonstrate that the MYB12 transcription factor plays an important role in regulating the flavonoid pathway in tomato fruit and suggest strongly that SlMYB12 is a likely candidate for the y mutation.

Methods of Development of Biparental Mapping Populations in Horticultural Crops.

Biparental mapping populations consist of a set of individuals derived from crosses between two parents often belonging to diverse species of a botanical genus and differing in terms of phenotype and traits to share. The development of such recombinant libraries represents a powerful strategy for dissection of the genetic basis of complex traits in crops and these are largely utilized to develop pre-breeding sources to use in crop improvement. This chapter provides an overview of methods and strategies to follow, for the construction of different types of populations, from a plant breeder point of view. Starting from the initial crossing between founder lines toward the further selection steps, here are described the populations commonly established in autogamous species including F2, double haploids, backcrosses and recombinant inbreds, and introgression lines.

High-Throughput DNA Isolation in Vegetable Crops for Genomics Applications.

Isolating high-quality DNA is essential for several applications in molecular biology and genomics. Performing whole-genome sequencing in crops and development of reduced representation genomic libraries for genotyping require precise standard on DNA in terms of concentration and purity. For screening large populations it is essential to increase the extraction throughput at affordable costs. In this chapter a homemade protocol is provided that is able to isolate in 96-well plates 198 samples of DNA in a single extraction. The method has been validated in tomato and pepper and can be applied in several vegetable species.