On the origin of European sheep as revealed by the diversity of the Balkan breeds and by optimizing population-genetic analysis tools.

BACKGROUND: In the Neolithic, domestic sheep migrated into Europe and subsequently spread in westerly and northwesterly directions. Reconstruction of these migrations and subsequent genetic events requires a more detailed characterization of the current phylogeographic differentiation. RESULTS: We collected 50 K single nucleotide polymorphism (SNP) profiles of Balkan sheep that are currently found near the major Neolithic point of entry into Europe, and combined these data with published genotypes from southwest-Asian, Mediterranean, central-European and north-European sheep and from Asian and European mouflons. We detected clines, ancestral components and admixture by using variants of common analysis tools: geography-informative supervised principal component analysis (PCA), breed-specific admixture analysis, across-breed [Formula: see text] profiles and phylogenetic analysis of regional pools of breeds. The regional Balkan sheep populations exhibit considerable genetic overlap, but are clearly distinct from the breeds in surrounding regions. The Asian mouflon did not influence the differentiation of the European domestic sheep and is only distantly related to present-day sheep, including those from Iran where the mouflons were sampled. We demonstrate the occurrence, from southeast to northwest Europe, of a continuously increasing ancestral component of up to 20% contributed by the European mouflon, which is assumed to descend from the original Neolithic domesticates. The overall patterns indicate that the Balkan region and Italy served as post-domestication migration hubs, from which wool sheep reached Spain and north Italy with subsequent migrations northwards. The documented dispersal of Tarentine wool sheep during the Roman period may have been part of this process. Our results also reproduce the documented 18th century admixture of Spanish Merino sheep into several central-European breeds. CONCLUSIONS: Our results contribute to a better understanding of the events that have created the present diversity pattern, which is relevant for the management of the genetic resources represented by the European sheep population.

Chloroplast and whole-genome sequencing shed light on the evolutionary history and phenotypic diversification of peanuts.

Cultivated peanut (Arachis hypogaea L.) is a widely grown oilseed crop worldwide; however, the events leading to its origin and diversification are not fully understood. Here by combining chloroplast and whole-genome sequence data from a large germplasm collection, we show that the two subspecies of A. hypogaea (hypogaea and fastigiata) likely arose from distinct allopolyploidization and domestication events. Peanut genetic clusters were then differentiated in relation to dissemination routes and breeding efforts. A combination of linkage mapping and genome-wide association studies allowed us to characterize genes and genomic regions related to main peanut morpho-agronomic traits, namely flowering pattern, inner tegument color, growth habit, pod/seed weight and oil content. Together, our findings shed light on the evolutionary history and phenotypic diversification of peanuts and might be of broad interest to plant breeders.

High-density linkage mapping and genetic dissection of resistance to broomrape (Orobanche crenata Forsk.) in pea (Pisum sativum L.).

Pea (Pisum sativum L.) is a widely cultivated legume of major importance for global food security and agricultural sustainability. Crenate broomrape (Orobanche crenata Forsk.) (Oc) is a parasitic weed severely affecting legumes, including pea, in the Mediterranean Basin and the Middle East. Previously, the identification of the pea line "ROR12", displaying resistance to Oc, was reported. Two-year field trials on a segregant population of 148 F7 recombinant inbred lines (RILs), originating from a cross between "ROR12" and the susceptible cultivar "Sprinter", revealed high heritability (0.84) of the "ROR12" resistance source. Genotyping-by-sequencing (GBS) on the same RIL population allowed the construction of a high-density pea linkage map, which was compared with the pea reference genome and used for quantitative trait locus (QTL) mapping. Three QTLs associated with the response to Oc infection, named PsOcr-1, PsOcr-2, and PsOcr-3, were identified, with PsOcr-1 explaining 69.3% of the genotypic variance. Evaluation of the effects of different genotypic combinations indicated additivity between PsOcr-1 and PsOcr-2, and between PsOcr-1 and PsOcr-3, and epistasis between PsOcr-2 and PsOcr-3. Finally, three Kompetitive Allele Specific PCR (KASP) marker assays were designed on the single-nucleotide polymorphisms (SNPs) associated with the QTL significance peaks. Besides contributing to the development of pea genomic resources, this work lays the foundation for the obtainment of pea cultivars resistant to Oc and the identification of genes involved in resistance to parasitic Orobanchaceae.

Detection and distribution of two dominant alleles associated with the sweet kernel phenotype in almond cultivated germplasm.

Almond [Prunus dulcis Miller (D. A. Webb), syn. Prunus amygdalus L.)] is the major tree nut crop worldwide in terms of production and cultivated area. Almond domestication was enabled by the selection of individuals bearing sweet kernels, which do not accumulate high levels of the toxic cyanogenic glucoside amygdalin. Previously, we showed that the Sweet kernel (Sk) gene, controlling the kernel taste in almond, encodes a basic helix loop helix (bHLH) transcription factor regulating the amygdalin biosynthetic pathway. In addition, we characterized a dominant allele of this gene, further referred to as Sk-1, which originates from a C1036→T missense mutation and confers the sweet kernel phenotype. Here we provide evidence indicating that the allele further referred to as Sk-2, originally detected in the cultivar "Atocha" and arising from a T989→G missense mutation, is also dominantly inherited and confers the sweet kernel phenotype in almond cultivated germplasm. The use of single nucleotide polymorphism (SNP) data from genotyping by sequencing (GBS) for population structure and hierarchical clustering analyses indicated that Sk-2 occurs in a group of related genotypes, including the widespread cultivar "Texas", descending from the same ancestral population. KASP and dual label functional markers were developed for the accurate and high-throughput selection of the Sk-1 and Sk-2 alleles, and the genotyping of a panel of 134 almond cultivars. Overall, our results provide further insights on the understanding of the almond cultivation history. In addition, molecular marker assays and genotypic data presented in this study are expected to be of major interest for the conduction of almond breeding programs, which often need to select sweet kernel individuals in segregant populations.

Genotyping-by-Sequencing Defines Genetic Structure within the "Acquaviva" Red Onion Landrace.

Genetic structure and distinctive features of landraces, such as adaptability to local agro-ecosystems and specific qualitative profiles, can be substantially altered by the massive introduction of allochthonous germplasm. The landrace known as "Cipolla rossa di Acquaviva" (Acquaviva red onion, further referred to as ARO) is traditionally cultivated and propagated in a small area of the Apulia region (southern Italy). However, the recent rise of its market value and cultivation area is possibly causing genetic contamination with foreign propagating material. In this work, genotyping-by-sequencing (GBS) was used to characterize genetic variation of seven onion populations commercialized as ARO, as well as one population of the landrace "Montoro" (M), which is phenotypically similar, but originates from another cultivation area and displays different qualitative features. A panel of 5011 SNP markers was used to perform parametric and non-parametric genetic structure analyses, which supported the hypothesis of genetic contamination of germplasm commercialized as ARO with a gene pool including the M landrace. Four ARO populations formed a core genetic group, homogeneous and clearly distinct from the other ARO and M populations. Conversely, the remaining three ARO populations did not display significant differences with the M population. A set of private alleles for the ARO core genetic group was identified, indicating the possibility to trace the ARO landrace by means of a SNP-based molecular barcode. Overall, the results of this study provide a framework for further breeding activities and the traceability of the ARO landrace.

Merging genotyping-by-sequencing data from two ex situ collections provides insights on the pea evolutionary history.

Pea (Pisum sativum L. subsp. sativum) is one of the oldest domesticated species and a widely cultivated legume. In this study, we combined next generation sequencing (NGS) data referring to two genotyping-by-sequencing (GBS) libraries, each one prepared from a different Pisum germplasm collection. The selection of single nucleotide polymorphism (SNP) loci called in both germplasm collections caused some loss of information; however, this did not prevent the obtainment of one of the largest datasets ever used to explore pea biodiversity, consisting of 652 accessions and 22 127 markers. The analysis of population structure reflected genetic variation based on geographic patterns and allowed the definition of a model for the expansion of pea cultivation from the domestication centre to other regions of the world. In genetically distinct populations, the average decay of linkage disequilibrium (LD) ranged from a few bases to hundreds of kilobases, thus indicating different evolutionary histories leading to their diversification. Genome-wide scans resulted in the identification of putative selective sweeps associated with domestication and breeding, including genes known to regulate shoot branching, cotyledon colour and resistance to lodging, and the correct mapping of two Mendelian genes. In addition to providing information of major interest for fundamental and applied research on pea, our work describes the first successful example of integration of different GBS datasets generated from ex situ collections - a process of potential interest for a variety of purposes, including conservation genetics, genome-wide association studies, and breeding.

Genotyping-by-Sequencing in Vigna unguiculata Landraces and Its Utility for Assessing Taxonomic Relationships.

Genotyping by sequencing (GBS) was used to analyze relationships among cowpea and asparagus bean landraces from southern Italy and to assess the utility of this technology to study taxonomy in a wider panel, including V. unguiculata cultigroups, subspecies, and other Vigna species. The analysis of SNPs derived from GBS highlighted that, among the cowpea landraces, the African samples were separated from the other material, while, for the Italian landraces, a certain clustering depending on seed color/pattern was observed in the dendrogram. When examining the V. unguiculata species complex, a clear separation between the two groups of wild subspecies, i.e., the allogamous wild perennials and the perennial out/inbreds, could be observed, the former representing the more ancestral wild progenitors of V. unguiculata. The species V. vexillata appeared more closely related to V. unguiculata than to the other Vigna species analyzed.

Almond diversity and homozygosity define structure, kinship, inbreeding, and linkage disequilibrium in cultivated germplasm, and reveal genomic associations with nut and seed weight.

Almond [Prunus dulcis Miller (D.A. Webb)] is the main tree nut species worldwide. Here, genotyping-by-sequencing (GBS) was applied to 149 almond cultivars from the ex situ collections of the Italian Council for Agricultural Research (CREA) and the Spanish National Research Council (CSIC), leading to the detection of 93,119 single-nucleotide polymorphisms (SNPs). The study of population structure outlined four distinct genetic groups and highlighted diversification between the Mediterranean and Californian gene pools. Data on SNP diversity and runs of homozygosity (ROHs) allowed the definition of kinship, inbreeding, and linkage disequilibrium (LD) decay in almond cultivated germplasm. Four-year phenotypic observations, gathered on 98 cultivars of the CREA collection, were used to perform a genome-wide association study (GWAS) and, for the first time in a crop species, homozygosity mapping (HM), resulting in the identification of genomic associations with nut, shell, and seed weight. Both GWAS and HM suggested that loci controlling nut and seed weight are mostly independent. Overall, this study provides insights on the almond cultivation history and delivers information of major interest for almond genetics and breeding. In a broader perspective, our results encourage the use of ROHs in crop science to estimate inbreeding, choose parental combinations minimizing the risk of inbreeding depression, and identify genomic footprints of selection for specific traits.

Screening of Olive Biodiversity Defines Genotypes Potentially Resistant to Xylella fastidiosa.

The recent outbreak of the Olive Quick Decline Syndrome (OQDS), caused by Xylella fastidiosa subsp. pauca (Xf), is dramatically altering ecosystem services in the peninsula of Salento (Apulia Region, southeastern Italy). Here we report the accomplishment of several exploratory missions in the Salento area, resulting in the identification of thirty paucisymptomatic or asymptomatic plants in olive orchards severely affected by the OQDS. The genetic profiles of such putatively resistant plants (PRPs), assessed by a selection of ten simple sequence repeat (SSR) markers, were compared with those of 141 Mediterranean cultivars. Most (23) PRPs formed a genetic cluster (K1) with 22 Italian cultivars, including 'Leccino' and 'FS17', previously reported as resistant to Xf. The remaining PRPs displayed relatedness with genetically differentiated germplasm, including a cluster of Tunisian cultivars. Markedly lower colonization levels were observed in PRPs of the cluster K1 with respect to control plants. Field evaluation of four cultivars related to PRPs allowed the definition of partial resistance in the genotypes 'Frantoio' and 'Nocellara Messinese'. Some of the PRPs identified in this study might be exploited in cultivation, or as parental clones of breeding programs. In addition, our results indicate the possibility to characterize resistance to Xf in cultivars genetically related to PRPs.

Recommendations for Choosing the Genotyping Method and Best Practices for Quality Control in Crop Genome-Wide Association Studies.

High-throughput genotyping boosts genome-wide association studies (GWAS) in crop species, leading to the identification of single-nucleotide polymorphisms (SNPs) associated with economically important traits. Choosing a cost-effective genotyping method for crop GWAS requires careful examination of several aspects, namely, the purpose and the scale of the study, crop-specific genomic features, and technical and economic matters associated with each genotyping option. Once genotypic data have been obtained, quality control (QC) procedures must be applied to avoid bias and false signals in genotype-phenotype association tests. QC for human GWAS has been extensively reviewed; however, QC for crop GWAS may require different actions, depending on the GWAS population type. Here, we review most popular genotyping methods based on next-generation sequencing (NGS) and array hybridization, and report observations that should guide the investigator in the choice of the genotyping method for crop GWAS. We provide recommendations to perform QC in crop species, and deliver an overview of bioinformatics tools that can be used to accomplish all needed tasks. Overall, this work aims to provide guidelines to harmonize those procedures leading to SNP datasets ready for crop GWAS.