Wild Progenitor Research Articles

Profiling the selected hotspots for ear traits in two maize-teosinte populations.

Eight selected hotspots related to ear traits were identified from two maize-teosinte populations. Throughout the history of maize cultivation, ear-related traits have been selected. However, little is known about the specific genes involved in shaping these traits from their origins in the wild progenitor, teosinte, to the characteristics observed in modern maize. In this study, five ear traits (kernel row number [KRN], ear length [EL], kernel number per row [KNR], cob diameter [CD], and ear diameter [ED]) were investigated, and eight quantitative trait loci (QTL) hotspots were identified in two maize-teosinte populations. Notably, our findings revealed a significant enrichment of genes showing a selection signature and expressed in the ear in qbdCD1.1, qbdCD5.1, qbpCD2.1, qbdED1.1, qbpEL1.1, qbpEL5.1, qbdKNR1.1, and qbdKNR10.1, suggesting that these eight QTL are selected hotspots involved in shaping the maize ear. By combining the results of the QTL analysis with data from previous genome-wide association study (GWAS) involving two natural panels, we identified eight candidate selected genes related to KRN, KNR, CD, and ED. Among these, considering their expression pattern and sequence variation, Zm00001d025111, encoding a WD40/YVTN protein, was proposed as a positive regulator of KNR. This study presents a framework for understanding the genomic distribution of selected loci crucial in determining ear-related traits.

Designing future peanut: the power of genomics-assisted breeding.

Integrating GAB methods with high-throughput phenotyping, genome editing, and speed breeding hold great potential in designing future smart peanut cultivars to meet market and food supply demands. Cultivated peanut (Arachis hypogaea L.), a legume crop greatly valued for its nourishing food, cooking oil, and fodder, is extensively grown worldwide. Despite decades of classical breeding efforts, the actual on-farm yield of peanut remains below its potential productivity due to the complicated interplay of genotype, environment, and management factors, as well as their intricate interactions. Integrating modern genomics tools into crop breeding is necessary to fast-track breeding efficiency and rapid progress. When combined with speed breeding methods, this integration can substantially accelerate the breeding process, leading to faster access of improved varieties to farmers. Availability of high-quality reference genomes for wild diploid progenitors and cultivated peanuts has accelerated the process of gene/quantitative locus discovery, developing markers and genotyping assays as well as a few molecular breeding products with improved resistance and oil quality. The use of new breeding tools, e.g., genomic selection, haplotype-based breeding, speed breeding, high-throughput phenotyping, and genome editing, is probable to boost genetic gains in peanut. Moreover, renewed attention to efficient selection and exploitation of targeted genetic resources is also needed to design high-quality and high-yielding peanut cultivars with main adaptation attributes. In this context, the combination of genomics-assisted breeding (GAB), genome editing, and speed breeding hold great potential in designing future improved peanut cultivars to meet market and food supply demands.

Evolutionary relationships, hybridization and diversification under domestication of the locoto chile (Capsicum pubescens) and its wild relatives.

Patterns of genetic variation in crops are the result of multiple processes that have occurred during their domestication and improvement, and are influenced by their wild progenitors that often remain understudied. The locoto chile, Capsicum pubescens, is a crop grown mainly in mid-highlands of South-Central America. This species is not known from the wild and exists only as a cultigen. The evolutionary affinities and exact origin of C. pubescens have still not been elucidated, with hypotheses suggesting its genetic relatedness and origin to two wild putative ancestral Capsicum species from the Central Andes, C.eximium and C. cardenasii. In the current study, RAD-sequencing was applied to obtain genome-wide data for 48 individuals of C. pubescens and its wild allies representing different geographical areas. Bayesian, Maximum Likelihood and coalescent-based analytical approaches were used to reconstruct population genetic patterns and phylogenetic relationships of the studied species. The results revealed that C. pubescens forms a well-defined monotypic lineage closely related to wild C. cardenasii and C. eximium, and also to C. eshbaughii. The primary lineages associated with the diversification under domestication of C. pubescens were also identified. Although direct ancestor-descendant relationship could not be inferred within this group of taxa, hybridization events were detected between C. pubescens and both C.cardenasii and C. eximium. Therefore, although hybrid origin of C. pubescens could not be inferred, gene flow involving its wild siblings was shown to be an important factor contributing to its contemporary genetic diversity. The data allowed for the inference of the center of origin of C. pubescens in central-western Bolivia highlands and for better understanding of the dynamics of its gene pool. The results of this study are essential for germplasm conservation and breeding purposes, and provide excellent basis for further research of the locoto chile and its wild relatives.

Porous borders at the wild-crop interface promote weed adaptation in Southeast Asia

High reproductive compatibility between crops and their wild relatives can provide benefits for crop breeding but also poses risks for agricultural weed evolution. Weedy rice is a feral relative of rice that infests paddies and causes severe crop losses worldwide. In regions of tropical Asia where the wild progenitor of rice occurs, weedy rice could be influenced by hybridization with the wild species. Genomic analysis of this phenomenon has been very limited. Here we use whole genome sequence analyses of 217 wild, weedy and cultivated rice samples to show that wild rice hybridization has contributed substantially to the evolution of Southeast Asian weedy rice, with some strains acquiring weed-adaptive traits through introgression from the wild progenitor. Our study highlights how adaptive introgression from wild species can contribute to agricultural weed evolution, and it provides a case study of parallel evolution of weediness in independently-evolved strains of a weedy crop relative.

Demography as a confounding factor to explain highly diverged loci between cultivated and wild rice

AbstractThe domestication of rice increases the divergence between cultivated rice and its wild progenitor because of artificial selection. However, it remains unknown whether highly diverged loci in rice can be explained by neutral demographic scenarios alone. In this study, we genotyped 45 InDels (insertion/deletion) in two subspecies of Asian cultivated rice (Oryza sativa ssp. japonica and Oryza sativa ssp. indica) and their wild progenitor (O. rufipogon/O. nivara). Among them, 17 loci are highly diverged (FST > 0.4) between rice cultivars and their ancestor. We performed coalescent-based simulations on neutral demographic scenarios and found that neutral demography alone could explain the polymorphic profiles on those highly diverged loci between cultivated and wild rice. Therefore, more signatures of selection should be considered when detecting artificial selection in rice.

Genomic evidence for domestication selection in three hatchery populations of Chinook salmon, Oncorhynchus tshawytscha.

Fish hatcheries are widely used to enhance fisheries and supplement declining wild populations. However, substantial evidence suggests that hatchery fish are subject to differential selection pressures compared to their wild counterparts. Domestication selection, or adaptation to the hatchery environment, poses a risk to wild populations if traits specific to success in the hatchery environment have a genetic component and there is subsequent introgression between hatchery and wild fish. Few studies have investigated domestication selection in hatcheries on a genomic level, and even fewer have done so in parallel across multiple hatchery-wild population pairs. In this study, we used low-coverage whole-genome sequencing to investigate signals of domestication selection in three separate hatchery populations of Chinook salmon, Oncorhynchus tshawytscha, after approximately seven generations of divergence from their corresponding wild progenitor populations. We sequenced 192 individuals from populations across Southeast Alaska and estimated genotype likelihoods at over six million loci. We discovered a total of 14 outlier peaks displaying high genetic differentiation (F ST) between hatchery-wild pairs, although no peaks were shared across the three comparisons. Peaks were small (53 kb on average) and often displayed elevated absolute genetic divergence (D xy) and linkage disequilibrium, suggesting some level of domestication selection has occurred. Our study provides evidence that domestication selection can lead to genetic differences between hatchery and wild populations in only a few generations. Additionally, our data suggest that population-specific adaptation to hatchery environments likely occurs through different genetic pathways, even for populations with similar standing genetic variation. These results highlight the need to collect paired genotype-phenotype data to understand how domestication may be affecting fitness and to identify potential management practices that may mitigate genetic risks despite multiple pathways of domestication.

Population comparative genomics discovers gene gain and loss during grapevine domestication.

Plant domestication are evolutionary experiments conducted by early farmers since thousands years ago, during which the crop wild progenitors are artificially selected for desired agronomic traits along with dramatic genomic variation in the course of moderate to severe bottlenecks. However, previous investigations are mainly focused on small-effect variants, while changes in gene contents are rarely investigated due to the lack of population-level assemblies for both the crop and its wild relatives. Here, we applied comparative genomic analyses to discover gene gain and loss during grapevine domestication using long-read assemblies of representative population samples for both domesticated grapevines (V. vinifera ssp. vinifera) and their wild progenitors (V. vinifera ssp. sylvestris). Only ∼7% of gene families were shared by 16 Vitis genomes while ∼8% of gene families were specific to each accession, suggesting dramatic variations of gene contents in grapevine genomes. Compared to wild progenitors, the domesticated accessions exhibited an increased presence of genes associated with asexual reproduction, while the wild progenitors showcased a higher abundance of genes related to pollination, revealing the transition from sexual reproduction to clonal propagation during domestication processes. Moreover, the domesticated accessions harbored fewer disease-resistance genes than wild progenitors. The SVs occurred frequently in aroma and disease-resistance related genes between domesticated grapevines and wild progenitors, indicating the rapid diversification of these genes during domestication. Our study provides insights and resources for biological studies and breeding programs in grapevine.

Agricultural intensification reduces selection of putative plant growth-promoting rhizobacteria in wheat.

The complex evolutionary history of wheat has shaped its associated root microbial community. However, consideration of impacts from agricultural intensification has been limited. This study investigated how endogenous (genome polyploidization) and exogenous (introduction of chemical fertilizers) factors have shaped beneficial rhizobacterial selection. We combined culture-independent and -dependent methods to analyze rhizobacterial community composition and its associated functions at the root-soil interface from a range of ancestral and modern wheat genotypes, grown with and without the addition of chemical fertilizer. In controlled pot experiments, fertilization and soil compartment (rhizosphere, rhizoplane) were the dominant factors shaping rhizobacterial community composition, whereas the expansion of the wheat genome from diploid to allopolyploid caused the next greatest variation. Rhizoplane-derived culturable bacterial collections tested for plant growth-promoting (PGP) traits revealed that fertilization reduced the abundance of putative plant growth-promoting rhizobacteria in allopolyploid wheats but not in wild wheat progenitors. Taxonomic classification of these isolates showed that these differences were largely driven by reduced selection of beneficial root bacteria representative of the Bacteroidota phylum in allopolyploid wheats. Furthermore, the complexity of supported beneficial bacterial populations in hexaploid wheats was greatly reduced in comparison to diploid wild wheats. We therefore propose that the selection of root-associated bacterial genera with PGP functions may be impaired by crop domestication in a fertilizer-dependent manner, a potentially crucial finding to direct future plant breeding programs to improve crop production systems in a changing environment.

Early human selection of crops' wild progenitors explains the acquisitive physiology of modern cultivars.

Crops have resource-acquisitive leaf traits, which are usually attributed to the process of domestication. However, early choices of wild plants amenable for domestication may also have played a key role in the evolution of crops' physiological traits. Here we compiled data on 1,034 annual herbs to place the ecophysiological traits of 69 crops' wild progenitors in the context of global botanical variation, and we conducted a common-garden experiment to measure the effects of domestication on crop ecophysiology. Our study found that crops' wild progenitors already had high leaf nitrogen, photosynthesis, conductance and transpiration and soft leaves. After domestication, ecophysiological traits varied little and in idiosyncratic ways. Crops did not surpass the trait boundaries of wild species. Overall, the resource-acquisitive strategy of crops is largely due to the inheritance from their wild progenitors rather than to further breeding improvements. Our study concurs with recent literature highlighting constraints of crop breeding for faster ecophysiological traits.

Regulation of Seed Dormancy Genes in Triticeae Species.

Wild progenitors of Triticeae crops generally have long dormancy periods. Domesticated crops inherited these longer dormancy alleles from their wild progenitors, which have since been modified and selected during cultivation and utilization by humans. Thus, allelic combinations at different seed dormancy loci are currently represented in Triticeae germplasm preserved in seed repositories and gene banks as accessions and materials of breeding programs. Methods to evaluate seed dormancy are key to explore, analyze, and exploit optimal alleles in dormancy genes. Recent developments in genomics have accelerated the identification and analysis of seed dormancy loci in Triticeae species. Transgenic experiments have been conducted to validate if candidate genes affect seed dormancy and more recently have yielded an array of mutations derived from genome editing for practical applications. The information gathered on these seed dormancy loci provides a deeper knowledge of germplasm diversity and offers strategies to control seed dormancy in breeding programs in Triticeae crops.

A natural mutant kumquat fruit with abnormal oil glands shows lower volatile production, better fruit flavor and higher resistance to postharvest disease

Oil glands of citrus peel are known sites for synthesis and deposition of volatile organic compounds (VOCs). However, it is unclear how oil glands and VOCs affect fruit flavor and postharvest disease resistance in vivo. Here, we report characterization of a natural mutant ‘Huapi’ (HP) kumquat (Fortunella crassifolia Swingle) compared with its wild progenitor ‘Rongan’ (RA). HP fruit decreased in both size and number of oil glands from the onset of fruit development when compared to RA. The total VOCs concentration and the number of individual VOCs in mature HP fruit were lower than that in RA. Limonene, α-pinene and β-myrcene concentrations showed the greatest reduction (30-, 21- and 20-fold). Sensory evaluation showed that flavor of HP fruit was more acceptable to consumers compared to that of RA. Sweeter, less acidic features of HP fruit were likely due to higher soluble sugars content and lower organic acid content, respectively. Changes in HP fruit VOCs likely contribute to its less pungent and better aroma features. HP fruit displayed stronger resistance to the fungal pathogens Penicillium italicum and Geotrichum candidum compared to RA in in vivo inoculation experiments. Transcriptomic analysis and qPCR validation indicated that FcERF096, FcCHI, FcCXE, FcPi21L1, FcPi21L2, FcPIP and FcXYLL potentially involve in enhanced disease resistance of HP fruit. Taken together, these results reveal the potential of oil glands and terpenes in affecting fruit sensory attributes and postharvest disease resistance in planta in kumquat, providing new insights into the technologies improving these important traits in citrus.

Pangenome analysis reveals genomic variations associated with domestication traits in broomcorn millet

Broomcorn millet (Panicum miliaceum L.) is an orphan crop with the potential to improve cereal production and quality, and ensure food security. Here we present the genetic variations, population structure and diversity of a diverse worldwide collection of 516 broomcorn millet genomes. Population analysis indicated that the domesticated broomcorn millet originated from its wild progenitor in China. We then constructed a graph-based pangenome of broomcorn millet based on long-read de novo genome assemblies of 32 representative accessions. Our analysis revealed that the structural variations were highly associated with transposable elements, which influenced gene expression when located in the coding or regulatory regions. We also identified 139 loci associated with 31 key domestication and agronomic traits, including candidate genes and superior haplotypes, such as LG1, for panicle architecture. Thus, the study’s findings provide foundational resources for developing genomics-assisted breeding programs in broomcorn millet.

Genome scans capture key adaptation and historical hybridization signatures in tetraploid wheat.

Tetraploid wheats (Triticum turgidum L.), including durum wheat (T. turgidum ssp. durum (Desf.) Husn.), are important crops with high nutritional and cultural values. However, their production is constrained by sensitivity to environmental conditions. In search of adaptive genetic signatures tracing historical selection and hybridization events, we performed genome scans on two datasets: (1) Durum Global Diversity Panel comprising a total of 442 tetraploid wheat and wild progenitor accessions including durum landraces (n=286), domesticated emmer (T. turgidum ssp. dicoccum (Schrank) Thell.; n=103) and wild emmer (T. turgidum ssp. dicoccoides (Korn. ex Asch. & Graebn.) Thell.; n=53) wheats genotyped using the 90K single nucleotide polymorphism (SNP) array, and (2) a second dataset comprising a total 121 accessions of nine T. turgidum subspecies including wild emmer genotyped with>100M SNPs from whole-genome resequencing. The genome scan on the first dataset detected six outlier loci on chromosomes 1A, 1B, 3A (n=2), 6A, and 7A. These loci harbored important genes for adaptation to abiotic stresses, phenological responses, such as seed dormancy, circadian clock, flowering time, and key yield-related traits, including pleiotropic genes, such as HAT1, KUODA1, CBL1, and ZFN1. The scan on the second dataset captured a highly differentiated region on chromosome 2B that shows significant differentiation between two groups: one group consists of Georgian (T. turgidum ssp.paleocolchicum A. Love & D. Love) and Persian (T. turgidum ssp. carthlicum (Nevski) A. Love & D. Love) wheat accessions, while the other group comprises all the remaining tetraploids including wild emmer. This is consistent with a previously reported introgression in this genomic region from T. timopheevii Zhuk. which naturally cohabit in the Georgian and neighboring areas. This region harbored several adaptive genes, including the thermomorphogenesis gene PIF4, which confers temperature-resilient disease resistance and regulates other biological processes. Genome scans can be used to fast-track germplasm housed in gene banks and in situ; which helps to identify environmentally resilient accessions for breeding and/or to prioritize them for conservation.

Comparative Genomic Analysis of Asian Cultivated Rice and Its Wild Progenitor (Oryza rufipogon) Has Revealed Evolutionary Innovation of the Pentatricopeptide Repeat Gene Family through Gene Duplication.

The pentatricopeptide repeat (PPR) gene family is one of the largest gene families in land plants. However, current knowledge about the evolution of the PPR gene family remains largely limited. In this study, we performed a comparative genomic analysis of the PPR gene family in O. sativa and its wild progenitor, O. rufipogon, and outlined a comprehensive landscape of gene duplications. Our findings suggest that the majority of PPR genes originated from dispersed duplications. Although segmental duplications have only expanded approximately 11.30% and 13.57% of the PPR gene families in the O. sativa and O. rufipogon genomes, we interestingly obtained evidence that segmental duplication promotes the structural diversity of PPR genes through incomplete gene duplications. In the O. sativa and O. rufipogon genomes, 10 (~33.33%) and 22 pairs of gene duplications (~45.83%) had non-PPR paralogous genes through incomplete gene duplication. Segmental duplications leading to incomplete gene duplications might result in the acquisition of domains, thus promoting functional innovation and structural diversification of PPR genes. This study offers a unique perspective on the evolution of PPR gene structures and underscores the potential role of segmental duplications in PPR gene structural diversity.

Progress and future impacts on genomic dissection of soybean domestication and improvement

Soybean (Glycine max [L.] Merr.) was domesticated from the wild annual progenitor Glycine soja (Sieb. & Zucc.) in the temperate zone of China. Through domestication and improvement, soybean agronomic traits and adaptability have been significantly altered. In this review, we discuss the ways in which genetic changes were selected in soybean to enable adaptation to diverse environmental conditions. Challenges and strategies are discussed for breeding new elite varieties from existing elite varieties. Finally, we propose a strategy to break the current genetic bottleneck in soybean breeding: de novo domestication, which utilizes the excellent genetic resources available in wild soybean and provides a feasible strategy for accelerating the soybean improvement process. Overall, this review serves as a guide to understand the genetic factors that have driven soybean domestication and improvement over thousands of years, promoting future generation of soybean cultivars that are optimized for high yield under stressful environmental conditions.

Redefining the Evolutionary History of the Rock Dove, Columba livia, Using Whole Genome Sequences.

The domestic pigeon's exceptional phenotypic diversity was key in developing Darwin's Theory of Evolution and establishing the concept of artificial selection. However, unlike its domestic counterpart, its wild progenitor, the rock dove Columba livia has received considerably less attention. Therefore, questions regarding its domestication, evolution, taxonomy, and conservation status remain unresolved. We generated whole-genome sequencing data from 65 historical rock doves that represent all currently recognized subspecies and span the species' original geographic distribution. Our dataset includes 3 specimens from Darwin's collection, and the type specimens of 5 different taxa. We characterized their population structure, genomic diversity, and gene-flow patterns. Our results show the West African subspecies C. l. gymnocyclus is basal to rock doves and domestic pigeons, and suggests gene-flow between the rock dove's sister species C. rupestris, and the ancestor of rock doves after its split from West African populations. These genomes allowed us to propose a model for the evolution of the rock dove in light of the refugia theory. We propose that rock dove genetic diversity and introgression patterns derive from a history of allopatric cycles and dispersion waves during the Quaternary glacial and interglacial periods. To explore the rock dove domestication history, we combined our new dataset with available genomes from domestic pigeons. Our results point to at least 1 domestication event in the Levant that gave rise to all domestic breeds analysed in this study. Finally, we propose a species-level taxonomic arrangement to reflect the evolutionary history of the West African rock dove populations.

Conservation and molecular identification of traditional rice varieties collected from Kerala using matK and rbcL DNA barcode markers

Varietal identification of economically significant crops has acquired great importance in the global market especially in the terms of protection of plant varieties. Kerala is the home to several different specialty traditional rice varieties which is vital sources of nutritional and therapeutic properties. Traditional rice landraces are an integral part of the nutrition, medicine, and culture of Kerala. Most of these varieties possess similar morphological characteristics but differ in their nutritional and medicinal properties. It has become a challenge for a non-taxonomist to identify and recognize the traditional rice varieties accurately. DNA barcoding has been considered a standard method to identify species or variety which utilizes a unique pattern of variation in the DNA of the species. It can be adopted as a routine and faster approach to determine the molecular taxonomy when compared to morphological identification approaches. Currently, the most intensively applied two DNA barcode loci are maturase K (matK) and ribulose 1,5-biphosphate carboxylase (rbcL) for plant identification. But, the efficacy of these two loci in discriminating plant species may vary. Hence, in this study fifty-four traditional rice varieties collected from the Wayanad district of Kerala were used to generate and compare the two standard DNA barcode regions for plant matK and rbcL via PCR amplification. The varieties collected were varying in the husk color from black; red to brown and the brown rice varieties not display any morphological variation. The study identified that among the two selected markers rbcL was observed to have the potential to discriminate the intra and interspecific regions of the studied rice varieties and this would have been utilized to generate DNA barcodes for each variety. This study separates and identifies 28 traditional rice varieties among the 54 studied varieties as Oryza nivara, the wild progenitor of Asian cultivated rice. Along with other supporting information the data presented here could be used as a reference to check the authenticity and varietal purity of commercially significant rice varieties in the future.

Novel genes and alleles of the BTB/POZ protein family in Oryza rufipogon

The BTB/POZ family of proteins is widespread in plants and animals, playing important roles in development, growth, metabolism, and environmental responses. Although members of the expanded BTB/POZ gene family (OsBTB) have been identified in cultivated rice (Oryza sativa), their conservation, novelty, and potential applications for allele mining in O. rufipogon, the direct progenitor of O. sativa ssp. japonica and potential wide-introgression donor, are yet to be explored. This study describes an analysis of 110 BTB/POZ encoding gene loci (OrBTB) across the genome of O. rufipogon as outcomes of tandem duplication events. Phylogenetic grouping of duplicated OrBTB genes was supported by the analysis of gene sequences and protein domain architecture, shedding some light on their evolution and functional divergence. The O. rufipogon genome encodes nine novel BTB/POZ genes with orthologs in its distant cousins in the family Poaceae (Sorghum bicolor, Brachypodium distachyon), but such orthologs appeared to have been lost in its domesticated descendant, O. sativa ssp. japonica. Comparative sequence analysis and structure comparisons of novel OrBTB genes revealed that diverged upstream regulatory sequences and regulon restructuring are the key features of the evolution of this large gene family. Novel genes from the wild progenitor serve as a reservoir of potential new alleles that can bring novel functions to cultivars when introgressed by wide hybridization. This study establishes a foundation for hypothesis-driven functional genomic studies and their applications for widening the genetic base of rice cultivars through the introgression of novel genes or alleles from the exotic gene pool.

Detection of locally adapted genomic regions in wild rice (Oryza rufipogon) using environmental association analysis.

Oryza rufipogon is the wild progenitor of cultivated rice Oryza sativa and exhibits high levels of genetic diversity across its distribution, making it a useful resource for the identification of abiotic stress-tolerant varieties and genes that could limit future climate-changed-induced yield losses. To investigate local adaptation in O. rufipogon, we analyzed single nucleotide polymorphism (SNP) data from a panel of 286 samples located across a diverse range of climates. Environmental association analysis (EAA), a genome-wide association study (GWAS)-based method, was used and revealed 15 regions of the genome significantly associated with various climate factors. Genes within these environmentally associated regions have putative functions in abiotic stress response, phytohormone signaling, and the control of flowering time. This provides an insight into potential local adaptation in O. rufipogon and reveals possible locally adaptive genes that may provide opportunities for breeding novel rice varieties with climate change-resilient phenotypes.

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.