High-throughput discovery of rare human nucleotide polymorphisms by Ecotilling

Human individuals differ from one another at only ~0.1% of nucleotide positions, but these single nucleotide differences account for most heritable phenotypic variation. Large-scale efforts to discover and genotype human variation have been limited to common polymorphisms. However, these efforts overlook rare nucleotide changes that may contribute to phenotypic diversity and genetic disorders, including cancer. Thus, there is an increasing need for high-throughput methods to robustly detect rare nucleotide differences. Toward this end, we have adapted the mismatch discovery method known as Ecotilling for the discovery of human single nucleotide polymorphisms. To increase throughput and reduce costs, we developed a universal primer strategy and implemented algorithms for automated band detection. Ecotilling was validated by screening 90 human DNA samples for nucleotide changes in 5 gene targets and by comparing results to public resequencing data. To increase throughput for discovery of rare alleles, we pooled samples 8-fold and found Ecotilling to be efficient relative to resequencing, with a false negative rate of 5% and a false discovery rate of 4%. We identified 28 new rare alleles, including some that are predicted to damage protein function. The detection of rare damaging mutations has implications for models of human disease.     

Genotyping by Genome Reducing and Sequencing for Outbred Animals

Next-generation sequencing (NGS) approaches are widely used in genome-wide genetic marker discovery and genotyping. However, current NGS approaches are not easy to apply to general outbred populations (human and some major farm animals) for SNP identification because of the high level of heterogeneity and phase ambiguity in the haplotype. Here, we reported a new method for SNP genotyping, called genotyping by genome reducing and sequencing (GGRS) to genotype outbred species. Through an improved procedure for library preparation and a marker discovery and genotyping pipeline, the GGRS approach can genotype outbred species cost-effectively and high-reproducibly. We also evaluated the efficiency and accuracy of our approach for high-density SNP discovery and genotyping in a large genome pig species (2.8 Gb), for which more than 70,000 single nucleotide polymorphisms (SNPs) can be identified for an expenditure of only $80 (USD)/sample.     

Nitric oxide (NO): a key player in the senescence of Medicago truncatula root nodules

The ectomycorrhizal fungus Tricholoma populinum is host-specific with Populus species. T. populinum has wind-dispersed progagules and may be capable of long-distance dispersal. In this study, we tested the hypothesis of a panmictic population between Scandinavia and North America. DNA sequences from five nuclear loci were used to assess phylogeographic structure and nucleotide divergence between continents. Tricholoma populinum was composed of Scandinavian and North American lineages with complete absence of shared haplotypes and only one shared nucleotide mutation. Divergence of these lineages was estimated at approx. 1.7-1.0 million yr ago (Ma), which occurred after the estimated divergence of host species Populus tremula and Populus balsamifera/Populus trichocarpa at 5 Ma. Phylogeographic structure was not observed within Scandinavian or North American lineages of T. populinum. Intercontinental divergence appears to have resulted from either allopatric isolation; a recent, rare long-distance dispersal founding event followed by genetic drift; or the response in an obligate mycorrhizal fungus with a narrow host range to contractions and expansion of host distribution during glacial and interglacial episodes within continents. Understanding present genetic variation in populations is important for predicting how obligate symbiotic fungi will adapt to present and future changing climatic conditions.     

A 34K SNP genotyping array for Populus trichocarpa: Design,application to the study of natural populations and transferability to other Populus species

Genetic mapping of quantitative traits requires genotypic data for large numbers of markers in many individuals. For such studies, the use of large single nucleotide polymorphism (SNP) genotyping arrays still offers the most cost‐effective solution. Herein we report on the design and performance of a SNP genotyping array for Populus trichocarpa (black cottonwood). This genotyping array was designed with SNPs pre‐ascertained in 34 wild accessions covering most of the species latitudinal range. We adopted a candidate gene approach to the array design that resulted in the selection of 34 131 SNPs, the majority of which are located in, or within 2 kb of, 3543 candidate genes. A subset of the SNPs on the array (539) was selected based on patterns of variation among the SNP discovery accessions. We show that more than 95% of the loci produce high quality genotypes and that the genotyping error rate for these is likely below 2%. We demonstrate that even among small numbers of samples (n = 10) from local populations over 84% of loci are polymorphic. We also tested the applicability of the array to other species in the genus and found that the number of polymorphic loci decreases rapidly with genetic distance, with the largest numbers detected in other species in section Tacamahaca. Finally, we provide evidence for the utility of the array to address evolutionary questions such as intraspecific studies of genetic differentiation, species assignment and the detection of natural hybrids.     

Genetic variation of the bud and leaf phenology of seventeen poplar clones in a short rotation coppice culture

Abstract: Leaf phenology of 17 poplar ( Populus spp.) clones, encompassing spring phenology, length of growth period and end-of-year phenology, was examined over several years of different rotations. The 17 poplar clones differed in their latitude of origin (45°30'N to 51°N) and were studied on a short rotation experimental field plantation, situated in Boom (province of Antwerpen, Belgium; 51°05'N, 04°22'E). A similar, clear pattern of bud burst was observed during the different years of study for all clones. Clones Columbia River, Fritzi Pauley, Trichobel (Populus trichocarpa) and Balsam Spire (Populus trichocarpa × Populus balsamifera) from 45°30'N to 49°N reached bud burst (expressed as day of the year or degree day sums) almost every year earlier than clones Wolterson (Populus nigra), Gaver, Gibecq and Primo (Populus deltoides × Populus nigra) (50°N to 51°N). This observation could not be generalised to end-of-season phenology, for which a yearly returning pattern for all clones was lacking. Late bud burst and early leaf fall of some clones (Beaupré, Boelare, IBW1, IBW2, IBW3) was brought about by increasing rust incidence during the years of observation. For these clones, the variability in leaf phenology was reflected in high coefficients of variation among years. The patterns of genetic variation in leaf phenology have implications for short rotation intensive culture forestry and management of natural populations. Moreover, the variation in phenology reported here is relevant with regard to the genetic mapping of poplar.     

单核苷酸多态性在林木中的研究进展

摘要: 单核苷酸多态性(Single nucleotide polymorphisms, SNPs)是许多生物体最丰富的遗传变异形式。林木是重要的植物类群和陆地植物生态系统的重要组成部分, SNP作为新的分子标记已应用于松、杨、黄杉、桉和云杉等属的多个树种的遗传育种学研究, 获得了包括核苷酸多样性、连锁不平衡及群体结构等相关的遗传信息, 这些研究主要建立在对候选基因序列进行测序分析的基础上。基于SNP的关联遗传学分析或连锁不平衡(Linkage disequilibrium, LD)作图, 已成为研究林木复杂数量性状的理想工具, 对桉树和火炬松的关联遗传学研究发现, 多个基因内的SNP位点与不同的木材性状相关联。利用SNP标记对林木遗传参数的估算从不同程度上揭示了林木群体进化规律及其生态学意义。SNP标记在林木中应用的不断深入, 必将极大地推动林木遗传育种学研究的发展。     

Patterns of DNA sequence variation at candidate gene loci in black poplar (<Emphasis Type="Italic">Populus nigra</Emphasis> L.) as revealed by single nucleotide polymorphisms

Black poplar (Populus nigra L.) is an economically and ecologically important tree species and an ideal organism for studies of genetic variation. In the present work, we use a candidate gene approach to infer the patterns of DNA variation in natural populations of this species. A total of 312 single nucleotide polymorphisms (SNPs) are found among 8,056 bp sequenced from nine drought-adaptation and photosynthesis-related gene loci. The median SNP frequency is one site per 26 bp. The average nucleotide diversity is calculated to be θ_W = 0.01074 and π_T = 0.00702, higher values than those observed in P. tremula, P. trichocarpa and most conifer species. Tests of neutrality for each gene reveal a general excess of low-frequency mutations, a greater number of haplotypes than expected and an excess of high-frequency derived variants in P. nigra, which is consistent with previous findings that genetic hitchhiking has occurred in this species. Linkage disequilibrium is low, decaying rapidly from 0.45 to 0.20 or less within a distance of 300 bp, although the declines of r ² are variable among different loci. This is similar to the rate of decay reported in most other tree species. Our dataset is expected to enhance understanding of how evolutionary forces shape genetic variation, and it will contribute to molecular breeding in black poplar.     

Alternative ecotilling protocol for rapid, cost-effective single-nucleotide polymorphism discovery and genotyping in rice (Oryza sativa L.)

Ecotilling is a high-throughput method of discovery and analysis of single-nucleotide polymorphism (SNP) variations in natural populations, but it requires a substantial investment in sophisticated equipment, costly reagents, and specialized software programs and implementation of several time-consuming steps that limit its use in laboratories with modest financial resources. Moreover, labeling efficiency of PCR primers with fluorescent dyes during Ecotilling can be reduced by unwanted exonuclease activity of single strand-specific nucleases. A new alternative protocol involving a simplified gel system, unlabeled primers, DNA staining after single strand-specific nuclease digestion, and standard gel data analysis was optimized to address these constraints. Using this alternative protocol, we successfully identified four new SNPs verified by sequencing in a collection of 57 diverse rice accessions along with 2 previously reported SNPs in a 922-bp DNA region from thealk gene. An SNP cluster containing a deletion within a 472-bp fragment of thewaxy gene was also characterized. In addition, 4 previously reported SNPs in thealk andwaxy genes were faithfully genotyped among the 57 accessions based on comparisons with sequencing results. Associations between the genotyped SNPs and amylose class and starch gelatinization temperature were as anticipated. These results, along with detailed time and cost comparisons between the 2 methods, suggest that alternative Ecotilling is a simple and reproducible method for SNP discovery and genotyping in rice that leads to substantial savings in equipment, reagents, software, and time compared with the standard Ecotilling procedure. Approved for publication by the Director of the Louisiana Agricultural Experiment Station as paper No. 06-14-0237.     

TILLING - a shortcut in functional genomics

Recent advances in large-scale genome sequencing projects have opened up new possibilities for the application of conventional mutation techniques in not only forward but also reverse genetics strategies. TILLING (Targeting Induced Local Lesions IN Genomes) was developed a decade ago as an alternative to insertional mutagenesis. It takes advantage of classical mutagenesis, sequence availability and high-throughput screening for nucleotide polymorphisms in a targeted sequence. The main advantage of TILLING as a reverse genetics strategy is that it can be applied to any species, regardless of its genome size and ploidy level. The TILLING protocol provides a high frequency of point mutations distributed randomly in the genome. The great mutagenic potential of chemical agents to generate a high rate of nucleotide substitutions has been proven by the high density of mutations reported for TILLING populations in various plant species. For most of them, the analysis of several genes revealed 1 mutation/200–500 kb screened and much higher densities were observed for polyploid species, such as wheat. High-throughput TILLING permits the rapid and low-cost discovery of new alleles that are induced in plants. Several research centres have established a TILLING public service for various plant species. The recent trends in TILLING procedures rely on the diversification of bioinformatic tools, new methods of mutation detection, including mismatch-specific and sensitive endonucleases, but also various alternatives for LI-COR screening and single nucleotide polymorphism (SNP) discovery using next-generation sequencing technologies. The TILLING strategy has found numerous applications in functional genomics. Additionally, wide applications of this throughput method in basic and applied research have already been implemented through modifications of the original TILLING strategy, such as Ecotilling or Deletion TILLING.     

Discovery of nucleotide polymorphisms in the Musa gene pool by Ecotilling

Musa (banana and plantain) is an important genus for the global export market and in local markets where it provides staple food for approximately 400 million people. Hybridization and polyploidization of several (sub)species, combined with vegetative propagation and human selection have produced a complex genetic history. We describe the application of the Ecotilling method for the discovery and characterization of nucleotide polymorphisms in diploid and polyploid accessions of Musa. We discovered over 800 novel alleles in 80 accessions. Sequencing and band evaluation shows Ecotilling to be a robust and accurate platform for the discovery of polymorphisms in homologous and homeologous gene targets. In the process of validating the method, we identified two single nucleotide polymorphisms that may be deleterious for the function of a gene putatively important for phototropism. Evaluation of heterozygous polymorphism and haplotype blocks revealed a high level of nucleotide diversity in Musa accessions. We further applied a strategy for the simultaneous discovery of heterozygous and homozygous polymorphisms in diploid accessions to rapidly evaluate nucleotide diversity in accessions of the same genome type. This strategy can be used to develop hypotheses for inheritance patterns of nucleotide polymorphisms within and between genome types. We conclude that Ecotilling is suitable for diversity studies in Musa, that it can be considered for functional genomics studies and as tool in selecting germplasm for traditional and mutation breeding approaches.     

Single nucleotide polymorphisms in Cryptomeria japonica: their discovery and validation for genome mapping and diversity studies

In order to develop a large set of single-nucleotide polymorphisms (SNPs) in Cryptomeria japonica, for a wide range of applications, we adopted a systematic EST (expressed sequence tags) re-sequencing approach. We examined a group of four genotypes comprising parents of a mapping population as well as representatives of two main lines from natural populations. We sequenced 5,170 gene fragments, representing analysis of over 1.3?Mb of DNA sequences in C. japonica. This analysis leads to the discovery of 13,413 SNPs in 3,744 amplicons, with an average of one SNP for every 101.0?bp (one SNP for every 78.3?bp in introns and for every 106.7?bp in exon regions). Nucleotide diversity in C. japonica (???=?0.0045) was found to be similar to values recorded in highly polymorphic forest tree species such as pine. We also validated the use of the SNPs as molecular markers for genetic diversity studies using the high throughput SNP genotyping platform GoldenGate. From 1,536 candidate SNP sites tested, 1,164 (75.8?%) were confirmed to be polymorphic. We anticipate that the genome-wide SNP markers reported here will be useful for evaluating the species?? range-wide genetic structure and in marker-assisted selection used as part of the C. japonica tree improvement program.     

The genetics and genomics of the drought response in Populus

The genetic nature of tree adaptation to drought stress was examined by utilizing variation in the drought response of a full-sib second generation (F(2)) mapping population from a cross between Populus trichocarpa (93-968) and P. deltoides Bart (ILL-129) and known to be highly divergent for a vast range of phenotypic traits. We combined phenotyping, quantitative trait loci (QTL) analysis and microarray experiments to demonstrate that 'genetical genomics' can be used to provide information on adaptation at the species level. The grandparents and F(2) population were subjected to soil drying, and contrasting responses to drought across genotypes, including leaf coloration, expansion and abscission, were observed, and QTL for these traits mapped. A subset of extreme genotypes exhibiting extreme sensitivity and insensitivity to drought on the basis of leaf abscission were defined, and microarray experiments conducted on these genotypes and the grandparent species. The extreme genotype groups induced a different set of genes: 215 and 125 genes differed in their expression response between groups in control and drought, respectively, suggesting species adaptation at the gene expression level. Co-location of differentially expressed genes with drought-specific and drought-responsive QTLs was examined, and these may represent candidate genes contributing to the variation in drought response.     

Plant eco-devo: the potential of poplar as a model organism

Ecological developmental genetics is the study of how ecologically significant traits originate in the genome and how the allelic combinations responsible are maintained in populations and species. Plant development involves a continuous feedback between growth and environment and the success of individual genotype x environment interactions determines the passage of alleles to the next generation: the adaptive recursion. Outbreeding plants contain a large amount of genetic variation, mostly in the form of single nucleotide polymorphisms (SNPs). One of the challenges of eco-devo is to distinguish neutral SNPs from those with ecological consequences. The complete genome sequence of Populus trichocarpa Torr. & A. Gray will be a significant aid in this endeavour. Occurring from California to Alaska, this is the first ecologically 'keystone' species to be sequenced. It has a rich natural history and is an obligate outbreeder. The individual sequenced, Nisqually-1, appears to be heterozygous on average about every 100 bp over the c. 500 million bp of the genome. Overlaid on this within-individual variation is some ecologically based between-individual genotypic variation evident across the distribution of the species. The synthesis of information from genomics and ecology is now in prospect. This 'ecomolecular synthesis' is likely to provide a rich insight into the genomic basis of plant adaptation.     

Climate‐driven reduction of genetic variation in plant phenology alters soil communities and nutrient pools

We examined the hypothesis that climate‐driven evolution of plant traits will influence associated soil microbiomes and ecosystem function across the landscape. Using a foundation tree species, Populus angustifolia, observational and common garden approaches, and a base population genetic collection that spans 17 river systems in the western United States, from AZ to MT, we show that (a) as mean annual temperature (MAT) increases, genetic and phenotypic variation for bud break phenology decline; (b) soil microbiomes, soil nitrogen (N), and soil carbon (C) vary in response to MAT and conditioning by trees; and (c) with losses of genetic variation due to warming, population‐level regulation of community and ecosystem functions strengthen. These results demonstrate a relationship between the potential evolutionary response of populations and subsequent shifts in ecosystem function along a large temperature gradient.     

Whole genome SNP discovery and analysis of genetic diversity in Turkey (Meleagris gallopavo)

ABSTRACT: BACKGROUND: The turkey (Meleagris gallopavo) is an important agricultural species and the second largest contributor to the world's poultry meat production. Genetic improvement is attributed largely to selective breeding programs that rely on highly heritable phenotypic traits, such as body size and breast muscle development. Commercial breeding with small effective population sizes and epistasis can result in loss of genetic diversity, which in turn can lead to reduced individual fitness and reduced response to selection. The presence of genomic diversity in domestic livestock species therefore, is of great importance and a prerequisite for rapid and accurate genetic improvement of selected breeds in various environments, as well as to facilitate rapid adaptation to potential changes in breeding goals. Genomic selection requires a large number of genetic markers such as e.g. single nucleotide polymorphisms (SNPs) the most abundant source of genetic variation within the genome. RESULTS: Alignment of next generation sequencing data of 32 individual turkeys from different populations was used for the discovery of 5.49 million SNPs, which subsequently were used for the analysis of genetic diversity among the different populations. All of the commercial lines branched from a single node relative to the heritage varieties and the South Mexican turkey population. Heterozygosity of all individuals from the different turkey populations ranged from 0.17-2.73 SNPs/Kb, while heterozygosity of populations ranged from 0.73-1.64 SNPs/Kb. The average frequency of heterozygous SNPs in individual turkeys was 1.07 SNPs/Kb. Five genomic regions with very low nucleotide variation were identified in domestic turkeys that showed state of fixation towards alleles different than wild alleles. CONCLUSION: The turkey genome is much less diverse with a relatively low frequency of heterozygous SNPs as compared to other livestock species like chicken and pig. The whole genome SNP discovery study in turkey resulted in the detection of 5.49 million putative SNPs compared to the reference genome. All commercial lines appear to share a common origin. Presence of different alleles/haplotypes in the SM population highlights that specific haplotypes have been selected in the modern domesticated turkey.     

High population differentiation and unusual haplotype structure in a shade-intolerant pioneer tree species, Zanthoxylum ailanthoides (Rutaceae) revealed by analysis of DNA polymorphism at four nuclear loci

Differences in demographic history, life-history traits, and breeding systems affect nucleotide variation patterns. It is expected that shade-intolerant pioneer tree species have different patterns of genetic polymorphism and population structure than climax species. We studied patterns of nucleotide polymorphism at four putative starch pathway loci (agpSA, agpSB, agpL, and GBSSI) in Zanthoxylum ailanthoides , a shade-intolerant pioneer tree species that occupies forest gaps in warm-temperate forests of East Asia. Genetic diversity was lower within each population than among populations, and differentiation among populations was high across the loci ( F _ST = 0.32–0.64), as expected from the insect-pollinated breeding system and the metapopulation structure of this pioneer species. Numbers of haplotypes were smaller than those expected from the observed numbers of segregating sites. Single haplotypes accounted for more than 47% of all the sampled genes at the respective loci. These variation patterns were incompatible with neutral predictions for populations of a finite island model. Complex population dynamics, such as bottleneck and/or admixture, in the history of this pioneer tree species might have resulted in the observed patterns of genetic variation and population structure, which are different from those of climax wind-pollinated tree species, such as conifers. In contrast to the other loci investigated in this study, agpL showed nearly no variation in Z. ailanthoides (one singleton only), but there was some extent of variation in a closely related species, Zanthoxylum schinifolium . This suggests possibly a recent selective sweep at or near the locus in Z. ailanthoides .     

Identification, characterization and interpretation of single-nucleotide sequence variation in allopolyploid crop species

An understanding of nature and extent of nucleotide sequence variation is required for programmes of discovery and characterization of single nucleotide polymorphisms (SNPs), which provide the most versatile class of molecular genetic marker. A majority of higher plant species are polyploids, and allopolyploidy, because of hybrid formation between closely related taxa, is very common. Mutational variation may arise both between allelic (homologous) sequences within individual subgenomes and between homoeologous sequences among subgenomes, in addition to paralogous variation between duplicated gene copies. Successful SNP validation in allopolyploids depends on differentiation of the sequence variation classes. A number of biological factors influence the feasibility of discrimination, including degree of gene family complexity, inbreeding or outbreeding reproductive habit, and the level of knowledge concerning progenitor diploid species. In addition, developments in high-throughput DNA sequencing and associated computational analysis provide general solutions for the genetic analysis of allopolyploids. These issues are explored in the context of experience from a range of allopolyploid species, representing grain (wheat and canola), forage (pasture legumes and grasses), and horticultural (strawberry) crop. Following SNP discovery, detection in routine genotyping applications also presents challenges for allopolyploids. Strategies based on either design of subgenome-specific SNP assays through homoeolocus-targeted polymerase chain reaction (PCR) amplification, or detection of incremental changes in nucleotide variant dosage, are described.     

High‐density SNP assay development for genetic analysis in maritime pine (Pinus pinaster)

Maritime pine provides essential ecosystem services in the south‐western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three‐generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene‐based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies.     

Geographical barriers and climate influence demographic history in narrowleaf cottonwoods

Studies of genetic variation can clarify the role of geography and spatio-temporal variation of climate in shaping demography, particularly in temperate zone tree species with large latitudinal ranges. Here, we examined genetic variation in narrowleaf cottonwood, Populus angustifolia, a dominant riparian tree. Using multi-locus surveys of polymorphism in 363 individuals across the species'' 1800 km latitudinal range, we found that, first, P. angustifolia has stronger neutral genetic structure than many forest trees (simple sequence repeat (SSR) F_ST=0.21), with major genetic groups corresponding to large apparent geographical barriers to gene flow. Second, using SSRs and putatively neutral sequenced loci, coalescent simulations indicated that populations diverged before the last glacial maximum (LGM), suggesting the presence of population structure before the LGM. Third, the LGM and subsequent warming appear to have had different influences on each of these distinct populations, with effective population size reduction in the southern extent of the range but major expansion in the north. These results are consistent with the hypothesis that climate and geographic barriers have jointly affected the demographic history of P. angustifolia, and point the importance of both factors as being instrumental in shaping genetic variation and structure in widespread forest trees.     

Single‐nucleotide polymorphism discovery and diversity in the model legume Medicago truncatula

Extensive genomic resources are available in the model legume Medicago truncatula. Here, we present the discovery and design of the first array of single‐nucleotide polymorphism (SNP) markers in M. truncatula through large‐scale Sanger resequencing of genomic fragments spanning the genome, in a diverse panel of 16 M. truncatula accessions. Both anonymous fragments and fragments targeting candidate genes for flowering phenology and symbiosis were surveyed for nucleotide variation in almost 230 kb of unique genomic regions. A set of 384 SNP markers was designed for an Illumina's GoldenGate assay, genotyped on a collection of 192 inbred lines (CC192) representing the geographical range of the species and used to survey the diversity of two natural populations. Finally, 86% of the tested SNPs were of high quality and exhibited polymorphism in the CC192 collection. Even at the population level, we detected polymorphism for more than 50% of the selected SNPs. Analysis of the allele frequency spectrum in the CC192 showed a reduced ascertainment bias, mostly limited to very rare alleles (frequency <0.01). The substantial polymorphism detected at the species and population levels, the high marker quality and the potential to survey large samples of individuals make this set of SNP markers a valuable tool to improve our understanding of the effect of demographic and selective factors that shape the natural genetic diversity within the selfing species Medicago truncatula.