Population structure, genetic diversity and linkage disequilibrium in elite winter wheat assessed with SNP and SSR markers

Modern genomics approaches rely on the availability of high-throughput and high-density genotyping platforms. A major breakthrough in wheat genotyping was the development of an SNP array. In this study, we used a diverse panel of 172 elite European winter wheat lines to evaluate the utility of the SNP array for genomic analyses in wheat germplasm derived from breeding programs. We investigated population structure and genetic relatedness and found that the results obtained with SNP and SSR markers differ. This suggests that additional research is required to determine the optimum approach for the investigation of population structure and kinship. Our analysis of linkage disequilibrium (LD) showed that LD decays within approximately 5–10 cM. Moreover, we found that LD is variable along chromosomes. Our results suggest that the number of SNPs needs to be increased further to obtain a higher coverage of the chromosomes. Taken together, SNPs can be a valuable tool for genomics approaches and for a knowledge-based improvement of wheat.     

Detecting population structure using STRUCTURE software: effect of background linkage disequilibrium

STRUCTURE is the most widely used clustering software to detect population genetic structure. The last version of this software (STRUCTURE 2.1) has been enhanced recently to take into account the occurrence of linkage disequilibrium (LD) caused by admixture between populations. This last version, however, still does not consider the effects of strong background LD caused by genetic drift, and which may cause spurious results. STRUCTURE authors have, therefore, suggested a rough threshold value of the distance (1.0 cM) between two loci below which the pair of loci should not be used. Because of the sensitiveness of LD to demographic events, the distance between loci is not always a good indicator of the strength of LD. In this study, we examine the link between genomic distance and the strength of the correlation between loci (r(LD)) in a free-ranging population of mouflon (Ovis aries), and we present an empirical test of effect of r(LD) on the clustering results provided by the linkage model in STRUCTURE. We showed that a high r(LD) value increases the probability of detecting spurious clustering. We propose to use r(LD) as an index to base a decision on whether or not to use a pair of loci in a clustering analysis.     

Population structure and linkage disequilibrium in barley assessed by DArT markers

Diversity Array Technology (DArT) markers were used to investigate the genetic diversity, population structure, and extent of linkage disequilibrium (LD) on a genome-wide level in Canadian barley (Hordeum vulgare L.). Approximately 1,000 DArT markers were polymorphic and scored with high confidence among a collection of 170 barley lines composed mostly of Canadian cultivars and breeding lines. The reproducibility of DArT markers proved very high, as 99.9% of allele calls were identical among seven replicated samples. The polymorphism information content (PIC) of DArT markers ranged between 0.04 and 0.50 with an average of 0.38. Using principal coordinate analysis (PCoA), most lines fell into one of two major groups reflecting inflorescence type (two-row versus six-row). Within these two large groups, evidence of geographic clustering of genotypes was also observed. A cluster analysis Unweighted Pair Group Method with Algorithmic Mean suggested the existence of three subgroups within the two-row group and four subgroups within the six-row group. An analysis of molecular variance (AMOVA) revealed highly significant (P < 0.001) genetic variance within subgroups, among subgroups, and among groups. Values of LD, expressed as r ², declined with increasing genetic distance, and mean values of r ² fell below 0.2 for markers located 2.6 cM apart. Approximately 8% of marker pairs located on the same chromosome and 3.4% of pairs located on different chromosomes were in LD (r ²  > 0.2). Within both the subsets of two-row and six-row lines, LD extended slightly further (3.5 cM) than for the entire set, while 7.5% of intra-chromosomal locus pairs and <2% of inter-chromosomal pairs were in LD. We discuss the implications of these findings with regard to the prospects of association mapping of complex traits in barley.     

Molecular genetic diversity and population structure in Lycium accessions using SSR markers

This study was conducted to assess the genetic diversity and population structure of 139 Lycium chinense accessions using 18 simple sequence repeat (SSR) markers. In total, 108 alleles were detected. The number of alleles per marker locus ranged from two to 17, with an average of six. The gene diversity and polymorphism information content value averaged 0.3792 and 0.3296, with ranges of 0.0793 to 0.8023 and 0.0775 to 0.7734, respectively. The average heterozygosity was 0.4394. The model-based structure analysis revealed the presence of three subpopulations, which was consistent with clustering based on genetic distance. An AMOVA analysis showed that the between-population component of genetic variance was less than 15.3%, in contrast to 84.7% for the within-population component. The overall F_ST value was 0.1178, indicating a moderate differentiation among groups. The results could be used for future L. chinense allele mining, association mapping, gene cloning, germplasm conservation, and designing effective breeding programs.     

Genetic analysis of enhanced-axillary-branching-derived Eucalyptus tereticornis Smith and E. camaldulensis Dehn. plants

In a culture method for enhanced axillary branching functional plants of Eucalyptus tereticornis and E. camaldulensis are efficiently regenerated. To assess the genetic integrity among the regenerants, we employed multiple analytical tools including cytochemical and molecular assays. The 2C DNA amounts were estimated in the meristematic zones of root and shoot tips of 250 micropropagated plants, collected at various cycles of tissue culture from multiplication to field transfer, and compared to the corresponding mother plants. The culture conditions did not induce amplification or deletion of DNA sequences, nor were there drastic change(s) in chromosome number, since all the micropropagated plants of E. tereticornis (1.2 pg) and E. camaldulensis (1.4 pg) maintained the same DNA amounts as the mother plant. Total DNA of 46 micropropagated and mother plants digested with eight restriction enzymes and hybridized to 13 nuclear, mitochondrial, and synthetic oligonucleotide DNA probes yielded 82 bands. Hybridization patterns indicated that the variation observed was minor. To further confirm the genetic fidelity, 12 arbitrary 10-base primers and six synthetic oligonucleotide sequences, successfully used to amplify genomic DNA from in vivo and in vitro materials, produced 133 fragments that were monomorphic across the plants tested. The present results demonstrate that enhanced-axillary-branching culture of mature trees could be utilized commercially for mass clonal propagation of these two important Eucalyptus species that have been recalcitrant to vegetative propagation. The results also provide novel insights into the genetic differences between E. tereticornis and E. camaldulensis. Received: 8 October 1996 / Revision received: 22 July 1997 / Accepted: 30 July 1997     

SNP genotyping in melons: genetic variation, population structure, and linkage disequilibrium

Novel sequencing technologies were recently used to generate sequences from multiple melon (Cucumis melo L.) genotypes, enabling the in silico identification of large single nucleotide polymorphism (SNP) collections. In order to optimize the use of these markers, SNP validation and large-scale genotyping are necessary. In this paper, we present the first validated design for a genotyping array with 768 SNPs that are evenly distributed throughout the melon genome. This customized Illumina GoldenGate assay was used to genotype a collection of 74 accessions, representing most of the botanical groups of the species. Of the assayed loci, 91 % were successfully genotyped. The array provided a large number of polymorphic SNPs within and across accessions. This set of SNPs detected high levels of variation in accessions from this crop’s center of origin as well as from several other areas of melon diversification. Allele distribution throughout the genome revealed regions that distinguished between the two main groups of cultivated accessions (inodorus and cantalupensis). Population structure analysis showed a subdivision into five subpopulations, reflecting the history of the crop. A considerably low level of LD was detected, which decayed rapidly within a few kilobases. Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in melon. Since many of the genotyped accessions are currently being used as the parents of breeding populations in various programs, this set of mapped markers could be used for future mapping and breeding efforts.     

Selection of genetic markers for association analyses,using linkage disequilibrium and haplotypes

The genotyping of closely spaced single-nucleotide polymorphism (SNP) markers frequently yields highly correlated data, owing to extensive linkage disequilibrium (LD) between markers. The extent of LD varies widely across the genome and drives the number of frequent haplotypes observed in small regions. Several studies have illustrated the possibility that LD or haplotype data could be used to select a subset of SNPs that optimize the information retained in a genomic region while reducing the genotyping effort and simplifying the analysis. We propose a method based on the spectral decomposition of the matrices of pairwise LD between markers, and we select markers on the basis of their contributions to the total genetic variation. We also modify Clayton's "haplotype tagging SNP" selection method, which utilizes haplotype information. For both methods, we propose sliding window-based algorithms that allow the methods to be applied to large chromosomal regions. Our procedures require genotype information about a small number of individuals for an initial set of SNPs and selection of an optimum subset of SNPs that could be efficiently genotyped on larger numbers of samples while retaining most of the genetic variation in samples. We identify suitable parameter combinations for the procedures, and we show that a sample size of 50-100 individuals achieves consistent results in studies of simulated data sets in linkage equilibrium and LD. When applied to experimental data sets, both procedures were similarly effective at reducing the genotyping requirement while maintaining the genetic information content throughout the regions. We also show that haplotype-association results that Hosking et al. obtained near CYP2D6 were almost identical before and after marker selection.     

Evaluation of linkage disequilibrium measures between multi-allelic markers as predictors of linkage disequilibrium between markers and QTL

Effectiveness of marker-assisted selection (MAS) and quantitative trait loci (QTL) mapping using population-wide linkage disequilibrium (LD) between markers and QTL depends on the extent of LD and how it declines with distance in a population. Because marker-QTL LD cannot be observed directly, the objective of this study was to evaluate alternative measures of observable LD between multi-allelic markers as predictors of usable LD of multi-allelic markers with presumed biallelic QTL. Observable LD between marker pairs was evaluated using eight existing measures and one new measure. These consisted of two pooled and standardized measures of LD between pairs of alleles at two markers based on Lewontin's LD measure, two pooled measures of squared correlations between alleles, one standardized measure using Hardy-Weinberg heterozygosities, and four measures based on the chi-square statistic for testing for association between alleles at two loci. In simulated populations with a range of LD generated by drift and a range of marker polymorphism, marker-marker LD measured by a standardized chi-square statistic (denoted chi(2')) was found to be the best predictor of useable marker-QTL LD for a group of multi-allelic markers. Estimates of the level and decline of marker-marker LD with distance obtained from chi(2') were linearly and highly correlated with usable LD of those markers with QTL across population structures and marker polymorphism. Corresponding relationships were poorer for the other marker-marker LD measures. Therefore, when LD is generated by drift, chi(2') is recommended to quantify the amount and extent of usable LD in a population for QTL mapping and MAS based on multi-allelic markers.     

Analysis of molecular genetic diversity and population structure in Amaranthus germplasm using SSR markers

We assessed the molecular genetic diversity and population structure of Amaranthus species accessions using 11 simple sequence repeat markers. A total of 122 alleles were detected, and the number of alleles per marker (N_A) ranged from 6 to 21 with an average of 11.1 alleles. The frequency of major alleles per locus ranged from 0.148 to 0.695, with an average value of 0.496 per marker. The overall polymorphic information content values were 0.436–0.898, with an average value of 0.657. The observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged from 0.056 to 0.876 and from 0.480 to 0.907, with average values of 0.287 and 0.698, respectively. The average H_O (0.240) was lower than the H_E and gene flow (Nm), and showed substantial genetic variability among all populations of amaranth accessions. The sample groupings did not strictly follow the geographic affiliations of the accessions. A similar pattern was obtained using model-based structure analysis without grouping by species type. Knowledge of the genetic diversity and population structure of amaranth can be used to select representative genotypes and manage Amaranthus germplasm breeding programs.     

Genetic variation, population structure, and linkage disequilibrium in European elite germplasm of perennial ryegrass

Perennial ryegrass (Lolium perenne L.) is a highly valued temperate climate grass species grown as forage crop and for amenity uses. Due to its outbreeding nature and recent domestication, a high degree of genetic diversity is expected among cultivars. The aim of this study was to assess the extent of linkage disequilibrium (LD) within European elite germplasm and to evaluate the appropriate methodology for genetic association mapping in perennial ryegrass. A high level of genetic diversity was observed in a set of 380 perennial ryegrass elite genotypes when genotyped with 40 SSRs and 2 STS markers. A Bayesian structure analysis identified two subpopulations, which were confirmed by principal coordinate analysis (PCoA). One subpopulation consisted mainly of genotypes originating from the UK, while germplasm mostly from Continental Europe was grouped into the second subpopulation. LD (r²) decay was rapid and occurred within 0.4 cM across European varieties, when population structure was taken into consideration. However, an extended LD of up to 6.6 cM was detected within the variety Aberdart. High genetic diversity and rapid LD decay provide means for high resolution association mapping in elite materials of perennial ryegrass. However, different strategies need to be applied depending on the material used. Genome-wide association study (GWAS) with several hundred markers can be applied within synthetic varieties to identify large (up to 10 cM) genomic regions affecting trait variation. A combination of available and novel DNA markers is needed to achieve resolution required for GWAS in elite breeding materials. An even higher marker density of several million SNPs might be needed for GWAS in diverse ecotype collections, potentially resulting in quantitative trait polymorphism (QTP) identification.     

Changes in growth performance and fecundity of Eucalyptus camaldulensis and E. tereticornis during domestication in southern India

Bulk seedlots of two unpedigreed multiprovenance seed production areas (SPAs) each of Eucalyptus camaldulensis and Eucalyptus tereticornis and one pedigreed seedling seed orchard (SSO) of E. tereticornis were planted in genetic gain trials at three southern Indian trial sites. At the time of seed collection, fewer than 30% trees flowered in these orchards, except in one E. camaldulensis SPA where 73% of the trees flowered, which had an estimated outcrossing rate of 86%. The E. tereticornis SSO was dominated by pollen from five highly fecund families of the Indian Mysore gum land race, which contributed 59% of the fruits produced. The SPA and SSO seedlots were compared with a bulked natural-provenance seedlot of E. camaldulensis (Morehead, Laura, and Kennedy Rivers, Queensland), another natural-provenance seedlot (Petford, Queensland), commercial eucalypt clones at two sites, and a Mysore gum seedlot at one site. At 3 years, progeny from all the four SPAs displayed good survival (79–93%) and performance similar to that of the natural provenances and the commercial clones. Progeny from the E. tereticornis SSO had significantly lower growth (at two sites) and lower survival at all three test sites. The Mysore gum seedlot displayed high fecundity and lower growth but better survival than the SSO progeny. Seed orchard genetic composition and flowering contributions thus affected progeny performance and the extent to which orchard genetic diversity was captured in the progeny. SPA progeny displayed greater fecundity than the natural provenances, indicating a response to selection for fertility.     

Species discrimination and population differentiation in ants using microsatellites

This study was conducted to establish the regional scale of population differentiation of ants in the wheat belt of central western New South Wales. Microsatellite variation was surveyed at five loci in two morphologically similar ant species (designated "A" and "B") from the Camponotus ephippium complex. Three of the five scored microsatellite loci were highly variable with totals, in the two species, of 11, 13 and 42 alleles. The other loci had two and three alleles. The mean number of alleles per locus per sample ranged from 2.0 to 4.6 for species A and from 1.4 to 3.8 in species B. Mean observed heterozygosity was 0.385 for species A and 0.363 for species B. The geographic distribution of genotypes was significantly non-random for all tested loci in both species. Eight of 47 alleles in species A and 15 of 28 in species B were restricted to a single site. Allelic accumulation percentages were calculated for several orderings of samples - level of heterozygosity, sample size and geographic position. In all orderings three or more samples must be included for more than three-quarters of alleles to be represented.     

Towards linkage analysis with markers in linkage disequilibrium by graphical modelling

We review recent developments of MCMC integration methods for computations on graphical models for two applications in statistical genetics: modelling allelic association and pedigree based linkage analysis. We discuss and illustrate estimation of graphical models from haploid and diploid genotypes, and the importance of MCMC updating schemes beyond what is strictly necessary for irreducibility. We then outline an approach combining these methods to compute linkage statistics when alleles at the marker loci are in linkage disequilibrium. Other extensions suitable for analysis of SNP genotype data in pedigrees are also discussed and programs that implement these methods, and which are available from the author's web site, are described. We conclude with a discussion of how this still experimental approach might be further developed.     

Genetic diversity,population structure,and linkage disequilibrium of elite and local apple accessions from Belgium using the IRSC array

The identification of molecular markers associated with economic and quality traits will help improve breeding for new apple (Malus × domestica Borkh.) cultivars. Tools such as the 8K apple SNP array developed by the RosBREED consortium allow for high-throughput genotyping of SNP polymorphisms within collections. However, genetic characterization and the identification of population stratification and kinship within germplasm collections is a fundamental prerequisite for identifying robust marker–trait associations. In this study, a collection of apple germplasm originally developed for plant architectural studies and consisting of both non-commercial/local and elite accessions was genotyped using the 8K apple SNP array to identify cryptic relationships between accessions, to analyze population structure and to calculate the linkage disequilibrium (LD). A total of nine pairs of synonyms and several triploids accessions were identified within the 130 accessions genotyped. In addition, most of the known parent-child relations were confirmed, and several putative, previously unknown parent-child relations were identified among the local accessions. No clear subgroups could be identified although some separation between local and elite accessions was evident. The study of LD showed a rapid decay in our collection, indicating that a larger number of SNPs is necessary to perform whole genome association mapping. Finally, an association mapping effort for architectural traits was carried out on a small number of accessions to estimate the feasibility of this approach.     

Markers for ornamental traits in Phalaenopsis orchids: population structure, linkage disequilibrium and association mapping

Although Phalaenopsis orchids are among the most economically important potted plants, little is known about either the genetic diversity among varieties or the genetic complexity of key ornamental traits. Therefore, we analysed the genetic diversity of a broad collection of Phalaenopsis varieties and selected wild species by means of molecular markers. The marker data were used to obtain genetic distances, estimates of the degree of linkage disequilibrium and population structure for the genotypes under study. With a total of 492 markers, the genotypes clustered according to their horticultural classification (for example, old hybrids vs. more recent hybrids) but not according to their origin, indicating extensive exchange of germplasm among breeders. Linkage disequilibrium was found to decrease relatively slowly, most likely due to the small number of generations that have occurred since the first hybrids were generated. Based on the most likely estimates for the population structure (ranging from 10 to 12 subpopulations), associations between ornamental traits like flower size, flower colour, flower type, flower texture, stem length and leaf shape were calculated. These results can now serve as starting points for detailed analyses of the genetic architecture of these traits.     

Genetic diversity and linkage disequilibrium in Chinese bread wheat (Triticum aestivum L.) revealed by SSR markers

Two hundred and fifty bread wheat lines, mainly Chinese mini core accessions, were assayed for polymorphism and linkage disequilibrium (LD) based on 512 whole-genome microsatellite loci representing a mean marker density of 5.1 cM. A total of 6,724 alleles ranging from 1 to 49 per locus were identified in all collections. The mean PIC value was 0.650, ranging from 0 to 0.965. Population structure and principal coordinate analysis revealed that landraces and modern varieties were two relatively independent genetic sub-groups. Landraces had a higher allelic diversity than modern varieties with respect to both genomes and chromosomes in terms of total number of alleles and allelic richness. 3,833 (57.0%) and 2,788 (41.5%) rare alleles with frequencies of <5% were found in the landrace and modern variety gene pools, respectively, indicating greater numbers of rare variants, or likely new alleles, in landraces. Analysis of molecular variance (AMOVA) showed that A genome had the largest genetic differentiation and D genome the lowest. In contrast to genetic diversity, modern varieties displayed a wider average LD decay across the whole genome for locus pairs with r(2)>0.05 (P<0.001) than the landraces. Mean LD decay distance for the landraces at the whole genome level was <5 cM, while a higher LD decay distance of 5-10 cM in modern varieties. LD decay distances were also somewhat different for each of the 21 chromosomes, being higher for most of the chromosomes in modern varieties (<5 ～ 25 cM) compared to landraces (<5 ～ 15 cM), presumably indicating the influences of domestication and breeding. This study facilitates predicting the marker density required to effectively associate genotypes with traits in Chinese wheat genetic resources.     

Early detection of population fragmentation using linkage disequilibrium estimation of effective population size

Population subdivision due to habitat loss and modification, exploitation of wild populations and altered spatial population dynamics is of increasing concern in nature. Detecting population fragmentation is therefore crucial for conservation management. Using computer simulations, we show that a single sample estimator of N _e based on linkage disequilibrium is a highly sensitive and promising indicator of recent population fragmentation and bottlenecks, even with some continued gene flow. For example, fragmentation of a panmictic population of N _e = 1,000 into demes of N _e = 100 can be detected with high probability after a single generation when estimates from this method are compared to prefragmentation estimates, given data for ~20 microsatellite loci in samples of 50 individuals. We consider a range of loci (10–40) and individuals (25–100) typical of current studies of natural populations and show that increasing the number of loci gives nearly the same increase in precision as increasing the number of individuals sampled. We also evaluated effects of incomplete fragmentation and found this N _e-reduction signal is still apparent in the presence of considerable migration (m ~ 0.10–0.25). Single-sample genetic estimates of N _e thus show considerable promise for early detection of population fragmentation and decline.     

Visualization of pairwise and multilocus linkage disequilibrium structure using latent forests

Linkage disequilibrium study represents a major issue in statistical genetics as it plays a fundamental role in gene mapping and helps us to learn more about human history. The linkage disequilibrium complex structure makes its exploratory data analysis essential yet challenging. Visualization methods, such as the triangular heat map implemented in Haploview, provide simple and useful tools to help understand complex genetic patterns, but remain insufficient to fully describe them. Probabilistic graphical models have been widely recognized as a powerful formalism allowing a concise and accurate modeling of dependences between variables. In this paper, we propose a method for short-range, long-range and chromosome-wide linkage disequilibrium visualization using forests of hierarchical latent class models. Thanks to its hierarchical nature, our method is shown to provide a compact view of both pairwise and multilocus linkage disequilibrium spatial structures for the geneticist. Besides, a multilocus linkage disequilibrium measure has been designed to evaluate linkage disequilibrium in hierarchy clusters. To learn the proposed model, a new scalable algorithm is presented. It constrains the dependence scope, relying on physical positions, and is able to deal with more than one hundred thousand single nucleotide polymorphisms. The proposed algorithm is fast and does not require phase genotypic data.     

Sex-specific population structure,natural selection,and linkage disequilibrium in a wild bird population as revealed by genome-wide microsatellite analyses

Background  

Sexual dimorphism in ecologically important traits is widespread, yet the differences in the genomic architecture between the two sexes are largely unexplored. We employed a genome-wide multilocus approach to examine the sexual differences in population subdivision, natural selection and linkage disequilibrium (LD) in a wild Siberian jay (Perisoreus infaustus) population, using genotypes at a total of 107 autosomal and Z-chromosomal microsatellites.     

Using multilocus sequence data to assess population structure, natural selection, and linkage disequilibrium in wild tomatoes

We employed a multilocus approach to examine the effects of population subdivision and natural selection on DNA polymorphism in 2 closely related wild tomato species (Solanum peruvianum and Solanum chilense), using sequence data for 8 nuclear loci from populations across much of the species' range. Both species exhibit substantial levels of nucleotide variation. The species-wide level of silent nucleotide diversity is 18% higher in S. peruvianum (pi(sil) approximately 2.50%) than in S. chilense (pi(sil) approximately 2.12%). One of the loci deviates from neutral expectations, showing a clinal pattern of nucleotide diversity and haplotype structure in S. chilense. This geographic pattern of variation is suggestive of an incomplete (ongoing) selective sweep, but neutral explanations cannot be entirely dismissed. Both wild tomato species exhibit moderate levels of population differentiation (average F(ST) approximately 0.20). Interestingly, the pooled samples (across different demes) exhibit more negative Tajima's D and Fu and Li's D values; this marked excess of low-frequency polymorphism can only be explained by population (or range) expansion and is unlikely to be due to population structure per se. We thus propose that population structure and population/range expansion are among the most important evolutionary forces shaping patterns of nucleotide diversity within and among demes in these wild tomatoes. Patterns of population differentiation may also be impacted by soil seed banks and historical associations mediated by climatic cycles. Intragenic linkage disequilibrium (LD) decays very rapidly with physical distance, suggesting high recombination rates and effective population sizes in both species. The rapid decline of LD seems very promising for future association studies with the purpose of mapping functional variation in wild tomatoes.