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.     

Development and evaluation of a genome‐wide Coffee 8.5K SNP array and its application for high‐density genetic mapping and for investigating the origin of Coffea arabica L.

Coffee species such as Coffea canephora P. (Robusta) and C. arabica L. (Arabica) are important cash crops in tropical regions around the world. C. arabica is an allotetraploid (2n = 4x = 44) originating from a hybridization event of the two diploid species C. canephora and C. eugenioides (2n = 2x = 22). Interestingly, these progenitor species harbour a greater level of genetic variability and are an important source of genes to broaden the narrow Arabica genetic base. Here, we describe the development, evaluation and use of a single‐nucleotide polymorphism (SNP) array for coffee trees. A total of 8580 unique and informative SNPs were selected from C. canephora and C. arabica sequencing data, with 40% of the SNP located in annotated genes. In particular, this array contains 227 markers associated to 149 genes and traits of agronomic importance. Among these, 7065 SNPs (~82.3%) were scorable and evenly distributed over the genome with a mean distance of 54.4 Kb between markers. With this array, we improved the Robusta high‐density genetic map by adding 1307 SNP markers, whereas 945 SNPs were found segregating in the Arabica mapping progeny. A panel of C. canephora accessions was successfully discriminated and over 70% of the SNP markers were transferable across the three species. Furthermore, the canephora‐derived subgenome of C. arabica was shown to be more closely related to C. canephora accessions from northern Uganda than to other current populations. These validated SNP markers and high‐density genetic maps will be useful to molecular genetics and for innovative approaches in coffee breeding.     

Single‐nucleotide polymorphism discovery and validation in high‐density SNP array for genetic analysis in European white oaks

An Illumina Infinium SNP genotyping array was constructed for European white oaks. Six individuals of Quercus petraea and Q. robur were considered for SNP discovery using both previously obtained Sanger sequences across 676 gene regions (1371 in vitro SNPs) and Roche 454 technology sequences from 5112 contigs (6542 putative in silico SNPs). The 7913 SNPs were genotyped across the six parental individuals, full‐sib progenies (one within each species and two interspecific crosses between Q. petraea and Q. robur) and three natural populations from south‐western France that included two additional interfertile white oak species (Q. pubescens and Q. pyrenaica). The genotyping success rate in mapping populations was 80.4% overall and 72.4% for polymorphic SNPs. In natural populations, these figures were lower (54.8% and 51.9%, respectively). Illumina genotype clusters with compression (shift of clusters on the normalized x‐axis) were detected in ~25% of the successfully genotyped SNPs and may be due to the presence of paralogues. Compressed clusters were significantly more frequent for SNPs showing a priori incorrect Illumina genotypes, suggesting that they should be considered with caution or discarded. Altogether, these results show a high experimental error rate for the Infinium array (between 15% and 20% of SNPs potentially unreliable and 10% when excluding all compressed clusters), and recommendations are proposed when applying this type of high‐throughput technique. Finally, results on diversity levels and shared polymorphisms across targeted white oaks and more distant species of the Quercus genus are discussed, and perspectives for future comparative studies are proposed.     

Assessment of the functionality of genome‐wide canine SNP arrays and implications for canine disease association studies

Domestic dogs share a wide range of important disease conditions with humans, including cancers, diabetes and epilepsy. Many of these conditions have similar or identical underlying pathologies to their human counterparts and thus dogs represent physiologically relevant natural models of human disorders. Comparative genomic approaches whereby disease genes can be identified in dog diseases and then mapped onto the human genome are now recognized as a valid method and are increasing in popularity. The majority of dog breeds have been created over the past few hundred years and, as a consequence, the dog genome is characterized by extensive linkage disequilibrium (LD), extending usually from hundreds of kilobases to several megabases within a breed, rather than tens of kilobases observed in the human genome. Genome‐wide canine SNP arrays have been developed, and increasing success of using these arrays to map disease loci in dogs is emerging. No equivalent of the human HapMap currently exists for different canine breeds, and the LD structure for such breeds is far less understood than for humans. This study is a dedicated large‐scale assessment of the functionalities (LD and SNP tagging performance) of canine genome‐wide SNP arrays in multiple domestic dog breeds. We have used genotype data from 18 breeds as well as wolves and coyotes genotyped by the Illumina 22K canine SNP array and Affymetrix 50K canine SNP array. As expected, high tagging performance was observed with most of the breeds using both Illumina and Affymetrix arrays when multi‐marker tagging was applied. In contrast, however, large differences in population structure, LD coverage and pairwise tagging performance were found between breeds, suggesting that study designs should be carefully assessed for individual breeds before undertaking genome‐wide association studies (GWAS).     

netview p: a network visualization tool to unravel complex population structure using genome‐wide SNPs

Network‐based approaches are emerging as valuable tools for the analysis of complex genetic structure in wild and captive populations. netview p combines data quality control with the construction of population networks through mutual k‐nearest neighbours thresholds applied to genome‐wide SNPs. The program is cross‐platform compatible, open‐source and efficiently operates on data ranging from hundreds to hundreds of thousands of SNPs. The pipeline was used for the analysis of pedigree data from simulated (n = 750, SNPs = 1279) and captive silver‐lipped pearl oysters (n = 415, SNPs = 1107), wild populations of the European hake from the Atlantic and Mediterranean (n = 834, SNPs = 380) and grey wolves from North America (n = 239, SNPs = 78 255). The population networks effectively visualize large‐ and fine‐scale genetic structure within and between populations, including family‐level structure and relationships. netview p comprises a network‐based addition to other population analysis tools and provides user‐friendly access to a complex network analysis pipeline through implementation in python .     

Development,validation and genetic analysis of a large soybean SNP genotyping array

Cultivated soybean (Glycine max) suffers from a narrow germplasm relative to other crop species, probably because of under‐use of wild soybean (Glycine soja) as a breeding resource. Use of a single nucleotide polymorphism (SNP) genotyping array is a promising method for dissecting cultivated and wild germplasms to identify important adaptive genes through high‐density genetic mapping and genome‐wide association studies. Here we describe a large soybean SNP array for use in diversity analyses, linkage mapping and genome‐wide association analyses. More than four million high‐quality SNPs identified from high‐depth genome re‐sequencing of 16 soybean accessions and low‐depth genome re‐sequencing of 31 soybean accessions were used to select 180 961 SNPs for creation of the Axiom^® SoyaSNP array. Validation analysis for a set of 222 diverse soybean lines showed that 170 223 markers were of good quality for genotyping. Phylogenetic and allele frequency analyses of the validation set data indicated that accessions showing an intermediate morphology between cultivated and wild soybeans collected in Korea were natural hybrids. More than 90 unanchored scaffolds in the current soybean reference sequence were assigned to chromosomes using this array. Finally, dense average spacing and preferential distribution of the SNPs in gene‐rich chromosomal regions suggest that this array may be suitable for genome‐wide association studies of soybean germplasm. Taken together, these results suggest that use of this array may be a powerful method for soybean genetic analyses relating to many aspects of soybean breeding.     

Detection of genetic variants affecting cattle behaviour and their impact on milk production: a genome‐wide association study

Behaviour traits of cattle have been reported to affect important production traits, such as meat quality and milk performance as well as reproduction and health. Genetic predisposition is, together with environmental stimuli, undoubtedly involved in the development of behaviour phenotypes. Underlying molecular mechanisms affecting behaviour in general and behaviour and productions traits in particular still have to be studied in detail. Therefore, we performed a genome‐wide association study in an F₂ Charolais × German Holstein cross‐breed population to identify genetic variants that affect behaviour‐related traits assessed in an open‐field and novel‐object test and analysed their putative impact on milk performance. Of 37 201 tested single nucleotide polymorphism (SNPs), four showed a genome‐wide and 37 a chromosome‐wide significant association with behaviour traits assessed in both tests. Nine of the SNPs that were associated with behaviour traits likewise showed a nominal significant association with milk performance traits. On chromosomes 14 and 29, six SNPs were identified to be associated with exploratory behaviour and inactivity during the novel‐object test as well as with milk yield traits. Least squares means for behaviour and milk performance traits for these SNPs revealed that genotypes associated with higher inactivity and less exploratory behaviour promote higher milk yields. Whether these results are due to molecular mechanisms simultaneously affecting behaviour and milk performance or due to a behaviour predisposition, which causes indirect effects on milk performance by influencing individual reactivity, needs further investigation.     

Development of an integrated 200K SNP genotyping array and application for genetic mapping,genome assembly improvement and genome wide association studies in pear (Pyrus)

Pear (Pyrus; 2n = 34), the third most important temperate fruit crop, has great nutritional and economic value. Despite the availability of many genomic resources in pear, it is challenging to genotype novel germplasm resources and breeding progeny in a timely and cost‐effective manner. Genotyping arrays can provide fast, efficient and high‐throughput genetic characterization of diverse germplasm, genetic mapping and breeding populations. We present here 200K AXIOM^® PyrSNP, a large‐scale single nucleotide polymorphism (SNP) genotyping array to facilitate genotyping of Pyrus species. A diverse panel of 113 re‐sequenced pear genotypes was used to discover SNPs to promote increased adoption of the array. A set of 188 diverse accessions and an F₁ population of 98 individuals from ‘Cuiguan’ × ‘Starkrimson’ was genotyped with the array to assess its effectiveness. A large majority of SNPs (166 335 or 83%) are of high quality. The high density and uniform distribution of the array SNPs facilitated prediction of centromeric regions on 17 pear chromosomes, and significantly improved the genome assembly from 75.5% to 81.4% based on genetic mapping. Identification of a gene associated with flowering time and candidate genes linked to size of fruit core via genome wide association studies showed the usefulness of the array in pear genetic research. The newly developed high‐density SNP array presents an important tool for rapid and high‐throughput genotyping in pear for genetic map construction, QTL identification and genomic selection.     

MobiSeq: De novo SNP discovery in model and non‐model species through sequencing the flanking region of transposable elements

In recent years, the availability of reduced representation library (RRL) methods has catalysed an expansion of genome‐scale studies to characterize both model and non‐model organisms. Most of these methods rely on the use of restriction enzymes to obtain DNA sequences at a genome‐wide level. These approaches have been widely used to sequence thousands of markers across individuals for many organisms at a reasonable cost, revolutionizing the field of population genomics. However, there are still some limitations associated with these methods, in particular the high molecular weight DNA required as starting material, the reduced number of common loci among investigated samples, and the short length of the sequenced site‐associated DNA. Here, we present MobiSeq, a RRL protocol exploiting simple laboratory techniques, that generates genomic data based on PCR targeted enrichment of transposable elements and the sequencing of the associated flanking region. We validate its performance across 103 DNA extracts derived from three mammalian species: grey wolf (Canis lupus), red deer complex (Cervus sp.) and brown rat (Rattus norvegicus). MobiSeq enables the sequencing of hundreds of thousands loci across the genome and performs SNP discovery with relatively low rates of clonality. Given the ease and flexibility of MobiSeq protocol, the method has the potential to be implemented for marker discovery and population genomics across a wide range of organisms—enabling the exploration of diverse evolutionary and conservation questions.     

Insights into the genetic history of Green‐legged Partridgelike fowl: mtDNA and genome‐wide SNP analysis

The Green‐legged Partridgelike (GP) fowl, an old native Polish breed, is characterised by reseda green‐coloured shanks rather than yellow, white, slate or black commonly observed across most domestic breeds of chicken. Here, we investigate the origin, genetic relationships and structure of the GP fowl using mtDNA D‐loop sequencing and genome‐wide SNP analysis. Genome‐wide association analysis between breeds enables us to verify the genetic control of the reseda green shank phenotype, a defining trait for the breed. Two mtDNA D‐loop haplogroups and three autosomal genetic backgrounds are revealed. Significant associations of SNPs on chromosomes GGA24 and GGAZ indicate that the reseda green leg phenotype is associated with recessive alleles linked to the W and Id loci. Our results provide new insights into the genetic history of European chicken, indicating an admixd origin of East European traditional breeds of chicken on the continent, as supported by the presence of the reseda green phenotype and the knowledge that the GP fowl as a breed was developed before the advent of commercial stocks.     

The easy road to genome‐wide medium density SNP screening in a non‐model species: development and application of a 10 K SNP‐chip for the house sparrow (Passer domesticus)

With the advent of next generation sequencing, new avenues have opened to study genomics in wild populations of non‐model species. Here, we describe a successful approach to a genome‐wide medium density Single Nucleotide Polymorphism (SNP) panel in a non‐model species, the house sparrow (Passer domesticus), through the development of a 10 K Illumina iSelect HD BeadChip. Genomic DNA and cDNA derived from six individuals were sequenced on a 454 GS FLX system and generated a total of 1.2 million sequences, in which SNPs were detected. As no reference genome exists for the house sparrow, we used the zebra finch (Taeniopygia guttata) reference genome to determine the most likely position of each SNP. The 10 000 SNPs on the SNP‐chip were selected to be distributed evenly across 31 chromosomes, giving on average one SNP per 100 000 bp. The SNP‐chip was screened across 1968 individual house sparrows from four island populations. Of the original 10 000 SNPs, 7413 were found to be variable, and 99% of these SNPs were successfully called in at least 93% of all individuals. We used the SNP‐chip to demonstrate the ability of such genome‐wide marker data to detect population sub‐division, and compared these results to similar analyses using microsatellites. The SNP‐chip will be used to map Quantitative Trait Loci (QTL) for fitness‐related phenotypic traits in natural populations.     

SNP discovery in wild and domesticated populations of blue catfish,Ictalurus furcatus,using genotyping‐by‐sequencing and subsequent SNP validation

Blue catfish, Ictalurus furcatus, are valued in the United States as a trophy fishery for their capacity to reach large sizes, sometimes exceeding 45 kg. Additionally, blue catfish × channel catfish (I. punctatus) hybrid food fish production has recently increased the demand for blue catfish broodstock. However, there has been little study of the genetic impacts and interaction of farmed, introduced and stocked populations of blue catfish. We utilized genotyping‐by‐sequencing (GBS) to capture and genotype SNP markers on 190 individuals from five wild and domesticated populations (Mississippi River, Missouri, D&B, Rio Grande and Texas). Stringent filtering of SNP‐calling parameters resulted in 4275 SNP loci represented across all five populations. Population genetics and structure analyses revealed potential shared ancestry and admixture between populations. We utilized the Sequenom MassARRAY to validate two multiplex panels of SNPs selected from the GBS data. Selection criteria included SNPs shared between populations, SNPs specific to populations, number of reads per individual and number of individuals genotyped by GBS. Putative SNPs were validated in the discovery population and in two additional populations not used in the GBS analysis. A total of 64 SNPs were genotyped successfully in 191 individuals from nine populations. Our results should guide the development of highly informative, flexible genotyping multiplexes for blue catfish from the larger GBS SNP set as well as provide an example of a rapid, low‐cost approach to generate and genotype informative marker loci in aquatic species with minimal previous genetic information.     

A powerful tool for genome analysis in maize: development and evaluation of the high density 600 k SNP genotyping array

Background

High density genotyping data are indispensable for genomic analyses of complex traits in animal and crop species. Maize is one of the most important crop plants worldwide, however a high density SNP genotyping array for analysis of its large and highly dynamic genome was not available so far.

Results

We developed a high density maize SNP array composed of 616,201 variants (SNPs and small indels). Initially, 57 M variants were discovered by sequencing 30 representative temperate maize lines and then stringently filtered for sequence quality scores and predicted conversion performance on the array resulting in the selection of 1.2 M polymorphic variants assayed on two screening arrays. To identify high-confidence variants, 285 DNA samples from a broad genetic diversity panel of worldwide maize lines including the samples used for sequencing, important founder lines for European maize breeding, hybrids, and proprietary samples with European, US, semi-tropical, and tropical origin were used for experimental validation. We selected 616 k variants according to their performance during validation, support of genotype calls through sequencing data, and physical distribution for further analysis and for the design of the commercially available Affymetrix® Axiom® Maize Genotyping Array. This array is composed of 609,442 SNPs and 6,759 indels. Among these are 116,224 variants in coding regions and 45,655 SNPs of the Illumina® MaizeSNP50 BeadChip for study comparison. In a subset of 45,974 variants, apart from the target SNP additional off-target variants are detected, which show only a minor bias towards intermediate allele frequencies. We performed principal coordinate and admixture analyses to determine the ability of the array to detect and resolve population structure and investigated the extent of LD within a worldwide validation panel.

Conclusions

The high density Affymetrix® Axiom® Maize Genotyping Array is optimized for European and American temperate maize and was developed based on a diverse sample panel by applying stringent quality filter criteria to ensure its suitability for a broad range of applications. With 600 k variants it is the largest currently publically available genotyping array in crop species.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-823) contains supplementary material, which is available to authorized users.     

Estimating the inbreeding level and genetic relatedness in an isolated population of critically endangered Sichuan taimen (Hucho Bleekeri) using genome‐wide SNP markers

Sichuan taimen (Hucho bleekeri) is critically endangered fish listed in The Red List of Threatened Species compiled by the International Union for Conservation of Nature (IUCN). Specific locus amplified fragment sequencing (SLAF‐seq)‐based genotyping was performed for Sichuan taimen with 43 yearling individuals from three locations in Taibai River (a tributary of Yangtze River) that has been sequestered from its access to the ocean for more than 30 years since late 1980s. Applying the inbreeding level and genetic relatedness estimation using 15,396 genome‐wide SNP markers, we found that the inbreeding level of this whole isolated population was at a low level (2.6 × 10^?3 ± 0.079), and the means of coancestry coefficients within and between the three sampling locations were all very low (close to 0), too. Genomic differentiation was negatively correlated with the geographical distances between the sampling locations (p < .001), and the 43 individuals could be considered as genetically independent two groups. The low levels of genomic inbreeding and relatedness indicated a relatively large number of sexually mature individuals were involved in reproduction in Taibai River. This study suggested a genomic‐relatedness‐guided breeding and conservation strategy for wild fish species without pedigree information records.     

A genome‐wide search for local adaptation in a terrestrial‐breeding frog reveals vulnerability to climate change

Terrestrial‐breeding amphibians are likely to be vulnerable to warming and drying climates, as their embryos require consistent moisture for successful development. Adaptation to environmental change will depend on sufficient genetic variation existing within or between connected populations. Here, we use Single Nucleotide Polymorphism (SNP) data to investigate genome‐wide patterns in genetic diversity, gene flow and local adaptation in a terrestrial‐breeding frog (Pseudophryne guentheri) subject to a rapidly drying climate and recent habitat fragmentation. The species was sampled across 12 central and range‐edge populations (192 samples), and strong genetic structure was apparent, as were high inbreeding coefficients. Populations showed differences in genetic diversity, and one population lost significant genetic diversity in a decade. More than 500 SNP loci were putatively under directional selection, and 413 of these loci were correlated with environmental variables such as temperature, rainfall, evaporation and soil moisture. One locus showed homology to a gene involved in the activation of maturation in Xenopus oocytes, which may facilitate rapid development of embryos in drier climates. The low genetic diversity, strong population structuring and presence of local adaptation revealed in this study shows why management strategies such as targeted gene flow may be necessary to assist isolated populations to adapt to future climates.     

Metabolome‐genome‐wide association study dissects genetic architecture for generating natural variation in rice secondary metabolism

Plants produce structurally diverse secondary (specialized) metabolites to increase their fitness for survival under adverse environments. Several bioactive compounds for new drugs have been identified through screening of plant extracts. In this study, genome‐wide association studies (GWAS) were conducted to investigate the genetic architecture behind the natural variation of rice secondary metabolites. GWAS using the metabolome data of 175 rice accessions successfully identified 323 associations among 143 single nucleotide polymorphisms (SNPs) and 89 metabolites. The data analysis highlighted that levels of many metabolites are tightly associated with a small number of strong quantitative trait loci (QTLs). The tight association may be a mechanism generating strains with distinct metabolic composition through the crossing of two different strains. The results indicate that one plant species produces more diverse phytochemicals than previously expected, and plants still contain many useful compounds for human applications.     

Small‐ and large‐scale heterogeneity in genetic variation across the collard flycatcher genome: implications for estimating genetic diversity in nonmodel organisms

Population genetic studies in nonmodel organisms are often hampered by a lack of reference genomes that are essential for whole‐genome resequencing. In the light of this, genotyping methods have been developed to effectively eliminate the need for a reference genome, such as genotyping by sequencing or restriction site‐associated DNA sequencing (RAD‐seq). However, what remains relatively poorly studied is how accurately these methods capture both average and variation in genetic diversity across an organism's genome. In this issue of Molecular Ecology Resources, Dutoit et al. (2016) use whole‐genome resequencing data from the collard flycatcher to assess what factors drive heterogeneity in nucleotide diversity across the genome. Using these data, they then simulate how well different sequencing designs, including RAD sequencing, could capture most of the variation in genetic diversity. They conclude that for evolutionary and conservation‐related studies focused on the estimating genomic diversity, researchers should emphasize the number of loci analysed over the number of individuals sequenced.     

Inferences of genetic architecture of bill morphology in house sparrow using a high‐density SNP array point to a polygenic basis

Understanding the genetic architecture of quantitative traits can provide insights into the mechanisms driving phenotypic evolution. Bill morphology is an ecologically important and phenotypically variable trait, which is highly heritable and closely linked to individual fitness. Thus, bill morphology traits are suitable candidates for gene mapping analyses. Previous studies have revealed several genes that may influence bill morphology, but the similarity of gene and allele effects between species and populations is unknown. Here, we develop a custom 200K SNP array and use it to examine the genetic basis of bill morphology in 1857 house sparrow individuals from a large‐scale, island metapopulation off the coast of Northern Norway. We found high genomic heritabilities for bill depth and length, which were comparable with previous pedigree estimates. Candidate gene and genomewide association analyses yielded six significant loci, four of which have previously been associated with craniofacial development. Three of these loci are involved in bone morphogenic protein (BMP) signalling, suggesting a role for BMP genes in regulating bill morphology. However, these loci individually explain a small amount of variance. In combination with results from genome partitioning analyses, this indicates that bill morphology is a polygenic trait. Any studies of eco‐evolutionary processes in bill morphology are therefore dependent on methods that can accommodate polygenic inheritance of the phenotype and molecular‐scale evolution of genetic architecture.