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.     

New resources for genetic studies in Populus nigra: genome‐wide SNP discovery and development of a 12k Infinium array

Whole genome resequencing of 51 Populus nigra (L.) individuals from across Western Europe was performed using Illumina platforms. A total number of 1 878 727 SNPs distributed along the P. nigra reference sequence were identified. The SNP calling accuracy was validated with Sanger sequencing. SNPs were selected within 14 previously identified QTL regions, 2916 expressional candidate genes related to rust resistance, wood properties, water‐use efficiency and bud phenology and 1732 genes randomly spread across the genome. Over 10 000 SNPs were selected for the construction of a 12k Infinium Bead‐Chip array dedicated to association mapping. The SNP genotyping assay was performed with 888 P. nigra individuals. The genotyping success rate was 91%. Our high success rate was due to the discovery panel design and the stringent parameters applied for SNP calling and selection. In the same set of P. nigra genotypes, linkage disequilibrium throughout the genome decayed on average within 5–7 kb to half of its maximum value. As an application test, ADMIXTURE analysis was performed with a selection of 600 SNPs spread throughout the genome and 706 individuals collected along 12 river basins. The admixture pattern was consistent with genetic diversity revealed by neutral markers and the geographical distribution of the populations. These newly developed SNP resources and genotyping array provide a valuable tool for population genetic studies and identification of QTLs through natural‐population based genetic association studies in P. nigra.     

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.     

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.     

Development of SNP‐genotyping arrays in two shellfish species

Use of SNPs has been favoured due to their abundance in plant and animal genomes, accompanied by the falling cost and rising throughput capacity for detection and genotyping. Here, we present in vitro (obtained from targeted sequencing) and in silico discovery of SNPs, and the design of medium‐throughput genotyping arrays for two oyster species, the Pacific oyster, Crassostrea gigas, and European flat oyster, Ostrea edulis. Two sets of 384 SNP markers were designed for two Illumina GoldenGate arrays and genotyped on more than 1000 samples for each species. In each case, oyster samples were obtained from wild and selected populations and from three‐generation families segregating for traits of interest in aquaculture. The rate of successfully genotyped polymorphic SNPs was about 60% for each species. Effects of SNP origin and quality on genotyping success (Illumina functionality Score) were analysed and compared with other model and nonmodel species. Furthermore, a simulation was made based on a subset of the C. gigas SNP array with a minor allele frequency of 0.3 and typical crosses used in shellfish hatcheries. This simulation indicated that at least 150 markers were needed to perform an accurate parental assignment. Such panels might provide valuable tools to improve our understanding of the connectivity between wild (and selected) populations and could contribute to future selective breeding programmes.     

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.     

Population structure and genetic basis of the agronomic traits of upland cotton in China revealed by a genome‐wide association study using high‐density SNPs

Gossypium hirsutum L. represents the largest source of textile fibre, and China is one of the largest cotton‐producing and cotton‐consuming countries in the world. To investigate the genetic architecture of the agronomic traits of upland cotton in China, a diverse and nationwide population containing 503 G. hirsutum accessions was collected for a genome‐wide association study (GWAS) on 16 agronomic traits. The accessions were planted in four places from 2012 to 2013 for phenotyping. The CottonSNP63K array and a published high‐density map based on this array were used for genotyping. The 503 G. hirsutum accessions were divided into three subpopulations based on 11 975 quantified polymorphic single‐nucleotide polymorphisms (SNPs). By comparing the genetic structure and phenotypic variation among three genetic subpopulations, seven geographic distributions and four breeding periods, we found that geographic distribution and breeding period were not the determinants of genetic structure. In addition, no obvious phenotypic differentiations were found among the three subpopulations, even though they had different genetic backgrounds. A total of 324 SNPs and 160 candidate quantitative trait loci (QTL) regions were identified as significantly associated with the 16 agronomic traits. A network was established for multieffects in QTLs and interassociations among traits. Thirty‐eight associated regions had pleiotropic effects controlling more than one trait. One candidate gene, Gh_D08G2376, was speculated to control the lint percentage (LP). This GWAS is the first report using high‐resolution SNPs in upland cotton in China to comprehensively investigate agronomic traits, and it provides a fundamental resource for cotton genetic research and breeding.     

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.     

Fast and cost‐effective single nucleotide polymorphism (SNP) detection in the absence of a reference genome using semideep next‐generation Random Amplicon Sequencing (RAMseq)

Biodiversity has suffered a dramatic global decline during the past decades, and monitoring tools are urgently needed providing data for the development and evaluation of conservation efforts both on a species and on a genetic level. However, in wild species, the assessment of genetic diversity is often hampered by the lack of suitable genetic markers. In this article, we present Random Amplicon Sequencing (RAMseq), a novel approach for fast and cost‐effective detection of single nucleotide polymorphisms (SNPs) in nonmodel species by semideep sequencing of random amplicons. By applying RAMseq to the Eurasian otter (Lutra lutra), we identified 238 putative SNPs after quality filtering of all candidate loci and were able to validate 32 of 77 loci tested. In a second step, we evaluated the genotyping performance of these SNP loci in noninvasive samples, one of the most challenging genotyping applications, by comparing it with genotyping results of the same faecal samples at microsatellite markers. We compared (i) polymerase chain reaction (PCR) success rate, (ii) genotyping errors and (iii) Mendelian inheritance (population parameters). SNPs produced a significantly higher PCR success rate (75.5% vs. 65.1%) and lower mean allelic error rate (8.8% vs. 13.3%) than microsatellites, but showed a higher allelic dropout rate (29.7% vs. 19.8%). Genotyping results showed no deviations from Mendelian inheritance in any of the SNP loci. Hence, RAMseq appears to be a valuable tool for the detection of genetic markers in nonmodel species, which is a common challenge in conservation genetic studies.     

Genome‐wide detection of genetic loci and candidate genes for teat number and body conformation traits at birth in Chinese Sushan pigs

Body conformation at birth and teat number are economically important traits in the pig industry, as these traits are usually explored to evaluate the growth and reproductive potential of piglets. To detect genetic loci and candidate genes for these traits, we performed a GWAS on 269 pigs from a recently developed Chinese breed (Sushan) using 38  128 informative SNPs on the Affymetrix Porcine SNP 55K Array. In total, we detected one genome‐wide significant (P = 1.31e‐6) SNP for teat number on chromosome X and 15 chromosome‐wide significant SNPs for teat number, body weight, body length, chest circumference and cannon circumference at birth on chromosomes 1, 3, 4, 6, 7, 9, 10, 13, 14, 15, 17 and 18. The most significant SNP had an additive effect of 0.74 × total teat number, explaining 20% of phenotypic variance. Five significant SNPs resided in the previously reported quantitative trait loci for these traits and seven significant SNPs had a pleiotropic effect on multiple traits. Intriguingly, 12 of the genes nearest to the significant SNPs are functionally related to body conformation and teat number traits, including SPRED2, MKX, TMSB4X and ESR1. GO analysis revealed that candidate genes proximal to the significant SNPs were enriched in the G‐protein coupled receptor and steroid hormone‐mediated signaling pathway. Our findings shed light on the genetic basis of the measured traits and provide molecular markers especially for the genetic improvement of teat number in Sushan and related pigs.     

Two highly validated SSR multiplexes (8‐plex) for Euphrates' poplar,Populus euphratica (Salicaceae)

Multiplex PCR amplification of microsatellites has significantly increased the throughput and decreased the costs of genotyping. We have developed two highly polymorphic microsatellite multiplexes for Populus euphratica, the only tree species found in desert regions of Western China and adjacent Central Asian countries. The first of these multiplex kits comprises an eight‐Plex of genomic SSRs (gSSRs) obtained from published databases. The second comprises an eight‐plex of newly designed EST‐SSRs (eSSRs) based on expressed sequence tags for P. euphratica. Both kits were tested on a sample of 170 individuals from four populations. The gSSRs exhibited slightly more polymorphism than the eSSRs. The new multiplex protocols yielded consistent results in the hands of multiple researchers, demonstrating their robustness. The 16 loci used in the kits exhibited a high transferability rate (82.0%) in eight other poplar species belonging to five different sections of the genus. Both kits should therefore be useful for further investigations of population genetics in P. euphratica and related species. Our results indicate that it is essential to follow recently established recommendations when developing microsatellite markers, including verifying the amplification efficiency, detecting null alleles and carefully measuring error rates.     

Development of high‐density SNP genotyping arrays for white spruce (Picea glauca) and transferability to subtropical and nordic congeners

High‐density SNP genotyping arrays can be designed for any species given sufficient sequence information of high quality. Two high‐density SNP arrays relying on the Infinium iSelect technology (Illumina) were designed for use in the conifer white spruce (Picea glauca). One array contained 7338 segregating SNPs representative of 2814 genes of various molecular functional classes for main uses in genetic association and population genetics studies. The other one contained 9559 segregating SNPs representative of 9543 genes for main uses in population genetics, linkage mapping of the genome and genomic prediction. The SNPs assayed were discovered from various sources of gene resequencing data. SNPs predicted from high‐quality sequences derived from genomic DNA reached a genotyping success rate of 64.7%. Nonsingleton in silico SNPs (i.e. a sequence polymorphism present in at least two reads) predicted from expressed sequenced tags obtained with the Roche 454 technology and Illumina GAII analyser resulted in a similar genotyping success rate of 71.6% when the deepest alignment was used and the most favourable SNP probe per gene was selected. A variable proportion of these SNPs was shared by other nordic and subtropical spruce species from North America and Europe. The number of shared SNPs was inversely proportional to phylogenetic divergence and standing genetic variation in the recipient species, but positively related to allele frequency in P. glauca natural populations. These validated SNP resources should open up new avenues for population genetics and comparative genetic mapping at a genomic scale in spruce species.     

Genome‐wide association analysis and gene set enrichment analysis with SNP data identify genes associated with 305‐day milk yield in Holstein dairy cows

Milk production traits, such as 305‐day milk yield (305MY), have been under direct selection to improve production in dairy cows. Over the past 50 years, the average milk yield has nearly doubled, and over 56% of the increase is attributable to genetic improvement. As such, additional improvements in milk yield are still possible as new loci are identified. The objectives of this study were to detect SNPs and gene sets associated with 305MY in order to identify new candidate genes contributing to variation in milk production. A population of 781 primiparous Holstein cows from six central Washington dairies with records of 305MY and energy corrected milk were used to perform a genome‐wide association analysis (GWAA) using the Illumina BovineHD BeadChip (777 962 SNPs) to identify QTL associated with 305MY (P < 1.0 × 10^?5). A gene set enrichment analysis with SNP data (GSEA‐SNP) was performed to identify gene sets (normalized enrichment score > 3.0) and leading edge genes (LEGs) influencing 305MY. The GWAA identified three QTL comprising 34 SNPs and 30 positional candidate genes. In the GSEA‐SNP, five gene sets with 58 unique and 24 shared LEGs contributed to 305MY. Identification of QTL and LEGs associated with 305MY can provide additional targets for genomic selection to continue to improve 305MY in dairy cattle.     

Spatial dynamics and mixing of bluefin tuna in the Atlantic Ocean and Mediterranean Sea revealed using next‐generation sequencing

The Atlantic bluefin tuna is a highly migratory species emblematic of the challenges associated with shared fisheries management. In an effort to resolve the species’ stock dynamics, a genomewide search for spatially informative single nucleotide polymorphisms (SNPs) was undertaken, by way of sequencing reduced representation libraries. An allele frequency approach to SNP discovery was used, combining the data of 555 larvae and young‐of‐the‐year (LYOY) into pools representing major geographical areas and mapping against a newly assembled genomic reference. From a set of 184,895 candidate loci, 384 were selected for validation using 167 LYOY. A highly discriminatory genotyping panel of 95 SNPs was ultimately developed by selecting loci with the most pronounced differences between western Atlantic and Mediterranean Sea LYOY. The panel was evaluated by genotyping a different set of LYOY (n = 326), and from these, 77.8% and 82.1% were correctly assigned to western Atlantic and Mediterranean Sea origins, respectively. The panel revealed temporally persistent differentiation among LYOY from the western Atlantic and Mediterranean Sea (F_ST = 0.008, p = .034). The composition of six mixed feeding aggregations in the Atlantic Ocean and Mediterranean Sea was characterized using genotypes from medium (n = 184) and large (n = 48) adults, applying population assignment and mixture analyses. The results provide evidence of persistent population structuring across broad geographic areas and extensive mixing in the Atlantic Ocean, particularly in the mid‐Atlantic Bight and Gulf of St. Lawrence. The genomic reference and genotyping tools presented here constitute novel resources useful for future research and conservation efforts.     

Single nucleotide polymorphism discovery in albacore and Atlantic bluefin tuna provides insights into worldwide population structure

The optimal management of the commercially important, but mostly over‐exploited, pelagic tunas, albacore (Thunnus alalunga Bonn., 1788) and Atlantic bluefin tuna (BFT; Thunnus thynnus L., 1758), requires a better understanding of population structure than has been provided by previous molecular methods. Despite numerous studies of both species, their population structures remain controversial. This study reports the development of single nucleotide polymorphisms (SNPs) in albacore and BFT and the application of these SNPs to survey genetic variability across the geographic ranges of these tunas. A total of 616 SNPs were discovered in 35 albacore tuna by comparing sequences of 54 nuclear DNA fragments. A panel of 53 SNPs yielded F_ST values ranging from 0.0 to 0.050 between samples after genotyping 460 albacore collected throughout the distribution of this species. No significant heterogeneity was detected within oceans, but between‐ocean comparisons (Atlantic, Pacific and Indian oceans along with Mediterranean Sea) were significant. Additionally, a 17‐SNP panel was developed in Atlantic BFT by cross‐species amplification in 107 fish. This limited number of SNPs discriminated between samples from the two major spawning areas of Atlantic BFT (F_ST = 0.116). The SNP markers developed in this study can be used to genotype large numbers of fish without the need for standardizing alleles among laboratories.     

Haplotype‐based genotyping‐by‐sequencing in oat genome research

In a de novo genotyping‐by‐sequencing (GBS) analysis of short, 64‐base tag‐level haplotypes in 4657 accessions of cultivated oat, we discovered 164741 tag‐level (TL) genetic variants containing 241224 SNPs. From this, the marker density of an oat consensus map was increased by the addition of more than 70000 loci. The mapped TL genotypes of a 635‐line diversity panel were used to infer chromosome‐level (CL) haplotype maps. These maps revealed differences in the number and size of haplotype blocks, as well as differences in haplotype diversity between chromosomes and subsets of the diversity panel. We then explored potential benefits of SNP vs. TL vs. CL GBS variants for mapping, high‐resolution genome analysis and genomic selection in oats. A combined genome‐wide association study (GWAS) of heading date from multiple locations using both TL haplotypes and individual SNP markers identified 184 significant associations. A comparative GWAS using TL haplotypes, CL haplotype blocks and their combinations demonstrated the superiority of using TL haplotype markers. Using a principal component‐based genome‐wide scan, genomic regions containing signatures of selection were identified. These regions may contain genes that are responsible for the local adaptation of oats to Northern American conditions. Genomic selection for heading date using TL haplotypes or SNP markers gave comparable and promising prediction accuracies of up to r = 0.74. Genomic selection carried out in an independent calibration and test population for heading date gave promising prediction accuracies that ranged between r = 0.42 and 0.67. In conclusion, TL haplotype GBS‐derived markers facilitate genome analysis and genomic selection in oat.