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 of a 135K SNP genotyping array for Actinidia arguta and its applications for genetic mapping and QTL analysis in kiwifruit

Kiwifruit (Actinidia spp) is a woody, perennial and deciduous vine. In this genus, there are multiple ploidy levels but the main cultivated cultivars are polyploid. Despite the availability of many genomic resources in kiwifruit, SNP genotyping is still a challenge given these different levels of polyploidy. Recent advances in SNP array technologies have offered a high-throughput genotyping platform for genome-wide DNA polymorphisms. In this study, we developed a high-density SNP genotyping array to facilitate genetic studies and breeding applications in kiwifruit. SNP discovery was performed by genome-wide DNA sequencing of 40 kiwifruit genotypes. The identified SNPs were stringently filtered for sequence quality, predicted conversion performance and distribution over the available Actinidia chinensis genome. A total of 134 729 unique SNPs were put on the array. The array was evaluated by genotyping 400 kiwifruit individuals. We performed a multidimensional scaling analysis to assess the diversity of kiwifruit germplasm, showing that the array was effective to distinguish kiwifruit accessions. Using a tetraploid F1 population, we constructed an integrated linkage map covering 3060.9 cM across 29 linkage groups and performed QTL analysis for the sex locus that has been identified on Linkage Group 3 (LG3) in Actinidia arguta. Finally, our dataset presented evidence of tetrasomic inheritance with partial preferential pairing in A. arguta. In conclusion, we developed and evaluated a 135K SNP genotyping array for kiwifruit. It has the advantage of a comprehensive design that can be an effective tool in genetic studies and breeding applications in this high-value crop.     

Development and validation of the Axiom®Apple480K SNP genotyping array

Cultivated apple (Malus × domestica Borkh.) is one of the most important fruit crops in temperate regions, and has great economic and cultural value. The apple genome is highly heterozygous and has undergone a recent duplication which, combined with a rapid linkage disequilibrium decay, makes it difficult to perform genome‐wide association (GWA) studies. Single nucleotide polymorphism arrays offer highly multiplexed assays at a relatively low cost per data point and can be a valid tool for the identification of the markers associated with traits of interest. Here, we describe the development and validation of a 487K SNP Affymetrix Axiom^® genotyping array for apple and discuss its potential applications. The array has been built from the high‐depth resequencing of 63 different cultivars covering most of the genetic diversity in cultivated apple. The SNPs were chosen by applying a focal points approach to enrich genic regions, but also to reach a uniform coverage of non‐genic regions. A total of 1324 apple accessions, including the 92 progenies of two mapping populations, have been genotyped with the Axiom^®Apple480K to assess the effectiveness of the array. A large majority of SNPs (359 994 or 74%) fell in the stringent class of poly high resolution polymorphisms. We also devised a filtering procedure to identify a subset of 275K very robust markers that can be safely used for germplasm surveys in apple. The Axiom^®Apple480K has now been commercially released both for public and proprietary use and will likely be a reference tool for GWA studies in apple.     

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 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.     

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.     

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.     

Potential of genomic selection for improvement of resistance to ostreid herpesvirus in Pacific oyster (Crassostrea gigas)

In genomic selection (GS), genome-wide SNP markers are used to generate genomic estimated breeding values for selection candidates. The application of GS in shellfish looks promising and has the potential to help in dealing with one of the main issues currently affecting Pacific oyster production worldwide, which is the ‘summer mortality syndrome’. This causes periodic mass mortality in farms worldwide and has mainly been attributed to a specific variant of the ostreid herpesvirus (OsHV-1). In the current study, we evaluated the potential of genomic selection for host resistance to OsHV-1 in Pacific oysters, and compared it with pedigree-based approaches. An OsHV-1 disease challenge was performed using an immersion-based virus exposure treatment for oysters for 7 days. A total of 768 samples were genotyped using the medium-density SNP array for oysters. A GWAS was performed for the survival trait using a GBLUP approach in blupf 90 software. Heritability ranged from 0.25 ± 0.05 to 0.37 ± 0.05 (mean ± SE) based on pedigree and genomic information respectively. Genomic prediction was more accurate than pedigree prediction, and SNP density reduction had little impact on prediction accuracy until marker densities dropped below approximately 500 SNPs. This demonstrates the potential for GS in Pacific oyster breeding programmes, and importantly, demonstrates that a low number of SNPs might suffice to obtain accurate genomic estimated breeding values, thus potentially making the implementation of GS more cost effective.     

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.     

Association between putative functional variants in the PSMB9 gene and risk of melanoma – re‐analysis of published melanoma genome‐wide association studies

To mine possibly hidden causal single‐nucleotide polymorphisms (SNPs) of melanoma, we investigated the association of SNPs in 76 M/G1 transition genes with melanoma risk using our published genome‐wide association study (GWAS) data set with 1804 melanoma cases and 1026 cancer‐free controls. We found multiple SNPs with P < 0.01 and performed validation studies for 18 putative functional SNPs in PSMB9 in two other GWAS data sets. Two SNPs (rs1351383 and rs2127675) were associated with melanoma risk in the GenoMEL data set (P = 0.013 and 0.004, respectively), but failed in validation using the Australian data set. Genotype–phenotype analysis revealed these two SNPs were significantly correlated with mRNA expression level of PSMB9. Further experiments revealed that SNP rs2071480, which is in high LD with rs1351383 and rs2127675, may have a weak effect on the promoter activity of PSMB9. Taken together, our data suggested that functional variants in PSMB9 may contribute to melanoma susceptibility.     

Population genomics reveals a fine‐scale recombination landscape for genetic improvement of cotton

Recombination breaks up ancestral linkage disequilibrium, creates combinations of alleles, affects the efficiency of natural selection, and plays a major role in crop domestication and improvement. However, there is little knowledge regarding the variation in the population‐scaled recombination rate in cotton. We constructed recombination maps and characterized the difference in the genomic landscape of the population‐scaled recombination rate between Gossypium hirsutum and G. arboreum and sub‐genomes based on the 381 sequenced G. hirsutum and 215 G. arboreum accessions. Comparative genomics identified large structural variations and syntenic genes in the recombination regions, suggesting that recombination was related to structural variation and occurred preferentially in the distal chromosomal regions. Correlation analysis indicated that recombination was only slightly affected by geographical distribution and breeding period. A genome‐wide association study (GWAS) was performed with 15 agronomic traits using 267 cotton accessions and identified 163 quantitative trait loci (QTL) and an important candidate gene (Ghir_COL2) for early maturity traits. Comparative analysis of recombination and a GWAS revealed that the QTL of fibre quality traits tended to be more common in high‐recombination regions than were those of yield and early maturity traits. These results provide insights into the population‐scaled recombination landscape, suggesting that recombination contributed to the domestication and improvement of cotton, which provides a useful reference for studying recombination in other species.     

A chromosome‐level genome assembly reveals the genetic basis of cold tolerance in a notorious rice insect pest,Chilo suppressalis

The rice stem borer, Chilo suppressalis, is one of the most damaging insect pests to rice production worldwide. Although C. suppressalis has been the focus of numerous studies examining cold tolerance and diapause, plant–insect interactions, pesticide targets and resistance, and the development of RNAi‐mediated pest management, the absence of a high‐quality genome has limited deeper insights. To address this limitation, we generated a draft C. suppressalis genome constructed from both Illumina and PacBio sequences. The assembled genome size was 824.35 Mb with a contig N₅₀ of 307 kb and a scaffold N₅₀ of 1.75 Mb. Hi‐C scaffolding assigned 99.2% of the bases to one of 29 chromosomes. Based on universal single‐copy orthologues (BUSCO), the draft genome assembly was estimated to be 97% complete and is predicted to encompass 15,653 protein‐coding genes. Cold tolerance is an extreme survival strategy found in animals. However, little is known regarding the genetic basis of the winter ecology of C. suppressalis. Here, we focused our orthologous analysis on those gene families associated with animal cold tolerance. Our finding provided the first genomic evidence revealing specific cold‐tolerant strategies in C. suppressalis, including those involved in glucose‐originated glycerol biosynthesis, triacylglycerol‐originated glycerol biosynthesis, fatty acid synthesis and trehalose transport‐intermediate cold tolerance. The high‐quality C. suppressalis genome provides a valuable resource for research into a broad range of areas in molecular ecology, and subsequently benefits developing modern pest control strategies.     

Genome of an iconic Australian bird: High‐quality assembly and linkage map of the superb fairy‐wren (Malurus cyaneus)

The superb fairy‐wren, Malurus cyaneus, is one of the most iconic Australian passerine species. This species belongs to an endemic Australasian clade, Meliphagides, which diversified early in the evolution of the oscine passerines. Today, the oscine passerines comprise almost half of all avian species diversity. Despite the rapid increase of available bird genome assemblies, this part of the avian tree has not yet been represented by a high‐quality reference. To rectify that, we present the first high‐quality genome assembly of a Meliphagides representative: the superb fairy‐wren. We combined Illumina shotgun and mate‐pair sequences, PacBio long‐reads, and a genetic linkage map from an intensively sampled pedigree of a wild population to generate this genome assembly. Of the final assembled 1.07‐Gb genome, 975 Mb (90.4%) was anchored onto 25 pseudochromosomes resulting in a final superscaffold N50 of 68.11 Mb. This high‐quality bird genome assembly is one of only a handful which is also accompanied by a genetic map and recombination landscape. In comparison to other pedigree‐based bird genetic maps, we find that the fairy‐wren genetic map more closely resembles those of Taeniopygia guttata and Parus major maps, unlike the Ficedula albicollis map which more closely resembles that of Gallus gallus. Lastly, we also provide a predictive gene and repeat annotation of the genome assembly. This new high‐quality, annotated genome assembly will be an invaluable resource not only regarding the superb fairy‐wren species and relatives but also broadly across the avian tree by providing a novel reference point for comparative genomic analyses.     

New resources for genetic studies in maize (Zea mays L.): a genome-wide Maize6H-60K single nucleotide polymorphism array and its application

Despite the availability of numerous molecular markers in maize, effective evaluation of all types of germplasm resources, accurate identification of varieties and analysis of a large number of materials in a timely, low-cost manner is challenging. Here, we present Maize6H-60K, a genome-wide single nucleotide polymorphism (SNP) array to facilitate maize genotyping. We first identified 160 million variants by sequencing data of 388 representative inbreds and then tiled 200 000 high-quality variants on a screening array. These variants were further narrowed down to 61 282 using stringent filtering criteria. Among the 60 000 markers, 21 460 SNPs (35%) were within genic regions and 12 835 (21%) were located in coding regions. To assess their effectiveness, 329 inbreds, 221 hybrids, 34 parent–offspring sets and six breeding samples were genotyped. Overall, 48 972 SNPs (80%) were categorized into the highest quality class, that of ‘poly high resolution’. A total of 54 658 (89.29%) and 53 091 (86.73%) SNPs had minor allele frequency values ≥ 0.20 in inbreds and hybrids respectively. A linkage disequilibrium (LD) analysis revealed that LD decline was in equilibrium when r² was between 0.10 and 0.15, which corresponds to a physical distance of 400–600 kb. UPGMA clustering analysis divided the 329 inbred lines into nine groups that were consistent with known pedigrees. A background analysis of breeding materials indicated that the 60 000 markers were suitable for evaluation of breeding populations constructed by materials between or within heterotic groups. The developed Maize6H-60K array should be an important tool in maize genetic studies, variety identification and molecular breeding.     

Genome‐wide association and genomic prediction for biomass yield in a genetically diverse Miscanthus sinensis germplasm panel phenotyped at five locations in Asia and North America

To improve the efficiency of breeding of Miscanthus for biomass yield, there is a need to develop genomics‐assisted selection for this long‐lived perennial crop by relating genotype to phenotype and breeding value across a broad range of environments. We present the first genome‐wide association (GWA) and genomic prediction study of Miscanthus that utilizes multilocation phenotypic data. A panel of 568 Miscanthus sinensis accessions was genotyped with 46,177 single nucleotide polymorphisms (SNPs) and evaluated at one subtropical and five temperate locations over 3 years for biomass yield and 14 yield‐component traits. GWA and genomic prediction were performed separately for different years of data in order to assess reproducibility. The analyses were also performed for individual field trial locations, as well as combined phenotypic data across groups of locations. GWA analyses identified 27 significant SNPs for yield, and a total of 504 associations across 298 unique SNPs across all traits, sites, and years. For yield, the greatest number of significant SNPs was identified by combining phenotypic data across all six locations. For some of the other yield‐component traits, greater numbers of significant SNPs were obtained from single site data, although the number of significant SNPs varied greatly from site to site. Candidate genes were identified. Accounting for population structure, genomic prediction accuracies for biomass yield ranged from 0.31 to 0.35 across five northern sites and from 0.13 to 0.18 for the subtropical location, depending on the estimation method. Genomic prediction accuracies of all traits were similar for single‐location and multilocation data, suggesting that genomic selection will be useful for breeding broadly adapted M. sinensis as well as M. sinensis optimized for specific climates. All of our data, including DNA sequences flanking each SNP, are publicly available. By facilitating genomic selection in M. sinensis and Miscanthus × giganteus, our results will accelerate the breeding of these species for biomass in diverse environments.     

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.     

Genome of ‘Charleston Gray’, the principal American watermelon cultivar,and genetic characterization of 1,365 accessions in the U.S. National Plant Germplasm System watermelon collection

Years of selection for desirable fruit quality traits in dessert watermelon (Citrullus lanatus) has resulted in a narrow genetic base in modern cultivars. Development of novel genomic and genetic resources offers great potential to expand genetic diversity and improve important traits in watermelon. Here, we report a high‐quality genome sequence of watermelon cultivar ‘Charleston Gray’, a principal American dessert watermelon, to complement the existing reference genome from ‘97103’, an East Asian cultivar. Comparative analyses between genomes of ‘Charleston Gray’ and ‘97103’ revealed genomic variants that may underlie phenotypic differences between the two cultivars. We then genotyped 1365 watermelon plant introduction (PI) lines maintained at the U.S. National Plant Germplasm System using genotyping‐by‐sequencing (GBS). These PI lines were collected throughout the world and belong to three Citrullus species, C. lanatus, C. mucosospermus and C. amarus. Approximately 25 000 high‐quality single nucleotide polymorphisms (SNPs) were derived from the GBS data using the ‘Charleston Gray’ genome as the reference. Population genomic analyses using these SNPs discovered a close relationship between C. lanatus and C. mucosospermus and identified four major groups in these two species correlated to their geographic locations. Citrullus amarus was found to have a distinct genetic makeup compared to C. lanatus and C. mucosospermus. The SNPs also enabled identification of genomic regions associated with important fruit quality and disease resistance traits through genome‐wide association studies. The high‐quality ‘Charleston Gray’ genome and the genotyping data of this large collection of watermelon accessions provide valuable resources for facilitating watermelon research, breeding and improvement.