Developing genomic resources for the common bottlenose dolphin (Tursiops truncatus): isolation and characterization of 153 single nucleotide polymorphisms and 53 genotyping assays

Although single nucleotide polymorphisms (SNPs) are commonly used in human genetics, they have only recently been incorporated into genetic studies of non‐model organisms, including cetaceans. SNPs have several advantages over other molecular markers for studies of population genetics: they are quicker and more straightforward to score, cross‐laboratory comparisons of data are less complicated, and they can be used successfully with low‐quality DNA. We screened portions of the genome of one of the most abundant cetaceans in U.S. waters, the common bottlenose dolphin (Tursiops truncatus), and identified 153 SNPs resulting in an overall average of one SNP every 463 base pairs. Custom TaqMan^® Assays were designed for 53 of these SNPs, and their performance was tested by genotyping a set of bottlenose dolphin samples, including some with low‐quality DNA. We found that in 19% of the loci examined, the minor allele frequency (MAF) estimated during initial SNP ascertainment using a DNA pool of 10 individuals differed significantly from the final MAF after genotyping over 100 individuals, suggesting caution when making inferences about MAF values based on small data sets. For two assays, we also characterized the basis for unusual clustering patterns to determine whether their data could still be utilized for further genetic studies. Overall results support the use of these SNPs for accurate analysis of both poor and good‐quality DNA. We report the first SNP markers and genotyping assays for use in population and conservation genetic studies of bottlenose dolphins.     

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.     

Use of genotyping by sequencing data to develop a high‐throughput and multifunctional SNP panel for conservation applications in Pacific lamprey

Next‐generation sequencing data can be mined for highly informative single nucleotide polymorphisms (SNPs) to develop high‐throughput genomic assays for nonmodel organisms. However, choosing a set of SNPs to address a variety of objectives can be difficult because SNPs are often not equally informative. We developed an optimal combination of 96 high‐throughput SNP assays from a total of 4439 SNPs identified in a previous study of Pacific lamprey (Entosphenus tridentatus) and used them to address four disparate objectives: parentage analysis, species identification and characterization of neutral and adaptive variation. Nine of these SNPs are F_ST outliers, and five of these outliers are localized within genes and significantly associated with geography, run‐timing and dwarf life history. Two of the 96 SNPs were diagnostic for two other lamprey species that were morphologically indistinguishable at early larval stages and were sympatric in the Pacific Northwest. The majority (85) of SNPs in the panel were highly informative for parentage analysis, that is, putatively neutral with high minor allele frequency across the species’ range. Results from three case studies are presented to demonstrate the broad utility of this panel of SNP markers in this species. As Pacific lamprey populations are undergoing rapid decline, these SNPs provide an important resource to address critical uncertainties associated with the conservation and recovery of this imperiled species.     

Rapid identification of homozygosity and site of wild relative introgressions in wheat through chromosome‐specific KASP genotyping assays

For future food security, it is important that wheat, one of the most widely consumed crops in the world, can survive the threat of abiotic and biotic stresses. New genetic variation is currently being introduced into wheat through introgressions from its wild relatives. For trait discovery, it is necessary that each introgression is homozygous and hence stable. Breeding programmes rely on efficient genotyping platforms for marker‐assisted selection (MAS). Recently, single nucleotide polymorphism (SNP)‐based markers have been made available on high‐throughput Axiom^® SNP genotyping arrays. However, these arrays are inflexible in their design and sample numbers, making their use unsuitable for long‐term MAS. SNPs can potentially be converted into Kompetitive allele‐specific PCR (KASP?) assays that are comparatively cost‐effective and efficient for low‐density genotyping of introgression lines. However, due to the polyploid nature of wheat, KASP assays for homoeologous SNPs can have difficulty in distinguishing between heterozygous and homozygous hybrid lines in a backcross population. To identify co‐dominant SNPs, that can differentiate between heterozygotes and homozygotes, we PCR‐amplified and sequenced genomic DNA from potential single‐copy regions of the wheat genome and compared them to orthologous copies from different wild relatives. A panel of 620 chromosome‐specific KASP assays have been developed that allow rapid detection of wild relative segments and provide information on their homozygosity and site of introgression in the wheat genome. A set of 90 chromosome‐nonspecific assays was also produced that can be used for genotyping introgression lines. These multipurpose KASP assays represent a powerful tool for wheat breeders worldwide.     

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.     

The Wheat 660K SNP array demonstrates great potential for marker‐assisted selection in polyploid wheat

The rapid development and application of molecular marker assays have facilitated genomic selection and genome‐wide linkage and association studies in wheat breeding. Although PCR‐based markers (e.g. simple sequence repeats and functional markers) and genotyping by sequencing have contributed greatly to gene discovery and marker‐assisted selection, the release of a more accurate and complete bread wheat reference genome has resulted in the design of single‐nucleotide polymorphism (SNP) arrays based on different densities or application targets. Here, we evaluated seven types of wheat SNP arrays in terms of their SNP number, distribution, density, associated genes, heterozygosity and application. The results suggested that the Wheat 660K SNP array contained the highest percentage (99.05%) of genome‐specific SNPs with reliable physical positions. SNP density analysis indicated that the SNPs were almost evenly distributed across the whole genome. In addition, 229 266 SNPs in the Wheat 660K SNP array were located in 66 834 annotated gene or promoter intervals. The annotated genes revealed by the Wheat 660K SNP array almost covered all genes revealed by the Wheat 35K (97.44%), 55K (99.73%), 90K (86.9%) and 820K (85.3%) SNP arrays. Therefore, the Wheat 660K SNP array could act as a substitute for other 6 arrays and shows promise for a wide range of possible applications. In summary, the Wheat 660K SNP array is reliable and cost‐effective and may be the best choice for targeted genotyping and marker‐assisted selection in wheat genetic improvement.     

Characterization of 13 single nucleotide polymorphism markers for chum salmon

We report the characterization of 13 single nucleotide polymorphism (SNP) genotyping assays for chum salmon (Oncorhynchus keta). These assays are based on the 5′‐nuclease reaction and thus facilitate high‐throughput genotyping with minimal optimization time. Because data generated using these markers may be transported and combined across laboratories, SNPs offer the potential to reduce the amount of redundant work being done in mixture and migratory studies of chum salmon.     

Discovery and development of exome‐based,co‐dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.)

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)‐based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP‐based wheat markers have been made available via the use of next‐generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial‐scale breeding programmes. To identify exome‐based co‐dominant SNP‐based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re‐sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co‐dominant. Validation of a subset of these putative co‐dominant markers confirmed that 96% were true polymorphisms and 65% were co‐dominant SNP assays. The new co‐dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web‐based database to facilitate their use on genotyping programmes worldwide.     

SNP discovery and population structure analysis in Lassi and Marecha camel breeds using a genotyping by sequencing method

Pakistani camels have been classified socio-geographically into 20 breeds, but they have not yet been subjected to substantial selective pressures and the genetic basis for these breeds is not understood. However, it should be possible to distinguish them by use of molecular data. This study investigated the genetic diversity and population structure within and between two major Pakistani camel breeds, Marecha and Lassi. As no SNP array is currently available, we first identified 63 619 SNPs using a genotyping by sequencing approach. After quality control, a panel of 36 926 SNPs was used in the analysis. Population structure was investigated with a principal coordinate analysis as well as a cluster analysis using NetView , and multilocus heterozygosity analysis to explore between- and within-breed genetic variation. In addition, between-breed variation was explored using the fixation index, F_ST. We also compared relationship matrices computed using the VanRaden SNP-based method and a method developed specifically for genotyping by sequencing data. Among the two camel breeds, Lassi showed a lower level of genetic diversity whereas Marecha showed a higher level. As a genotyping platform has not yet been developed for the camel, the SNPs discovered in this study will be useful in future genetic studies in camels.     

A whole‐genome SNP array (RICE6K) for genomic breeding in rice

The advances in genotyping technology provide an opportunity to use genomic tools in crop breeding. As compared to field selections performed in conventional breeding programmes, genomics‐based genotype screen can potentially reduce number of breeding cycles and more precisely integrate target genes for particular traits into an ideal genetic background. We developed a whole‐genome single nucleotide polymorphism (SNP) array, RICE6K, based on Infinium technology, using representative SNPs selected from more than four million SNPs identified from resequencing data of more than 500 rice landraces. RICE6K contains 5102 SNP and insertion–deletion (InDel) markers, about 4500 of which were of high quality in the tested rice lines producing highly repeatable results. Forty‐five functional markers that are located inside 28 characterized genes of important traits can be detected using RICE6K. The SNP markers are evenly distributed on the 12 chromosomes of rice with the average density of 12 SNPs per 1 Mb and can provide information for polymorphisms between indica and japonica subspecies as well as varieties within indica and japonica groups. Application tests of RICE6K showed that the array is suitable for rice germplasm fingerprinting, genotyping bulked segregating pools, seed authenticity check and genetic background selection. These results suggest that RICE6K provides an efficient and reliable genotyping tool for rice genomic breeding.     

Discovery,validation and characterization of 1039 cattle single nucleotide polymorphisms

We identified ～13 000 putative single nucleotide polymorphisms (SNPs) by comparison of repeat‐masked BAC‐end sequences from the cattle RPCI‐42 BAC library with whole‐genome shotgun contigs of cattle genome assembly Btau 1.0. Genotyping of a subset of these SNPs was performed on a panel containing 186 DNA samples from 18 cattle breeds including 43 trios. Of 1039 SNPs confirmed as polymorphic in the panel, 998 had minor allele frequency ≥0.25 among unrelated individuals of at least one breed. When Btau 4.0 became available, 974 of these validated SNPs were assigned in silico to known cattle chromosomes, while 41 SNPs were mapped to unassigned sequence scaffolds, yielding one SNP every ～3 Mbp on average. Twenty‐four SNPs identified in Btau 1.0 were not mapped to Btau 4.0. Of the 1015 SNPs mapped to Btau 4.0, 959 SNPs had nucleotide bases identical in Btau 4.0 and Btau 1.0 contigs, whereas 56 bases were changed, resulting in the loss of the in silico SNP in Btau 4.0. Because these 1039 SNPs were all directly confirmed by genotyping on the multi‐breed panel, it is likely that the original polymorphisms were correctly identified. The 1039 validated SNPs identified in this study represent a new and useful resource for genome‐wide association studies and applications in animal breeding.     

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.     

Identification of candidate genes and mutations in QTL regions for immune responses in chicken

There are two categories of immune responses – innate and adaptive immunity – both having polygenic backgrounds and a significant environmental component. In our study, adaptive immunity was represented by the specific antibody response toward keyhole limpet hemocyanin (KLH); innate immunity was represented by natural antibodies toward lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Defining genetic bases of immune responses leads from defining quantitative trait loci (QTL) toward a single mutation responsible for variation in the phenotypic trait. The goal of the reported study was to define candidate genes and mutations for the immune traits of interest in chicken by performing an association study of SNPs located in candidate genes defined in QTL regions. Candidate genes and SNPs in QTL regions were selected in silico. SNP association was based on a custom SNP panel, GoldenGate genotyping assay (Illumina) and two statistical models: random mixed model and CAR score. The most significant SNP for immune response toward KLH was located in the JMJD6 gene located on GGA18. Four SNPs in candidate genes FOXJ1 (GGA18), EPHB1 (GGA9), PTGER4 (GGAZ) and PRKCB (GGA14) showed association with natural antibodies for LPS. A single SNP in ITGB4 (GGA18) was associated with natural antibodies for LTA. All associated SNPs mentioned above showed additive effects.     

Discovery and characterization of single nucleotide polymorphisms in coho salmon,Oncorhynchus kisutch

Molecular population genetic analyses have become an integral part of ecological investigation and population monitoring for conservation and management. Microsatellites have been the molecular marker of choice for such applications over the last several decades, but single nucleotide polymorphism (SNP) markers are rapidly expanding beyond model organisms. Coho salmon (Oncorhynchus kisutch) is native to the north Pacific Ocean and its tributaries, where it is the focus of intensive fishery and conservation activities. As it is an anadromous species, coho salmon typically migrate across multiple jurisdictional boundaries, complicating management and requiring shared data collection methods. Here, we describe the discovery and validation of a suite of novel SNPs and associated genotyping assays which can be used in the genetic analyses of this species. These assays include 91 that are polymorphic in the species and one that discriminates it from a sister species, Chinook salmon. We demonstrate the utility of these SNPs for population assignment and phylogeographic analyses, and map them against the draft trout genome. The markers constitute a large majority of all SNP markers described for coho salmon and will enable both population‐ and pedigree‐based analyses across the southern part of the species native range.     

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 assessment of simple PCR markers for SNP genotyping in barley

Simple molecular marker assays underpin routine plant breeding and research activities in many laboratories worldwide. With the rapid growth of single nucleotide polymorphism (SNP) resources for many important crop plants, the availability of routine, low-tech marker assays for genotyping SNPs is of increased importance. In this study, we demonstrate that temperature-switch PCR (TSP) supports the rapid development of robust, allele-specific PCR markers for codominant SNP genotyping on agarose gel. A total of 87 TSP markers for assessing gene diversity in barley were developed and used to investigate the efficacy for marker development, assay reliably and genotyping accuracy. The TSP markers described provide good coverage of the barley genome, are simple to use, easy to interpret and score, and are amenable to assay automation. They provide a resource of informative SNP markers for assessing genetic relationships among individuals, populations and gene pools of cultivated barley (Hordeum vulgare L.) and its wild relative H. spontaneum K. Koch. TSP markers provide opportunities to use available SNP resources for marker-assisted breeding and plant genetic research, and to generate information that can be integrated with SNP data from different sources and studies. TSP markers are expected to provide similar advantages for any animal or plant species. M. J. Hayden and T. Tabone contributed equally to this work.     

Development of a SNP genotyping panel for genetic monitoring of the laboratory mouse

We have developed a genotyping system for detecting genetic contamination in the laboratory mouse based on assaying single-nucleotide polymorphism (SNP) markers positioned on all autosomes and the X chromosome. This system provides a fast, reliable, and cost-effective way for genetic monitoring, while maintaining a very high degree of confidence. We describe the allelic distribution of 235 SNPs in 48 mouse strains, thereby creating a database of polymorphisms useful for genotyping purposes. The SNP markers used in this study were chosen from publicly available SNP databases. Four genotyping methods were evaluated, and dynamic two-tube allele-specific PCR assays were developed for each marker and tested on a set of 48 inbred mouse strains. The minimal number of assays sufficient to distinguish groups consisting of different numbers of mouse strains was estimated, and a panel of 28 SNPs sufficient to distinguish virtually all of the inbred strains tested was selected. Amplifluor SNP detection assays were developed for these markers and tested on an extended list of 96 strains. This panel was used as a genetic quality control approach to monitor the genotypes of nearly 300 inbred, wild-derived, congenic, consomic, and recombinant inbred strains maintained at The Jackson Laboratory. We have concluded that this marker panel is sufficient for genetic contamination monitoring in colonies containing a large number of genetically diverse mouse strains and that reduced versions of the panel could be implemented in facilities housing a lower number of strains.     

A fair fight between molecular marker types in a seascape genetics setting

From its inception, population genetics has been nearly as concerned with the genetic data type—to which analyses are brought to bear—as it is with the analysis methods themselves. The field has traversed allozymes, microsatellites, segregating sites in multilocus alignments and, currently, single nucleotide polymorphisms (SNPs) generated by high‐throughput genomic sequencing methods, primarily whole genome sequencing and reduced representation library (RRL) sequencing. As each emerging data type has gained traction, it has been compared to existing methods, based on its relative ability to discern population structural complexity at increasing levels of resolution. However, this is usually done by comparing the gold standard in one data type to the gold standard in the new data type. These gold standards frequently differ in power and in sampling density, both across a genome and throughout a spatial range. In this issue of Molecular Ecology, D’Aloia et al. apply the high‐throughput approach as fully as possible to microsatellites, nuclear loci and SNPs genotyped through an RRL method; this is coupled with a spatially dense sampling scheme. Completing a battery of population genetics analyses across data types (including a series of down‐sampled data sets), the authors find that SNP data are slightly more sensitive to fine‐scale genetic structure, and the results are more resilient to down‐sampling than microsatellites and nonrepetitive nuclear loci. However, their results are far from an unqualified victory for RRL SNP data over all previous data types: the authors note that modest additions to the microsatellites and nuclear loci data sets may provide the necessary analytical power to delineate the fine‐scale genetic structuring identified by SNPs. As always, as the field begins to fully embrace the newest thing, good science reminds us that traditional data types are far from useless, especially when combined with a well‐designed sampling scheme.