High-throughput genotyping with the GoldenGate assay in the complex genome of soybean

Large numbers of single nucleotide polymorphism (SNP) markers are now available for a number of crop species. However, the high-throughput methods for multiplexing SNP assays are untested in complex genomes, such as soybean, that have a high proportion of paralogous genes. The Illumina GoldenGate assay is capable of multiplexing from 96 to 1,536 SNPs in a single reaction over a 3-day period. We tested the GoldenGate assay in soybean to determine the success rate of converting verified SNPs into working assays. A custom 384-SNP GoldenGate assay was designed using SNPs that had been discovered through the resequencing of five diverse accessions that are the parents of three recombinant inbred line (RIL) mapping populations. The 384 SNPs that were selected for this custom assay were predicted to segregate in one or more of the RIL mapping populations. Allelic data were successfully generated for 89% of the SNP loci (342 of the 384) when it was used in the three RIL mapping populations, indicating that the complex nature of the soybean genome had little impact on conversion of the discovered SNPs into usable assays. In addition, 80% of the 342 mapped SNPs had a minor allele frequency >10% when this assay was used on a diverse sample of Asian landrace germplasm accessions. The high success rate of the GoldenGate assay makes this a useful technique for quickly creating high density genetic maps in species where SNP markers are rapidly becoming available. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Mention of a trade name, proprietary product, or specific equipment does not constitute a guarantee or warranty by the USDA and does not imply approval of a product to the exclusion of others that may be suitable.     

Single nucleotide polymorphism genotyping in polyploid wheat with the Illumina GoldenGate assay

Single nucleotide polymorphisms (SNPs) are indispensable in such applications as association mapping and construction of high-density genetic maps. These applications usually require genotyping of thousands of SNPs in a large number of individuals. Although a number of SNP genotyping assays are available, most of them are designed for SNP genotyping in diploid individuals. Here, we demonstrate that the Illumina GoldenGate assay could be used for SNP genotyping of homozygous tetraploid and hexaploid wheat lines. Genotyping reactions could be carried out directly on genomic DNA without the necessity of preliminary PCR amplification. A total of 53 tetraploid and 38 hexaploid homozygous wheat lines were genotyped at 96 SNP loci. The genotyping error rate estimated after removal of low-quality data was 0 and 1% for tetraploid and hexaploid wheat, respectively. Developed SNP genotyping assays were shown to be useful for genotyping wheat cultivars. This study demonstrated that the GoldenGate assay is a very efficient tool for high-throughput genotyping of polyploid wheat, opening new possibilities for the analysis of genetic variation in wheat and dissection of genetic basis of complex traits using association mapping approach. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High‐density SNP assay development for genetic analysis in maritime pine (Pinus pinaster)

Maritime pine provides essential ecosystem services in the south‐western Mediterranean basin, where it covers around 4 million ha. Its scattered distribution over a range of environmental conditions makes it an ideal forest tree species for studies of local adaptation and evolutionary responses to climatic change. Highly multiplexed single nucleotide polymorphism (SNP) genotyping arrays are increasingly used to study genetic variation in living organisms and for practical applications in plant and animal breeding and genetic resource conservation. We developed a 9k Illumina Infinium SNP array and genotyped maritime pine trees from (i) a three‐generation inbred (F2) pedigree, (ii) the French breeding population and (iii) natural populations from Portugal and the French Atlantic coast. A large proportion of the exploitable SNPs (2052/8410, i.e. 24.4%) segregated in the mapping population and could be mapped, providing the densest ever gene‐based linkage map for this species. Based on 5016 SNPs, natural and breeding populations from the French gene pool exhibited similar level of genetic diversity. Population genetics and structure analyses based on 3981 SNP markers common to the Portuguese and French gene pools revealed high levels of differentiation, leading to the identification of a set of highly differentiated SNPs that could be used for seed provenance certification. Finally, we discuss how the validated SNPs could facilitate the identification of ecologically and economically relevant genes in this species, improving our understanding of the demography and selective forces shaping its natural genetic diversity, and providing support for new breeding strategies.     

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.     

Highly efficient genotyping of rice biparental populations by GoldenGate assays based on parental resequencing

Key message

A new time- and cost-effective strategy was developed for medium-density SNP genotyping of rice biparental populations, using GoldenGate assays based on parental resequencing.

Abstract

Since the advent of molecular markers, crop researchers and breeders have dedicated huge amounts of effort to detecting quantitative trait loci (QTL) in biparental populations for genetic analysis and marker-assisted selection (MAS). In this study, we developed a new time- and cost-effective strategy for genotyping a population of progeny from a rice cross using medium-density single nucleotide polymorphisms (SNPs). Using this strategy, 728,362 “high quality” SNPs were identified by resequencing Teqing and Lemont, the parents of the population. We selected 384 informative SNPs that were evenly distributed across the genome for genotyping the biparental population using the Illumina GoldenGate assay. 335 (87.2 %) validated SNPs were used for further genetic analyses. After removing segregation distortion markers, 321 SNPs were used for linkage map construction and QTL mapping. This strategy generated SNP markers distributed more evenly across the genome than previous SSR assays. Taking the GW5 gene that controls grain shape as an example, our strategy provided higher accuracy (0.8 Mb) and significance (LOD 5.5 and 10.1) in QTL mapping than SSR analysis. Our study thus provides a rapid and efficient strategy for genetic studies and QTL mapping using SNP genotyping assays.     

Genetic mapping of expressed sequence tag polymorphism (ESTP) markers in loblolly pine (Pinus taeda L.)

The development and mapping of genetic markers based upon expressed sequence tag polymorphisms (ESTPs) in loblolly pine (Pinus taeda L.) are reported. The new markers were generated by PCR-amplification of loblolly pine genomic DNAs with primers designed from sequenced cDNAs. The cDNA libraries were constructed from RNAs expressed in the needles of loblolly pine seedlings or in the xylem from young trees. DNA polymorphisms were identified by analyzing the amplified products for differences in fragment size or restriction sites, or by examining mobility differences using denaturing gradient gel electrophoresis (DGGE). DGGE revealed more DNA polymorphisms than the other two methods. Fifty six ESTPs were mapped using either of two mapping populations and positioned onto a loblolly pine consensus genetic map. Unlike many other markers commonly used in forestry, ESTPs can be used as orthologous markers for comparative mapping, to map genes of known function, or to identify candidate genes affecting important traits in loblolly pine. Received: 10 April 2000 / Accepted: 13 July 2000     

High-Throughput SNP Discovery and Genotyping for Constructing a Saturated Linkage Map of Chickpea (Cicer arietinum L.)

The present study reports the large-scale discovery of genome-wide single-nucleotide polymorphisms (SNPs) in chickpea, identified mainly through the next generation sequencing of two genotypes, i.e. Cicer arietinum ICC4958 and its wild progenitor C. reticulatum PI489777, parents of an inter-specific reference mapping population of chickpea. Development and validation of a high-throughput SNP genotyping assay based on Illumina''s GoldenGate Genotyping Technology and its application in building a high-resolution genetic linkage map of chickpea is described for the first time. In this study, 1022 SNPs were identified, of which 768 high-confidence SNPs were selected for designing the custom Oligo Pool All (CpOPA-I) for genotyping. Of these, 697 SNPs could be successfully used for genotyping, demonstrating a high success rate of 90.75%. Genotyping data of the 697 SNPs were compiled along with those of 368 co-dominant markers mapped in an earlier study, and a saturated genetic linkage map of chickpea was constructed. One thousand and sixty-three markers were mapped onto eight linkage groups spanning 1808.7 cM (centiMorgans) with an average inter-marker distance of 1.70 cM, thereby representing one of the most advanced maps of chickpea. The map was used for the synteny analysis of chickpea, which revealed a higher degree of synteny with the phylogenetically close Medicago than with soybean. The first set of validated SNPs and map resources developed in this study will not only facilitate QTL mapping, genome-wide association analysis and comparative mapping in legumes but also help anchor scaffolds arising out of the whole-genome sequencing of chickpea.     

A pipeline for high throughput detection and mapping of SNPs from EST databases

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic variation that can be used as molecular markers. The SNPs that are hidden in sequence databases can be unlocked using bioinformatic tools. For efficient application of these SNPs, the sequence set should be error-free as much as possible, targeting single loci and suitable for the SNP scoring platform of choice. We have developed a pipeline to effectively mine SNPs from public EST databases with or without quality information using QualitySNP software, select reliable SNP and prepare the loci for analysis on the Illumina GoldenGate genotyping platform. The applicability of the pipeline was demonstrated using publicly available potato EST data, genotyping individuals from two diploid mapping populations and subsequently mapping the SNP markers (putative genes) in both populations. Over 7000 reliable SNPs were identified that met the criteria for genotyping on the GoldenGate platform. Of the 384 SNPs on the SNP array approximately 12% dropped out. For the two potato mapping populations 165 and 185 SNPs segregating SNP loci could be mapped on the respective genetic maps, illustrating the effectiveness of our pipeline for SNP selection and validation.     

High-throughput genotyping in citrus accessions using an SNP genotyping array

We developed a 384 multiplexed SNP array, named CitSGA-1, for the genotyping of Citrus cultivars, and evaluated the performance and reliability of the genotyping. SNPs were surveyed by direct sequence comparison of the sequence tagged site (STS) fragment amplified from genomic DNA of cultivars representing the genetic diversity of citrus breeding in Japan. Among 1497 SNPs candidates, 384 SNPs for a high-throughput genotyping array were selected based on physical parameters of Illumina’s bead array criteria. The assay using CitSGA-1 was applied to a hybrid population of 88 progeny and 103 citrus accessions for breeding in Japan, which resulted in 73,726 SNP calls. A total of 351 SNPs (91 %) could call different genotypes among the DNA samples, resulting in a success rate for the assay comparable to previously reported rates for other plant species. To confirm the reliability of SNP genotype calls, parentage analysis was applied, and it indicated that the number of reliable SNPs and corresponding STSs were 276 and 213, respectively. The multiplexed SNP genotyping array reported here will be useful for the efficient construction of linkage map, for the detection of markers for marker-assisted breeding, and for the identification of cultivars.     

High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.)

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs.     

High-resolution, high-throughput SNP mapping in Drosophila melanogaster

Single nucleotide polymorphisms (SNPs) are useful markers for genetic mapping experiments in model organisms. Here we report the establishment of a high-density SNP map and high-throughput genotyping assays for Drosophila melanogaster. Our map comprises 27,367 SNPs in common laboratory Drosophila stocks. These SNPs were clustered within 2,238 amplifiable markers at an average density of 1 marker every 50.3 kb, or 6.3 genes. We have also constructed a set of 62 Drosophila stocks, each of which facilitates the generation of recombinants within a defined genetic interval of 1-2 Mb. For flexible, high-throughput SNP genotyping, we used fluorescent tag-array mini-sequencing (TAMS) assays. We designed and validated TAMS assays for 293 SNPs at an average resolution of 391.3 kb, and demonstrated the utility of these tools by rapidly mapping 14 mutations that disrupt embryonic muscle patterning. These resources enable high-resolution high-throughput genetic mapping in Drosophila.     

Association mapping in forest trees and fruit crops

Association mapping (AM), also known as linkage disequilibrium (LD) mapping, is a viable approach to overcome limitations of pedigree-based quantitative trait loci (QTL) mapping. In AM, genotypic and phenotypic correlations are investigated in unrelated individuals. Unlike QTL mapping, AM takes advantage of both LD and historical recombination present within the gene pool of an organism, thus utilizing a broader reference population. In plants, AM has been used in model species with available genomic resources. Pursuing AM in tree species requires both genotyping and phenotyping of large populations with unique architectures. Recently, genome sequences and genomic resources for forest and fruit crops have become available. Due to abundance of single nucleotide polymorphisms (SNPs) within a genome, along with availability of high-throughput resequencing methods, SNPs can be effectively used for genotyping trees. In addition to DNA polymorphisms, copy number variations (CNVs) in the form of deletions, duplications, and insertions also play major roles in control of expression of phenotypic traits. Thus, CNVs could provide yet another valuable resource, beyond those of microsatellite and SNP variations, for pursuing genomic studies. As genome-wide SNP data are generated from high-throughput sequencing efforts, these could be readily reanalysed to identify CNVs, and subsequently used for AM studies. However, forest and fruit crops possess unique architectural and biological features that ought to be taken into consideration when collecting genotyping and phenotyping data, as these will also dictate which AM strategies should be pursued. These unique features as well as their impact on undertaking AM studies are outlined and discussed.     

In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

Background

There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (∼23.8 Gb/C).

Methodology/Principal Findings

A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates).

Conclusions/Significance

This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome.     

High-throughput SNP genotyping by single-tube PCR with Tm-shift primers

Despite many recent advances in high-throughput single nucleotide polymorphism (SNP) genotyping technologies, there is still a great need for inexpensive and flexible methods with a reasonable throughput. Here we report substantial modifications and improvements to an existing homogenous allele-specific PCR-based SNP genotyping method, making it an attractive new option for researchers engaging in candidate gene studies or following up on genome-wide scans. In this advanced version of the melting temperature (Tm)-shift SNP genotyping method, we attach two GC-rich tails of different lengths to allele-specific PCR primers, such that SNP alleles in genomic DNA samples can be discriminated by the Tms of the PCR products. We have validated 306 SNP assays using this method and achieved a success rate in assay development of greater than 83% under uniform PCR conditions. We have developed a standalone software application to automatically assign genotypes directly from melting curve data. To demonstrate the accuracy of this method, we typed 592 individuals for 6 SNPs and showed a high call rate (>98%) and high accuracy (>99.9%). With this method, 6-10,000 samples can be genotyped per day using a single 384-well real-time thermal cycler with 2-4 standard 384-well PCR instruments.     

Genetic linkage maps of Populus nigra L. including AFLPs, SSRs, SNPs, and sex trait

Black poplar (Populus nigra L.) is a tree of ecological and economic interest. A better knowledge of P. nigra genome is needed for an effective protection and use of its genetic resources. The main objective of this study is the construction of a highly informative genetic map of P. nigra species including genes of adaptive and economic interest. Two genotypes originated from contrasted natural Italian populations were crossed to generate a F₁ mapping pedigree of 165 individuals. Amplification fragment length polymorphism (AFLP), simple sequence repeat (SSR), and single nucleotide polymorphism (SNP) markers were used to genotype 92 F₁ individuals, and the pseudo-test-cross strategy was applied for linkage analysis. The female parent map included 368 markers (274 AFLPs, 91 SSRs, and 3 SNPs) and spanned 2,104 cM with 20 linkage groups, and the male parent map, including 317 markers (205 AFLPs, 106 SSRs, 5 SNPs, and sex trait), spanned 2,453 cM with 23 main linkage groups. The sex, as morphological trait, was mapped on the linkage group XIX of the male parent map. The generated maps are among the most informative in SSRs when compared to the Populus maps published so far and allow a complete alignment with the 19 haploid chromosomes of Populus sequence genome. These genetic maps provide informative tools for a better understanding of P. nigra genome structure and genetic improvement of this ecologically and economically important European tree species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Further development of multiplex single nucleotide polymorphism typing method, the DigiTag2 assay

A number of single nucleotide polymorphisms (SNPs) are considered to be candidate susceptibility or resistance genetic factors for multifactorial disease. Genome-wide searches for disease susceptibility regions followed by high-resolution mapping of primary genes require cost-effective and highly reliable technology. To accomplish successful and low-cost typing for candidate SNPs, new technologies must be developed. We previously reported a multiplex SNP typing method, designated the DigiTag assay, that has the potential to analyze nearly any SNP with high accuracy and reproducibility. However, the DigiTag assay requires multiple washing steps in manipulation and uses genotyping probes modified with biotin for each target SNP. Here we describe the next version of the assay, DigiTag2, which works with simple protocols and uses unmodified genotyping probes. We investigated the feasibility of the DigiTag2 assay by genotyping 96 target SNPs spanning a 610-kb region of human chromosome 5. The DigiTag2 assay is suitable for genotyping an intermediate number of SNPs (tens to hundreds of sites) with a high conversion rate (>90%), high accuracy, and low cost.     

High-throughput single nucleotide polymorphism genotyping for breeding applications in rice using the BeadXpress platform

Multiplexed single nucleotide polymorphism (SNP) markers have the potential to increase the speed and cost-effectiveness of genotyping, provided that an optimal SNP density is used for each application. To test the efficiency of multiplexed SNP genotyping for diversity, mapping and breeding applications in rice (Oryza sativa L.), we designed seven GoldenGate VeraCode oligo pool assay (OPA) sets for the Illumina BeadXpress Reader. Validated markers from existing 1536 Illumina SNPs and 44?K Affymetrix SNP chips developed at Cornell University were used to select subsets of informative SNPs for different germplasm groups with even distribution across the genome. A 96-plex OPA was developed for quality control purposes and for assigning a sample into one of the five O. sativa population subgroups. Six 384-plex OPAs were designed for genetic diversity analysis, DNA fingerprinting, and to have evenly-spaced polymorphic markers for quantitative trait locus (QTL) mapping and background selection for crosses between different germplasm pools in rice: Indica/Indica, Indica/Japonica, Japonica/Japonica, Indica/O. rufipogon, and Japonica/O. rufipogon. After testing on a diverse set of rice varieties, two of the SNP sets were re-designed by replacing poor-performing SNPs. Pilot studies were successfully performed for diversity analysis, QTL mapping, marker-assisted backcrossing, and developing specialized genetic stocks, demonstrating that 384-plex SNP genotyping on the BeadXpress platform is a robust and efficient method for marker genotyping in rice.     

SNP high-throughput screening in grapevine using the SNPlex™ genotyping system

Background

Until recently, only a small number of low- and mid-throughput methods have been used for single nucleotide polymorphism (SNP) discovery and genotyping in grapevine (Vitis vinifera L.). However, following completion of the sequence of the highly heterozygous genome of Pinot Noir, it has been possible to identify millions of electronic SNPs (eSNPs) thus providing a valuable source for high-throughput genotyping methods.

Results

Herein we report the first application of the SNPlex? genotyping system in grapevine aiming at the anchoring of an eukaryotic genome. This approach combines robust SNP detection with automated assay readout and data analysis. 813 candidate eSNPs were developed from non-repetitive contigs of the assembled genome of Pinot Noir and tested in 90 progeny of Syrah × Pinot Noir cross. 563 new SNP-based markers were obtained and mapped. The efficiency rate of 69% was enhanced to 80% when multiple displacement amplification (MDA) methods were used for preparation of genomic DNA for the SNPlex assay.

Conclusion

Unlike other SNP genotyping methods used to investigate thousands of SNPs in a few genotypes, or a few SNPs in around a thousand genotypes, the SNPlex genotyping system represents a good compromise to investigate several hundred SNPs in a hundred or more samples simultaneously. Therefore, the use of the SNPlex assay, coupled with whole genome amplification (WGA), is a good solution for future applications in well-equipped laboratories.