High-throughput Single Nucleotide Polymorphism Genotyping Research Articles

Identification of growth-related genes based on BSA in Pacific white shrimp Litopenaeus vannamei

The development of SNP genotyping techniques has facilitated the application of genome-wide association studies (GWAS) and genomic selection (GS) in plants and animals. However, the cost of high-throughput SNP genotyping is particularly high in the case of aquaculture animals which can produce a large number of offspring. Consequently, a cost effective method for trait-associated SNP identification and application was urgently needed in aquaculture breeding. In this study, a Bulk Segregation Analysis sequencing (BSA-seq) method was employed to identify growth-related single nucleotide polymorphisms (SNPs) and genes in the Pacific white shrimp Litopenaeus vannamei. Two independent families were used for this analysis to reduce the false positive. The DNA of individuals with the highest and lowest body weights was pooled separately. Two methods including the ED (Euclidean distance) method and the chi-square test were applied to calculate the allele differences between high and low growth rate individuals. A total of 24,325,437 SNPs were identified and consequently 1,299 SNPs and 163 genes were identified to be associated with growth traits. KEGG enrichment analysis showed that these genes were significantly enriched in phosphatidylinositol signaling system and phospholipase D signaling pathway. The diacylglycerol kinase genes, phosphatidylinositol 3-kinase genes, inositol polyphosphate 5-phosphatase genes, mitogen-activated protein kinase genes, myotubularin-related protein genes and epidermal growth factor receptor genes in these pathways were considered as candidate growth genes. This study demonstrated that the BSA-seq based on whole genome resequencing is cost-effective and useful in aquaculture. The results not only provide useful information for understanding the genetic basis of growth traits, but also offer a source of SNPs for marker-assisted selection (MAS) and genomic selection (GS) in shrimp.

Plasmodium vivax populations in the western Greater Mekong Subregion evaluated using a genetic barcode.

An improved understanding of the Plasmodium vivax populations in the Great Mekong Subregion (GMS) is needed to monitor the progress of malaria elimination. This study aimed to use a P. vivax single nucleotide polymorphism (SNP) barcode to evaluate the population dynamics and explore the gene flow among P. vivax parasite populations in the western GMS (China, Myanmar and Thailand). A total of 315 P. vivax patient samples collected in 2011 and 2018 from four regions of the western GMS were genotyped for 42 SNPs using the high-throughput MassARRAY SNP genotyping technology. Population genetic analysis was conducted to estimate the genetic diversity, effective population size, and population structure among the P. vivax populations. Overall, 291 samples were successfully genotyped at 39 SNPs. A significant difference was observed in the proportion of polyclonal infections among the five P. vivax populations (P = 0.0012, Pearson Chi-square test, χ2 = 18.1), with western Myanmar having the highest proportion (96.2%, 50/52) in 2018. Likewise, the average complexity of infection was also highest in western Myanmar (1.31) and lowest in northeast Myanmar (1.01) in 2018. The older samples from western China in 2011 had the highest pairwise nucleotide diversity (π, 0.388 ± 0.046), expected heterozygosity (He, 0.363 ± 0.02), and the largest effective population size. In comparison, in the neighboring northeast Myanmar, the more recent samples in 2018 showed the lowest values (π, 0.224 ± 0.036; He, 0.220 ± 0.026). Furthermore, the 2018 northeast Myanmar parasites showed high and moderate genetic differentiation from other populations with FST values of 0.162-0.252, whereas genetic differentiation among other populations was relatively low (FST ≤ 0.059). Principal component analysis, phylogeny, and STRUCTURE analysis showed that the P. vivax population in northeast Myanmar in 2018 substantially diverged from other populations. Although the 42 SNP barcode is a valuable tool for tracking parasite origins of worldwide parasite populations, a more extended barcode with additional SNPs is needed to distinguish the more related parasite populations in the western GMS.

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

High‐throughput genotyping in estimating genetic resources and detecting pathogens in aquaculture

Genotyping by Sequencing (GBS) for Genome-Wide SNP Identification in Plants.

Marker-assisted selection has played a pivotal role in developing several elite varieties in the past two decades. Molecular markers employed in plant breeding programs have recently shifted from microsatellites or simple sequence repeats (SSRs) to single nucleotide polymorphisms (SNPs) due to the ubiquity of SNP markers in the genome and the availability of various high-throughput SNP genotyping platforms. Rapid advances in sequencing technologies and the reduction in sequencing cost have facilitated SNP discovery in several plant species including non-model organisms with little or no genomic resources. Despite the lower cost of sequencing, genome complexity reduction approaches are still useful for SNP identification because many applications do not require every base of the genome to be sequenced. Genotyping-by-sequencing (GBS) is a quick and affordable reduced representation method that can simultaneously identify and genotype a large number of SNPs that has been successfully applied to a wide range of plant species. This chapter describes a robust two-enzyme GBS method for SNP discovery and genotyping that has been verified in non-model plant species.

High-throughput single nucleotide polymorphism genotyping reveals population structure and genetic diversity of tall fescue (

Single nucleotide polymorphisms (SNPs) are the most abundant form of genetic variation in eukaryote genomes and may be useful for selection of genetically distant parents for crossing in breeding programs. In this study, genome-wide SNPs of tall fescue (Festuca arundinacea Schreb.) were genotyped by using double-digest restriction-site-associated DNA sequencing to determine the genetic variation and differentiation among and within forage, turf and hybrid populations. After filtering, 16 036 SNPs were used to investigate genetic diversity and for structure analysis. SNP markers clearly differentiated the populations from each other. However, the hybrid population had a higher genetic similarity with the turf population than with the forage population. The results were confirmed by genetic differentiation (fixation index, Fst) and gene flow (Nm) statistics, so that low Fst and high Nm were observed between turf and hybrid groups, indicating less genetic distance and a high similarity between them. Based on the results of the weighted pair group method with arithmetic mean clustering, discriminant analysis of principal components and analysis of molecular variance, greater genetic differentiations were found among diverse turf, forage and hybrid populations, especially between turf and forage ones. In conclusion, numerous informative SNPs, natural allelic diversity-led domestication patterns, basic genetic variation statistics (e.g. Fst and heterozygosity) and population structures have multidimensional applicability for tall fescue genomics-assisted breeding.

Deciphering the Genetic Diversity of Landraces With High-Throughput SNP Genotyping of DNA Bulks: Methodology and Application to the Maize 50k Array.

Genebanks harbor original landraces carrying many original favorable alleles for mitigating biotic and abiotic stresses. Their genetic diversity remains, however, poorly characterized due to their large within genetic diversity. We developed a high-throughput, cheap and labor saving DNA bulk approach based on single-nucleotide polymorphism (SNP) Illumina Infinium HD array to genotype landraces. Samples were gathered for each landrace by mixing equal weights from young leaves, from which DNA was extracted. We then estimated allelic frequencies in each DNA bulk based on fluorescent intensity ratio (FIR) between two alleles at each SNP using a two step-approach. We first tested either whether the DNA bulk was monomorphic or polymorphic according to the two FIR distributions of individuals homozygous for allele A or B, respectively. If the DNA bulk was polymorphic, we estimated its allelic frequency by using a predictive equation calibrated on FIR from DNA bulks with known allelic frequencies. Our approach: (i) gives accurate allelic frequency estimations that are highly reproducible across laboratories, (ii) protects against false detection of allele fixation within landraces. We estimated allelic frequencies of 23,412 SNPs in 156 landraces representing American and European maize diversity. Modified Roger’s genetic Distance between 156 landraces estimated from 23,412 SNPs and 17 simple sequence repeats using the same DNA bulks were highly correlated, suggesting that the ascertainment bias is low. Our approach is affordable, easy to implement and does not require specific bioinformatics support and laboratory equipment, and therefore should be highly relevant for large-scale characterization of genebanks for a wide range of species.

Physical mapping and InDel marker development for the restorer gene Rf2 in cytoplasmic male sterile CMS-D8 cotton

BackgroundCytoplasmic male sterile (CMS) with cytoplasm from Gossypium Trilobum (D8) fails to produce functional pollen. It is useful for commercial hybrid cotton seed production. The restore line of CMS-D8 containing Rf2 gene can restore the fertility of the corresponding sterile line. This study combined the whole genome resequencing bulked segregant analysis (BSA) with high-throughput SNP genotyping to accelerate the physical mapping of Rf2 locus in CMS-D8 cotton.MethodsThe fertility of backcross population ((sterile line×restorer line)×maintainer line) comprising of 1623 individuals was investigated in the field. The fertile pool (100 plants with fertile phenotypes, F-pool) and the sterile pool (100 plants with sterile phenotypes, S-pool) were constructed for BSA resequencing. The selection of 24 single nucleotide polymorphisms (SNP) through high-throughput genotyping and the development insertion and deletion (InDel) markers were conducted to narrow down the candidate interval. The pentapeptide repeat (PPR) family genes and upregulated genes in restore line in the candidate interval were analysed by qRT-PCR.ResultsThe fertility investigation results showed that fertile and sterile separation ratio was consistent with 1:1. BSA resequencing technology, high-throughput SNP genotyping, and InDel markers were used to identify Rf2 locus on candidate interval of 1.48 Mb on chromosome D05. Furthermore, it was quantified in this experiment that InDel markers co-segregated with Rf2 enhanced the selection of the restorer line. The qRT-PCR analysis revealed PPR family gene Gh_D05G3391 located in candidate interval had significantly lower expression than sterile and maintainer lines. In addition, utilization of anther RNA-Seq data of CMS-D8 identified that the expression level of Gh_D05G3374 encoding NB-ARC domain-containing disease resistance protein in restorer lines was significantly higher than that in sterile and maintainer lines.ConclusionsThis study not only enabled us to precisely locate the restore gene Rf2 but also evaluated the utilization of InDel markers for marker assisted selection in the CMS-D8 Rf2 cotton breeding line. The results of this study provide an important foundation for further studies on the mapping and cloning of restorer genes.

Effects of PPM1K rs1440581 and rs7678928 on serum branched-chain amino acid levels and risk of cardiovascular disease

Objective This study aimed to investigate the effects of PPM1K rs1440581 and rs7678928 single nucleotide polymorphisms (SNPs) on the serum branched-chain amino acids (BCAAs) levels and cardiovascular disease (CVD) risk. Methods Anthropometric and biochemical examinations were performed at baseline and the end of 4 years in 234 individuals who were randomly recruited from the Diabetes Prevention Programme in Huai’an and received lifestyle intervention and follow up for 4 years. Serum BCAAs (leucine, isoleucine and valine (Val)) levels were measured by hydrophilic interaction chromatography-tandem mass spectrometric method and the PPM1K rs1440581 and rs7678928 were detected by high-throughput SNP genotyping at baseline. The associations of rs1440581 and rs7678928 with serum BCAA levels and risk for CVD after 4 years were further evaluated. Results The distribution frequencies of PPM1K rs1440581 and rs7678928 met the Hardy-Weinberg equilibrium (p> .05). The baseline serum levels of Val (p = .022) and total BCAAs (p = .026) in subjects with rs1440581 CC genotype were higher than in those with TT genotype. There were no significant differences in the serum levels of BCAAs among subjects with different genotypes of rs7678928. After 4-year follow-up, the subjects with rs1440581 CC genotype had higher systolic blood pressure (SBP) (p = .027), diastolic blood pressure (DBP) (p = .019), triglycerides (TGs) (p = .019) and lower high-density lipoprotein cholesterol (HDL-c) (p = .008) than those with TT genotype, and had higher AST level than those with TT (p = .030) or TC (p = .003) genotype; the subjects with rs7678928 TT genotype had higher SBP (p = .039) and DBP (p = .019) and lower HDL-c than those with CC (p = .017) genotype. Lifestyle intervention had little influence on the serum levels of fasting plasma glucose (FPG), TG, HDL-c, alanine aminotransferase (ALT), AST and creatinine (CREA) in subjects with rs1440581 CC genotype or rs7678928 TT genotype (p> .05). The incidences of CVD and non-alcoholic fatty liver disease (NAFLD) in subjects with rs1440581 CC genotype were higher than in those with TT genotype; the incidence of CVD in subjects with rs7678928 TT genotype was higher than in those with CC (p < .05) genotype. Conclusions Allele C of PPM1K rs1440581 was associated with elevated serum Val, total BCAAs and CVD risks. rs1440581 CC genotype may be a better marker than baseline serum BCAAs in predicting the risk for CVD. Trial registration Diabetes Prevention Programme in Huai’an of Huai’an Second People's Hospital, ChiCTR-TRC-14005029. KEY MESSAGE Allele C of PPM1K rs1440581 was relevant to elevated serum Val and total BCAAs. PPM1K rs1440581 CC and rs7678928 TT genotypes were associated with CVD risk. PPM1K rs1440581 CC genotype carriers were more likely to have liver injury and develop NAFLD.

Identification of Polymorphic Markers by High-Resolution Melting (HRM) Assay for High-Throughput SNP Genotyping in Maize

The development of next generation sequencing (NGS) and high throughput genotyping are important techniques for the QTL mapping and genetic analysis of different crops. High-resolution melting (HRM) is an emerging technology used for detecting single-nucleotide polymorphisms (SNPs) in various species. However, its use is still limited in maize. The HRM analysis was integrated with SNPs to identify three types of populations (NIL population, RIL population and natural population), and the useful tags were screened. The patterns of temperature-shifted melting curves were investigated from the HRM analysis, and compared these with the kit. Among all 48 pairs of primers, 10 pairs of them were selected: six pairs of primers for the NIL population, three pairs of primers for the RIL population, and one pair of primer for the natural population. The marker for the natural population was developed with a matching rate of 80% for the plant height trait, based on the data of the phenotypic characteristics measured in the field. This study provides an effective method for maize genotyping in the classification of maize germplasm resources, which can be applied to other plants for high-throughput SNP genotyping or further mapping.

Specific-Locus Amplified Fragment Sequencing (SLAF-Seq) as High-Throughput SNP Genotyping Methods.

Most plant agronomic traits are quantitatively inherited. Identification of quantitative trait loci (QTL) is a challenging target for most scientists and crop breeders as large-scale genotyping is difficult. Molecular marker technology has continuously evolved from hybridization-based technology to PCR-based technology, and finally, sequencing-based high-throughput single-nucleotide polymorphisms (SNPs). High-throughput sequencing technologies can provide strategies for sequence-based SNP genotyping. Here we describe the SLAF-seq that can be applied as the SNP genotyping approach. The high-throughput SNP genotyping methods will prove useful for the construction of high-density genetic maps and identification of QTLs for their deployment in plant breeding and facilitate genome-wide selection (GWS) and genome-wide association studies (GWAS).

Rapid mapping of a stripe rust resistance gene YrZl31 using bulked segregant analysis combined with high-throughput single-nucleotide polymorphism genotyping arrays

Stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most important diseases of wheat. Identification and utilization of new resistance genes is one of the most effective way for achieving durable disease control. Zhongliang 31, a cultivar of common wheat (Triticum aestivum L.), is highly resistant to Chinese predominant Pst races and has been grown widely in the Longnan region in Gansu Province, China. Inheritance analysis of F1, F2, and F2:3 populations indicated that the stripe rust resistance of Zhongliang 31 was controlled by a single dominant gene, temporarily designated as YrZl31. Bulked segregant analysis (BSA) combined with wheat 660K single-nucleotide polymorphism (SNP) array showed the highest proportion of polymorphic SNPs located on chromosome 2B. One hundred forty-one kompetitive allele-specific PCR (KASP) and 165 simple sequence repeat (SSR) markers on chromosome 2B were used to map YrZl31. Linkage analysis indicated that 31 markers were linked to YrZl31, and the genetic distances of the two closest flanking KASP markers AX-111650565 and AX-110196738 were 2.8 and 4.1 cM, respectively. YrZl31 was located on wheat chromosome 2BL. Pedigree, resistance specificity, chromosome location, molecular and allelism tests suggested that YrZl31 may be a new gene and can be pyramided with other effective stripe rust resistance genes using the closely linked KASP markers developed in this study in wheat breeding programs.

Towards low cost, multiplex clinical genotyping: 4-fluorescent Kompetitive Allele-Specific PCR and its application on pharmacogenetics.

Single-nucleotide polymorphisms (SNPs) is associated with efficacy of specific drugs. Although there are several methods for SNP genotyping in clinical settings, alternative methods with lower cost, higher throughout and less complexity are still needed. In this study, we modified Kompetitive Allele Specific PCR to enable multiplex SNP genotyping by introducing additional fluorescent cassettes that specifically help to differentiate more amplification signals in a single reaction. This new format of assay achieved a limit of detection down to 310 copies/ reactions for simultaneous detection of 2 SNPs with only standard end-point PCR workflow for synthetic controls, and genotyped 117 clinical samples with results that were in 100% agreement with hospital reports. This study presented a simplified, cost-effective high-throughput SNP genotyping alternative for pharmacogenetic variants, and enabled easier access to pharmaceutical guidance when needed.

Association between GSTP1 gave polymorphisms and susceptibility to noise-induced hearing loss

Objective: To investigate the association between the single nucleotide polymorphisms (SNPS) at rs1695 and rs6591256 in glutathione S-transferase P1 (GSTP1) gene and susceptibility to noise-induced hearing loss in Chinese Han workers exposed to noise. Methods: A 1: 2 matched nested case-control study was performed, which based on the cohort of 6297 workers exposed to noise in an iron and steel plant in Henan, China, who were followed up from January 1, 2006 to December 31, 2015. According to the criteria of binaural average high-frequency hearing threshold ≥40 dB, a total of 292 workers were enrolled as hearing loss group; after the adjustment for sex, type of work, age (difference≤5 years) , and working years of noise exposure (difference≤2 years) , according to the criteria of binaural average high-frequency hearing threshold <35 dB, and the speech frequency hearing threshold of any ear at any frequency band ≤25 dB, a total of 584 workers were enrolled as control group. The single nucleotide polymorphisms (SNPs) of rs1695 and rs6591256 in GSTP1 were genotyped by high throughput SNP genotyping assay. Hardy-Weinberg equilibrium of control group was checked. The association between the SNPs at the two loci and susceptibility to NIHL was analyzed. Results: The L(Aeq, 8 h) range of workers exposed to noise was 80.2-98.8 dB (A) . The risk of NIHL in individuals with allele G of rs1695 was 1.291 times of those with allele A (95%CI: 1.042-1.598, P<0.05) . The risk of NIHL in individuals with allele G of rs6591256 was 1.390 times of those with allele A (95%CI: 1.119-1.728, P<0.05) . The risk of NIHL in individuals with AG and GG genotypes of rs6591256 was 1.437 times of those with AA genotype (95%CI: 1.057-1.952, P<0.05) . With the increase of noise exposure duration, individuals with AG and GG genotypes of rs6591256 had a higher risk of NIHL than those with AA genotype (HR=1.273, 95%CI: 1.002-1.616, P<0.05) . Conclusion: The allele G of rs1695 and rs6591256 may be risk factors of NIHL.

A direct PCR–based SNP marker–assisted selection system (D-MAS) for different crops

As more and more re-sequencing genome data in crops were released, SNPs (single nucleotide polymorphisms) are easily achieved for genotyping and show the highest abundance among all kinds of molecular markers. However, high-throughput SNP genotyping methods, such as KASP (Kompetitive Allele Specific PCR), TaqMan, or ARMS (amplification refractory mutation system), are always labor on DNA extraction and depending on expensive equipment. Therefore, most breeders are undergoing the bottleneck of lacking an easy, cost-saving, and stable genotyping system. Here, we report a direct PCR–based medium-throughput SNP marker–assisted selection (D-MAS) system suits available major crops including rice, wheat, maize, and rapeseed. The D-MAS system, which reduces the time of manual operation and result analysis dramatically, contains (1) seedling breeding in greenhouse; (2) high-throughput DNA extraction by alkaline lysis; and (3) gel-free SNP marker detection with 384-well by PARMS (penta-primer amplification refractory mutation) or KASP genotyping system. The stability of alkaline lysis DNA was validated by flexible dilution fold and long storing time under low temperatures. The alkaline lysis DNA from four F2 populations of rice, wheat, maize, and rapeseed showed equal efficiency in SNP calling relative to the ones from cetyl trimethylammonium bromide (CTAB) method. With the alkaline lysis DNA, PAMRS showed denser genotype cluster than KASP. Furthermore, the D-MAS system was adaptive with rice old leaves. The throughput and efficiency of the D-MAS system were validated in the hybrid rice seed purity test with a Xian-Geng-specific SNP marker which is also recommended to select ipa1-2d gene in the rice molecular breeding. In conclusion, we proposed a direct PCR–based SNP calling pipeline, which could be a simple, cheap, and robust standard operation procedure (SOP) of molecular breeding for different crops and get extensive use in most laboratory.

Genome sequencing and conservation genomics in the Scandinavian wolverine population.

Genetic approaches have proved valuable to the study and conservation of endangered populations, especially for monitoring programs, and there is potential for further developments in this direction by extending analyses to the genomic level. We assembled the genome of the wolverine (Gulo gulo), a mustelid that in Scandinavia has recently recovered from a significant population decline, and obtained a 2.42Gb draft sequence representing >85% of the genome and including >21,000protein-coding genes. We then performed whole-genome resequencing of 10Scandinavian wolverines for population genomic and demographic analyses. Genetic diversity was among the lowest detected in a red-listed population (mean genome-wide nucleotide diversity of 0.05%). Results of the demographic analyses indicated a long-term decline of the effective population size (Ne ) from 10,000well before the last glaciation to <500 after this period. Current Ne appeared even lower. The genome-wide FIS level was 0.089 (possibly signaling inbreeding), but this effect was not observed when analyzing a set of highly variable SNP markers, illustrating that such markers can give a biased picture of the overall character of genetic diversity. We found significant population structure, which has implications for population connectivity and conservation. We used an integrated microfluidic circuit chip technology to develop an SNP-array consisting of 96 highly informative markers that, together with a multiplex pre-amplification step, was successfully applied to low-quality DNA from scat samples. Our findings will inform management, conservation, and genetic monitoring of wolverines and serve as a genomic roadmap that can be applied to other endangered species. The approach used here can be generally utilized in other systems, but we acknowledge the trade-off between investing in genomic resources and direct conservation actions.

P077Imputing allelic variants for five human leukocyte antigen genes using a high-throughput genotyping array

Aim To assess the feasibility of imputing human leukocyte antigen (HLA) alleles in African cohorts based on high-throughput genotyping of single-nucleotide polymorphisms (SNPs) in the human major histocompatibility complex (MHC). Methods For two African cohorts representing eastern and southern Africa, genomic DNA samples extracted from peripheral blood were first used for genotyping HLA-A , HLA-B , HLA-C , HLA-DRB1 and HLA-DQB1 alleles, all with 4-digit resolution through a combination of PCR-based genotyping techniques, including direct sequencing of locus-specific amplicons. SNP genotypes were further defined by a widely-used commercial array (the Illumina ImmunoChip). Paired HLA and SNP data from the first 400 individuals in each cohort served as a reference (the training set) for validation in the rest of samples. All imputation procedures were done using the HIBAG algorithm. Results A total of 7539 SNPs spanning the extended human MHC passed various quality control procedures and then merged with fully-resolved HLA alleles in 490 Rwandans and 1100 Zambians. Overall, SNP-based imputation of 2-digit allele groups at the five target loci showed success rates between 93.9% and 98.3%, while success rates in imputing 4-digit alleles ranged from 85.4% to 97.2%. Two major HLA-DRB1 allele groups (DRB1*04 and DRB1*10) in Zambians were the only ones with suboptimal imputation probabilities ( Conclusions In the era of high-throughput genomics, SNP-based imputation of HLA variants may offer a quick and affordable alternative for initial assessment of 2-digit allele groups. However, reliable imputation of 4-digit allele remains difficult even for some common alleles of broad interest.

Programmable microfluidic genotyping of plant DNA samples for marker-assisted selection

As demands to maintain the global food production continue to mount, multinational seed companies are turning to new DNA marker technologies to accelerate the rate of plant breeding and crop improvement. The key to widespread adoption of molecular breeding is the availability of flexible and cost-effective tools that can perform combinatorial and high-throughput genotyping of single-nucleotide polymorphisms (SNPs) to guide the crop development process. Toward this end, we have developed a programmable, droplet-based microfluidic device for genotyping maize genomic DNA. A unique feature of the microfluidic platform is the nano sample processors (NSPs), which allow the device to sequentially load an unrestricted number of unique DNA samples using only two inlets, overcoming the current limitation to the number of sample inputs due to small device footprint. Direct and programmable droplet generation within the device allows each sample to be genotyped against a panel of markers on demand. Moreover, we have successfully implemented the Invader assay for SNP genotyping in flowing, 50-nL droplets, thus achieving significant reduction in consumption of reagents per reaction as compared with conventional genotyping platforms. As a demonstration, we performed 240 Invader reactions (testing 8 DNA samples against 10 SNP markers) and achieved greater than 93% accuracy in SNP calling of plant DNA samples in a single droplet-based experiment.

High throughput SNP discovery and genotyping in hexaploid wheat.

Because of their abundance and their amenability to high-throughput genotyping techniques, Single Nucleotide Polymorphisms (SNPs) are powerful tools for efficient genetics and genomics studies, including characterization of genetic resources, genome-wide association studies and genomic selection. In wheat, most of the previous SNP discovery initiatives targeted the coding fraction, leaving almost 98% of the wheat genome largely unexploited. Here we report on the use of whole-genome resequencing data from eight wheat lines to mine for SNPs in the genic, the repetitive and non-repetitive intergenic fractions of the wheat genome. Eventually, we identified 3.3 million SNPs, 49% being located on the B-genome, 41% on the A-genome and 10% on the D-genome. We also describe the development of the TaBW280K high-throughput genotyping array containing 280,226 SNPs. Performance of this chip was examined by genotyping a set of 96 wheat accessions representing the worldwide diversity. Sixty-nine percent of the SNPs can be efficiently scored, half of them showing a diploid-like clustering. The TaBW280K was proven to be a very efficient tool for diversity analyses, as well as for breeding as it can discriminate between closely related elite varieties. Finally, the TaBW280K array was used to genotype a population derived from a cross between Chinese Spring and Renan, leading to the construction a dense genetic map comprising 83,721 markers. The results described here will provide the wheat community with powerful tools for both basic and applied research.

Introduction of High Throughput and Cost Effective SNP Genotyping Platforms in Soybean

We presented here the application of two in-plate SNP (single nucleotide polymorphism) genotyping platforms for soybean plants [Glycine max (L.) Merr.], KASP® (Kompetitive Allele Specific PCR genotyping, LGC Genomics) and TaqMan® (Life Technologies) respectively. These two systems offer us an ability to determine the genotypes of 384 individual samples accurately and efficiently by allele specific PCR in a single plate using typical PCR conditions. Both of the systems require small quantity of genomic DNA obtained from a simple DNA extraction. The genomic sequences containing target SNPs can easily be used as a basic blueprint to design the probes and primers of KASP® and TaqMan® assays whether the sequences are obtained from the genome sequence of soybean William 82 (Wm82.a2.v1), Illumina Soy50k SNPs, or parallel resequencing. Moreover, we listed the pros and cons of the two systems and explained the principles behind the platforms. The high call rate and clear clustering separation of the SNPs can be readily obtained from these platforms without conducting any assay optimization processes. These platforms can routinely be performed on 96/384-well plate format with or without an automation procedure. Therefore, these platforms are especially suitable for the SNP genotyping on a particular trait with a large sample size, gene fine mapping, and marker assisted selection. Further, they require little hands-on experience and achieve per-site and per-individual costs below that of current SSR, AFLP, RFLP, and SNP chip technologies. The platforms can be used for genotyping on a wide range of organisms due to their simplicity and flexibility of handling. Meanwhile, we also especially presented some of the advantages using KASP® SNP genotyping pipeline, which was cost effective in the selection of allele specific assay and therefore, efficiently facilitated the soybean genotyping across large numbers (thousands or more) of individual lines for a great range of markers (hundreds to thousands) in our laboratory.

High-throughput SNP genotyping of modern and wild emmer wheat for yield and root morphology using a combined association and linkage analysis.

Durum wheat (Triticum turgidum var. durum Desf.) is a major world crop that is grown primarily in areas of the world that experience periodic drought, and therefore, breeding climate-resilient durum wheat is a priority. High-throughput single nucleotide polymorphism (SNP) genotyping techniques have greatly increased the power of linkage and association mapping analyses for bread wheat, but as yet there is no durum wheat-specific platform available. In this study, we evaluate the new 384HT Wheat Breeders Array for its usefulness in tetraploid wheat breeding by genotyping a breeding population of F6 hybrids, derived from multiple crosses between T. durum cultivars and wild and cultivated emmer wheat accessions. Using a combined linkage and association mapping approach, we generated a genetic map including 1345 SNP markers, and identified markers linked to 6 QTLs for coleoptile length (2), heading date (1), anthocyanin accumulation (1) and osmotic stress tolerance (2). We also developed a straightforward approach for combining genetic data from multiple families of limited size that will be useful for evaluating and mapping pre-existing breeding material.