High-throughput Genotyping System Research Articles

KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance.

Advances in plant molecular breeding have resulted in the development of new varieties with superior traits, thus improving the crop germplasm. Breeders can screen a large number of accessions without rigorous and time-consuming phenotyping by marker-assisted selection (MAS). Molecular markers are one of the most imperative tools in plant breeding programmes for MAS to develop new cultivars possessing multiple superior traits. Single nucleotide polymorphisms (SNPs) are ideal for MAS due to their low cost, low genotyping error rates, and reproducibility. Kompetitive Allele Specific PCR (KASP) is a globally recognized technology for SNP genotyping. KASP is an allele-specific oligo extension-based PCR assay that uses fluorescence resonance energy transfer (FRET) to detect genetic variations such as SNPs and insertions/deletions (InDels) at a specific locus. Additionally, KASP allows greater flexibility in assay design, which leads to a higher success rate and the capability to genotype a large population. Its versatility and ease of use make it a valuable tool in various fields, including genetics, agriculture, and medical research. KASP has been extensively used in various plant-breeding applications, such as the identification of germplasm resources, quality control (QC) analysis, allele mining, linkage mapping, quantitative trait locus (QTL) mapping, genetic map construction, trait-specific marker development, and MAS. This review provides an overview of the KASP assay and emphasizes its validation in crop improvement related to various biotic and abiotic stress tolerance and quality traits.

A Review of Rapid Generation Advancement (RGA) in Crop Improvement

The use of the speed breeding method is widely considered to be the wave of the future in plant breeding. The term "speed breeding" is used to describe a rapid generational advancement technique that is used to minimise the time it takes from seed to seed, therefore reducing the length of a crop plant's typical life cycle. Plants that are not sensitive to light may have as many as six generations in a single year using this method, whereas other plants can only expect two or three generations every year. With this technique, the photoperiodic and temperature needs of crops produced in controlled-poly homes may be altered. This methodology, when combined with other cutting-edge tools like genome editing and high-throughput genotyping systems, may help breed new kinds of crops at a much quicker pace. Spacefaring food producers: NASA first conceived of this notion. Breeder's equation may be used to determine whether speed breeding is applicable to a certain crop. Light, photoperiodic regime, temperature, and humidity modification make up the backbone of the fast-breeding formula. Accelerated breeding, expedited genomic selection, improved transgenic and CRISPR-Cas9 pipelines, and the investigation of critically important agricultural plant physiological properties are just some of the numerous uses for this approach.

Evaluation of two high-throughput genotyping systems for rapid identification of Canadian wheat varieties

In this study, we report an updated panel of 32 DNA markers used for identification of wheat varieties and assess their performance in the OpenArray and SmartChip high-throughput genotyping systems. While both systems are unique and offer different advantages and disadvantages, both systems can successfully identify Canadian wheat varieties.

Genome-wide analysis-based single nucleotide polymorphism marker sets to identify diverse genotypes in cabbage cultivars (Brassica oleracea var. capitata)

Plant variety protection is essential for breeders’ rights granted by the International Union for the Protection of New Varieties of Plants. Distinctness, uniformity, and stability (DUS) are necessary for new variety registration; to this end, currently, morphological traits are examined, which is time-consuming and laborious. Molecular markers are more effective, accurate, and stable descriptors of DUS. Advancements in next-generation sequencing technology have facilitated genome-wide identification of single nucleotide polymorphisms. Here, we developed a core set of single nucleotide polymorphism markers to identify cabbage varieties and traits of test guidance through clustering using the Fluidigm assay, a high-throughput genotyping system. Core sets of 87, 24, and 10 markers are selected based on a genome-wide association-based approach. All core markers could identify 94 cabbage varieties and determine 17 DUS traits. A genotypes database was validated using the Fluidigm platform for variety identification, population structure analysis, cabbage breeding, and DUS testing for plant cultivar protection.

Mungbean (Vigna radiata (L.) R. Wilczek): Progress in Breeding and Future Challenges

Mungbean (Vigna radiata (L.) R. Wilczek) is a short duration farmer preferred warm-season pulse crop. The crop has shown a balanced growth worldwide, especially in developing countries. Mungbean being a great source of protein with higher folate and iron levels attracts high demand and price on the market making the farmers happy and satisfied. Moreover, it can fix atmospheric nitrogen through symbiosis with nitrogen-fixing bacteria, making it perfect for rice based cropping systems and intercropping with other crops. Despite having so many benefits, mungbean has been a neglected crop compared to other pulses with limited efforts aiming at its breeding and development. Higher productivity, breeding for biotic and abiotic stresses resistance and nutritional quality improvement are future challenges for mungbean breeders. Several researchers are working in the direction of collecting and maintaining mungbean genetic resources. Mutation breeding and genetic engineering has also been enhanced in mungbean varietal improvement. Genomic information is limited compared to other legume species. However, the recent successful sequencing of mungbean genome has opened new vistas into the crop’s R&D. It is a self-pollinated pulse with small genome size, which could be used as a model for studying other legumes. Mungbean breeders at present times aim to identify useful alleles from diverse germplasm and markers closely associated with desirable traits. The high-throughput marker genotyping system has now made it feasible to pinpoint the exact gene locations and mutations contributing target phenotypes. In this review we present the current status of conventional and molecular breeding of the crop and summary of efforts in the utilization of genetic information and genomic resources for further mungbean improvement.

Genomic Variation in Korean japonica Rice Varieties.

Next-generation sequencing technologies have enabled the discovery of numerous sequence variations among closely related crop varieties. We analyzed genome resequencing data from 24 Korean temperate japonica rice varieties and discovered 954,233 sequence variations, including 791,121 single nucleotide polymorphisms (SNPs) and 163,112 insertions/deletions (InDels). On average, there was one variant per 391 base-pairs (bp), a variant density of 2.6 per 1 kbp. Of the InDels, 10,860 were longer than 20 bp, which enabled conversion to markers resolvable on an agarose gel. The effect of each variant on gene function was predicted using the SnpEff program. The variants were categorized into four groups according to their impact: high, moderate, low, and modifier. These groups contained 3524 (0.4%), 27,656 (2.9%), 24,875 (2.6%), and 898,178 (94.1%) variants, respectively. To test the accuracy of these data, eight InDels from a pre-harvest sprouting resistance QTL (qPHS11) target region, four highly polymorphic InDels, and four functional sequence variations in known agronomically important genes were selected and successfully developed into markers. These results will be useful to develop markers for marker-assisted selection, to select candidate genes in map-based cloning, and to produce efficient high-throughput genome-wide genotyping systems for Korean temperate japonica rice varieties.

Breeding synthetic varieties in annual caraway: observations on the outcrossing rate in a polycross using a high-throughput genotyping system

Caraway (Carum carvi) is an economically important spice and medicinal plant of the Apiaceae family (syn. Umbelliferrae). Farmers often favor annual cultivation of caraway. However, the annual varieties, which are currently available, do not provide satisfying seed yields. Employing heterosis can be a promising approach to increase yield. Breeding of synthetic varieties utilizes heterosis and may be the method of choice for future caraway breeding. Knowledge of the outcrossing rate is important to evaluate the effectiveness of this breeding method. However, the outcrossing rate of caraway is unknown so far. We estimated the outcrossing rate of seven inbred lines under field conditions in a neighbor-balanced polycross design. For this purpose, we implemented a high-throughput genotyping system (PACE), accompanied by a high-throughput method for DNA extraction adapted to caraway. In total, more than 1300 individual plants were genotyped. We found a high variability of lines regarding outcrossing rate and other traits associated with flowering. The outcrossing rate was on average 66.5% and ranged from 51.6 to 82%. We discussed implications of our findings on the targeted breeding method.

Marker integration and development of Fluidigm/KASP assays for high-throughput genotyping of radish

Radish (Raphanus sativus L.) is a representative root crop of the Brassicaceae family and is important to the vegetable seed industry in East Asia. Due to its agronomic importance, various molecular markers, genetic maps, genomic resources, and genome assemblies of radish have been developed during the past decade. Marker integration and comparative mapping using these resources will accelerate genetic improvements in radish cultivars. With the goal of establishing a marker-based high-throughput genetic analysis tool, we integrated 3765 nonredundant genetic markers into the Rs1.0 reference genome and converted them into 1182 single nucleotide polymorphism (SNP) markers via whole-genome resequencing data of the mapping parents ‘WK10039’ and ‘WK10024’. A genetic map covering 721.3 cM with 768 framework loci was constructed by analyzing these SNP conversion markers in the F2 mapping population, which was composed of 93 individuals. Comparison of this map with the Rs1.0 reference genome and other linkage maps showed the physical and genetic correlations of the markers. To develop a high-throughput genotyping system for large accessions or populations with smaller numbers of SNPs, 674 Fluidigm and 68 kompetitive allele-specific PCR (KASP) markers were validated. Application of the 68 KASP assays to 127 commercial cultivars enabled successful identification and classification of genotypes; 11 KASP markers constituted the minimum marker set. The SNP markers used to construct the genetic maps will be a useful resource in research on radish and should lead to low-cost, accurate, and high-throughput genotyping platforms.

Benchmarking database systems for Genomic Selection implementation.

MotivationWith high-throughput genotyping systems now available, it has become feasible to fully integrate genotyping information into breeding programs. To make use of this information effectively requires DNA extraction facilities and marker production facilities that can efficiently deploy the desired set of markers across samples with a rapid turnaround time that allows for selection before crosses needed to be made. In reality, breeders often have a short window of time to make decisions by the time they are able to collect all their phenotyping data and receive corresponding genotyping data. This presents a challenge to organize information and utilize it in downstream analyses to support decisions made by breeders. In order to implement genomic selection routinely as part of breeding programs, one would need an efficient genotyping data storage system. We selected and benchmarked six popular open-source data storage systems, including relational database management and columnar storage systems.ResultsWe found that data extract times are greatly influenced by the orientation in which genotype data is stored in a system. HDF5 consistently performed best, in part because it can more efficiently work with both orientations of the allele matrix.Availability http://gobiin1.bti.cornell.edu:6083/projects/GBM/repos/benchmarking/browse

A high-throughput genotyping system combining rapid DNA extraction and high-resolution melting analysis in allo-octoploid strawberry

A high-throughput genotyping system combining rapid DNA extraction and high-resolution melting analysis in allo-octoploid strawberry

A System for Distinguishing Octoploid Strawberry Cultivars Using High-Throughput SNP Genotyping

Cultivated strawberry (Fragaria × ananassa) is an important commercial berry crop grown throughout the world. Improved strawberry cultivars are developed to meet the needs of consumers and breeders. Strawberries are usually propagated through runners, which sometimes lead to mislabeling or misinterpretation of cultivars. However, perfect identification of strawberry cultivars is essential for germplasm maintenance and for breeding programs. Molecular marker technology has been widely used to distinguish cultivars of other crops, but marker development in octoploid strawberries is complicated. Therefore, SNP marker with high-density and even distribution in the genome has been used currently as efficient DNA markers. In this report, previously published high-quality poly high resolution (PHR) SNPs from the 90 K Axiom® SNP array were utilized to develop a Fluidigm 24 SNPs genotyping system. Hundred nine (109) octoploid strawberry cultivars were screened using this 24 SNPs chip set. In addition, 24 SNPs were mapped to six chromosomes of diploid strawberry (Fragaria vesca). Our developed SNPs fluidigm genotyping is automatable, easy and reliable for processing and interpretation of data. Thus, this high-throughput SNP genotyping system will be a useful tool for distinguishing strawberry cultivars and find out parent-offspring relationship.

Diversity analysis of cotton (Gossypium hirsutum L.) germplasm using the CottonSNP63K Array

BackgroundCotton germplasm resources contain beneficial alleles that can be exploited to develop germplasm adapted to emerging environmental and climate conditions. Accessions and lines have traditionally been characterized based on phenotypes, but phenotypic profiles are limited by the cost, time, and space required to make visual observations and measurements. With advances in molecular genetic methods, genotypic profiles are increasingly able to identify differences among accessions due to the larger number of genetic markers that can be measured. A combination of both methods would greatly enhance our ability to characterize germplasm resources. Recent efforts have culminated in the identification of sufficient SNP markers to establish high-throughput genotyping systems, such as the CottonSNP63K array, which enables a researcher to efficiently analyze large numbers of SNP markers and obtain highly repeatable results. In the current investigation, we have utilized the SNP array for analyzing genetic diversity primarily among cotton cultivars, making comparisons to SSR-based phylogenetic analyses, and identifying loci associated with seed nutritional traits.ResultsThe SNP markers distinctly separated G. hirsutum from other Gossypium species and distinguished the wild from cultivated types of G. hirsutum. The markers also efficiently discerned differences among cultivars, which was the primary goal when designing the CottonSNP63K array. Population structure within the genus compared favorably with previous results obtained using SSR markers, and an association study identified loci linked to factors that affect cottonseed protein content.ConclusionsOur results provide a large genome-wide variation data set for primarily cultivated cotton. Thousands of SNPs in representative cotton genotypes provide an opportunity to finely discriminate among cultivated cotton from around the world. The SNPs will be relevant as dense markers of genome variation for association mapping approaches aimed at correlating molecular polymorphisms with variation in phenotypic traits, as well as for molecular breeding approaches in cotton.

An in-house multilocus SNP genotyping assay for evaluation of complex genetic diseases

Background: With an increase in the discovery of newer genetic loci/polymorphisms in complex multifactorial diseases, there is also an increased need for methods that can simultaneously genotype multiple loci in a cost-effective manner. Using coronary artery disease (CAD) as a model, the study aimed to develop an in-house multilocus assay for simultaneous detection of 17 genetic variants in 11 genes implicated in CAD.Methods: A multiplex polymerase chain reaction (PCR)-based reverse line blot hybridization (MPCR-RLBH) approach was used, where each DNA sample was amplified using two separate MPCRs, and the alleles were genotyped using covalently immobilized, amino-linked sequence-specific oligonucleotide probes using an enhanced chemiluminescence system. The assay performance was tested on 75 healthy controls and 75 angiographically proven CAD cases. Validation was done by automated Sanger sequencing.Results: The assay could successfully discriminate both the alleles at CETP (I405V), LPL (D9N), NOS3 (T-786G and E298D), LIPC (C-514T), FGB (G-455A), ITGB3 (L33P), AGT (M235T), and MTR (A2756G) loci. Certain mutations included in this assay such as ins242G, ins397G, E387K, L393K in the LDLR; N291S in the LPL; D442G in the CETP; and T833C in the CBS genes were found to be absent. The genotype results obtained using this assay showed 100% concordance with sequencing.Conclusion: The study demonstrated development and validation of a multiplex SNP genotyping assay that can be used to assess genetic risk factors in CAD. The assay provides a cost-effective alternative to expensive high throughput genotyping systems in common molecular research laboratories.

Gen dan QTL Pengendali Toleransi Tanaman terhadap Keracunan Aluminium dan Aplikasinya untuk Pemuliaan Tanaman di Indonesia

<p>Genetic knowledge of loci controlling Al toxicity tolerance is the key for a successful breeding program in developing Al<br />tolerant cultivars. Tolerance level of crop plants to Al toxicity is genetically controlled. The gene inheritance pattern is mainly<br />resulted from intensive studies of cereal crops, such as wheat, sorghum, maize, and rice. The trait can be controlled by a<br />single dominant gene, a single dominant gene with many alleles, a pair of dominant genes, or by many genes (QTL). The<br />majority of the Al tolerance genes identified so far belongs to two independent groups of gene families, i.e. aluminumactivated<br />malate transporter (ALMT) and multidrug and toxic compound extrusion (MATE), both encoding transport proteins<br />involved in Al-activated organic acid release, mainly citrate and malate. The variations in Al toxicity tolerance phenotypes are<br />strongly correlated with the expressions of such genes in the root apical cells. Many Al tolerance QTLs have been mapped in<br />the genomes of various crop species and were found to be colocated with the ALMT and MATE genes. The genetic maps of<br />the Al tolerance genes and QTLs facilitate breeding programs for developing Al-tolerant cultivars through marker-assisted<br />breeding methods. Al tolerance genes that have been isolated from genetically unrelated species can be used in genetic<br />transformation studies of crop genotypes sexually incompatible to the gene source genotypes. The application of these<br />molecular breeding methods expedites breeding programs to develop crop cultivars tolerance to Al toxicity and acid soils.<br />Genomic technologies by using next-generation sequencing and high-throughput genotyping system accelerate Al toxicity<br />tolerance gene and QTL discoveries of various crop species. The modern genomic technologies also facilitate more<br />comprehensive PGR characterization and utilization to accelerate identification and isolation of the Al tolerance genes and<br />QTLs to be used in a more comprehensive breeding program to support national food self sufficiency and food security<br />programs.</p>

Development of an HRM-based, safe and high-throughput genotyping system for two low phytic acid mutations in soybean

Two low phytic acid (lpa) mutants, Gm-lpa-ZC-2 (ZC-lpa) and Gm-lpa-TW-1 (TW-lpa), resulting from a G → A mutation in GmIPK1 and a 2-bp deletion in GmMIPS1, respectively, were previously developed to increase the nutritional value and environmental friendliness of soybean meal. Two functional CAPS markers were subsequently developed for genotyping plants carrying the two mutant genes; however, both are costly and time consuming and hence unsuitable for large-scale breeding use. In the present work, by integrating a quick DNA extraction protocol with an optimized high-resolution melting curve (HRM) analysis, we developed a fast and high-throughput genotyping system for the two mutations. In this system, (1) DNAs are extracted within half an hour using a protocol that only requires freezing and heating of leaf disks in two non-toxic solutions and can be directly used for PCR; (2) for genotyping, asymmetric PCRs with competitive primers are performed, and the samples are then discriminated and grouped through HRM analysis; and (3) all steps are performed in a 96-well plate, and hence adaptable to high-throughput genotyping. Although the system was developed for two lpa mutations, the general principle should be applicable to any other genes in soybean.

Marker-assisted backcross breeding for development of pepper varieties (Capsicum annuum) containing capsinoids

Capsinoids have similar biological effects as capsaicinoids, including anticancer and anti-obesity properties. The Capsicum chinense ‘SNU11-001’ variety of pepper was previously reported to contain high levels of capsinoids due to a mutation in the putative aminotransferase (pAMT) gene, which leads to production of the capsinoid precursor vanillyl alcohol. Here, to develop Capsicum annuum pepper varieties with high capsinoid contents, marker-assisted backcrossing was performed during backcross breeding. For foreground selection, plants carrying the pAMT/pamt genotype were selected from BC1F1 and BC2F1 populations using SCAR markers derived from the unique pamt mutation of ‘SNU11-001.’ To obtain background selection markers, 412 single nucleotide polymorphism (SNP) markers were screened to identify SNP markers polymorphic between the ‘Shinhong’ paternal lines and ‘SNU11-001.’ Of the 412 SNP markers, 204 polymorphic SNP markers evenly distributed in pepper genome were selected. BC1F1 and BC2F1 plants carrying the pAMT/pamt genotype were subjected to background selection using these SNP markers. Multiple genotypes were analyzed using a high-throughput genotyping system. As a result, one BC1F1 plant 84 % similar to the recurrent parent and seven BC2F1 plants showing more than 96 % recovery of the recurrent parent genotype were selected. Genetic backgrounds of the selected BC2F1 plants were evaluated using the genotype-by-sequencing (GBS) method to confirm the background selection results from the SNP marker set. GBS results showed that the recovery rate and positions of introgressed segments were well matched between two methods, demonstrating MABC can be successfully performed with only ~200 SNP markers.

Abstract 4569: A simple method to screen patients for SNPs in NAT1 gene for prostate cancer risk

Abstract Single nucleotide polymorphisms (SNPs) are single base differences in DNA and are suitable for genotyping markers of risk in human disease. With emerging bio-technology and a cross-platform application of techniques improvements can be made in methodology. Although a number of high-throughput SNP genotyping systems are available the technology is expensive for it requires both trained personnel and dedicated platforms. The method reported here is a simple qPCR based High Resolution Method (HRM) based method and can be cost effectively used in screening patients in a general molecular biology and clinical laboratories. The NAT1 gene directs N-acetylation and O-acetylation of toxins including heterocyclic amines through the phase II xenobiotic metabolizing enzymes toxicity pathway. The purpose was to develop a screening tool to screen individuals with SNPs at locations 190, 445, 560 and 640. A modified qPCR approach was used with HRM analyses. Primers were designed using an in-house protocol; the rs sequence for the site-specific mutation was obtained from SNP database and fed to Primer Quest and having diligently identified the location of the mutation primers were designed and assessed using OligoAnalyzer Tool. The Tm for the forward and reverse were kept as close as possible and G was kept between -1 and -9 and the GC content greater than 50%. The following sequences were used rs58379106 (190C>T); rs4987076 (445G>A); rs4986782 (560G>A) and rs4986783 (640T>G). A RT qPCR 10μl reaction was designed using iTAQ Supermix (BioRad, CA), forward and reverse primers and genomic DNA obtained from prostate cancer cases and controls. Standard cycling conditions described for iTAQ were observed for 44 cycles. The data collected was analysed using HRM software (BioRad, CA) as per the guidelines described by the software. The data shows that when all the conditions are normalized and benchmarked the shape of the HRM curves are typical and characteristic of the SNP. The figures show the symmetry and similarity in the HRM. The similarity from the standard curve (Fig.1) and standard curve with unknown SNP (Fig. 2) and curves with a complementary SNP at 445 along with SNP at 640 (Fig. 3). Fig. 1 Std. curve with NAT1640 Fig. 2 Std. curve with unknown samples Fig. 3 Unknown samples with 640 and 445 This data shows that with this approach it is possible to identify site specific mutation and the presence of other mutation in the vicinity of the SNP of interest. The presence or absence of mutations among cases and controls can be assessed using this approach. Note: This abstract was not presented at the meeting. Citation Format: James Gomes, Melody Emaimem, Maja Zuric, Maitland Long. A simple method to screen patients for SNPs in NAT1 gene for prostate cancer risk. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4569. doi:10.1158/1538-7445.AM2015-4569

English

Accurate identification of individual genotypes is important for cacao ( Theobroma cacao L.) breeding, germplasm conservation and seed propagation. The development of single nucleotide polymorphism (SNP) markers in cacao offers an effective way to use a high-throughput genotyping system for cacao genotype verification. In the present study, high-throughput genotyping with SNP markers was used to fingerprint 160 cacao trees in the germplasm collection at the Cocoa Research Institute of Ghana (CRIG). These accessions had been originally introduced from international germplasm collections. The multilocus SNP profiles, generated by the Sequenom Mass Spectrometry platform, were compared with the SNP profiles of reference trees maintained in the international cacao collections. The comparison unambiguously identified mislabeled trees. For materials introduced as hybrid seeds without an available reference genotype, parentage analysis and model-based assignment were applied to verify their recorded parentage and genetic background. Our study shows that a small set of polymorphic SNP markers can provide a robust and accurate result for cacao genotype identification. This protocol can be applied for large-scale genotyping of cacao as well as for many other crops. Keywords: Cacao, conservation, chocolate, DNA fingerprint, molecular marker, tropical plant, off-type, true-to-type, West Africa. African Journal of Biotechnology , Vol 13(21), 2127-2136

Genomic regions involved in yield potential detected by genome-wide association analysis in Japanese high-yielding rice cultivars.

BackgroundHigh-yielding cultivars of rice (Oryza sativa L.) have been developed in Japan from crosses between overseas indica and domestic japonica cultivars. Recently, next-generation sequencing technology and high-throughput genotyping systems have shown many single-nucleotide polymorphisms (SNPs) that are proving useful for detailed analysis of genome composition. These SNPs can be used in genome-wide association studies to detect candidate genome regions associated with economically important traits. In this study, we used a custom SNP set to identify introgressed chromosomal regions in a set of high-yielding Japanese rice cultivars, and we performed an association study to identify genome regions associated with yield.ResultsAn informative set of 1152 SNPs was established by screening 14 high-yielding or primary ancestral cultivars for 5760 validated SNPs. Analysis of the population structure of high-yielding cultivars showed three genome types: japonica-type, indica-type and a mixture of the two. SNP allele frequencies showed several regions derived predominantly from one of the two parental genome types. Distinct regions skewed for the presence of parental alleles were observed on chromosomes 1, 2, 7, 8, 11 and 12 (indica) and on chromosomes 1, 2 and 6 (japonica). A possible relationship between these introgressed regions and six yield traits (blast susceptibility, heading date, length of unhusked seeds, number of panicles, surface area of unhusked seeds and 1000-grain weight) was detected in eight genome regions dominated by alleles of one parental origin. Two of these regions were near Ghd7, a heading date locus, and Pi-ta, a blast resistance locus. The allele types (i.e., japonica or indica) of significant SNPs coincided with those previously reported for candidate genes Ghd7 and Pi-ta.ConclusionsIntrogression breeding is an established strategy for the accumulation of QTLs and genes controlling high yield. Our custom SNP set is an effective tool for the identification of introgressed genome regions from a particular genetic background. This study demonstrates that changes in genome structure occurred during artificial selection for high yield, and provides information on several genomic regions associated with yield performance.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-346) contains supplementary material, which is available to authorized users.

HapRice, an SNP Haplotype Database and a Web Tool for Rice

Genome-wide single nucleotide polymorphism (SNP) analysis is a promising tool to examine the genetic diversity of rice populations and genetic traits of scientific and economic importance. Next-generation sequencing technology has accelerated the re-sequencing of diverse rice varieties and the discovery of genome-wide SNPs. Notably, validation of these SNPs by a high-throughput genotyping system, such as an SNP array, could provide a manageable and highly accurate SNP set. To enhance the potential utility of genome-wide SNPs for geneticists and breeders, analysis tools need to be developed. Here, we constructed an SNP haplotype database, which allows visualization of the allele frequency of all SNPs in the genome browser. We calculated the allele frequencies of 3,334 SNPs in 76 accessions from the world rice collection and 3,252 SNPs in 177 Japanese rice accessions; all these SNPs have been validated in our previous studies. The SNP haplotypes were defined by the allele frequency in each cultivar group (aus, indica, tropical japonica and temperate japonica) for the world rice accessions, and in non-irrigated and three irrigated groups (three variety registration periods) for Japanese rice accessions. We also developed web tools for finding polymorphic SNPs between any two rice accessions and for the primer design to develop cleaved amplified polymorphic sequence markers at any SNP. The 'HapRice' database and the web tools can be accessed at http://qtaro.abr.affrc.go.jp/index.html. In addition, we established a core SNP set consisting of 768 SNPs uniformly distributed in the rice genome; this set is of a practically appropriate size for use in rice genetic analysis.