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

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

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.     

Multiplexed,linkage group-specific SNP marker sets for rapid genetic mapping and fingerprinting of sugar beet (<Emphasis Type="Italic">Beta vulgaris</Emphasis> L.)

A set of single nucleotide polymorphism (SNP) markers has been developed for each of the nine linkage groups of sugar beet. Each set can monitor the polymorphic state at five to six linked marker loci. In each set, the loci selected for marker development are first amplified in a multiplexed reaction. These amplification products are the basis for sequence-specific elongation of primers adjacent to SNP positions. The extension step revealing SNP loci is based on fluorescently labelled nucleotides. In each set, primers developed to reveal SNP alleles differ in length to allow clear peak resolution in capillary electrophoresis. The nine linkage group (LG) –specific sets provide information on the polymorphism at a total of 52 SNP marker loci. Using the SNP-based tool, groups of concerned loci have been anchored to three different linkage maps of sugar beet. In a second experiment, sugar beet breeding lines have been fingerprinted. The use of the nine sets of LG-specific markers in sugar beet genetics and breeding is discussed. The information necessary to specify the 52 marker loci, as well as their map location, and all details concerning SNP assays, including allele type and nature of mutation, are reported.     

Mining single-nucleotide polymorphisms from hexaploid wheat ESTs.

Single-nucleotide polymorphisms (SNPs) represent a new form of functional marker, particularly when they are derived from expressed sequence tags (ESTs). A bioinformatics strategy was developed to discover SNPs within a large wheat EST database and to demonstrate the utility of SNPs in genetic mapping and genetic diversity applications. A collection of > 90000 wheat ESTs was assembled into contiguous sequences (contigs), and 45 random contigs were then visually inspected to identify primer pairs capable of amplifying specific alleles. We estimate that homoeologue sequence variants occurred 1 in 24 bp and the frequency of SNPs between wheat genotypes was 1 SNP/540 bp (theta = 0.0069). Furthermore, we estimate that one diagnostic SNP test can be developed from every contig with 10-60 EST members. Thus, EST databases are an abundant source of SNP markers. Polymorphism information content for SNPs ranged from 0.04 to 0.50 and ESTs could be mapped into a framework of microsatellite markers using segregating populations. The results showed that SNPs in wheat can be discovered in ESTs, validated, and be applied to conventional genetic studies.     

Development and validation of KASP markers for the greenbug resistance gene <Emphasis Type="Italic">Gb7</Emphasis> and the Hessian fly resistance gene <Emphasis Type="Italic">H32</Emphasis> in wheat

Key message

Greenbug and Hessian fly are important pests that decrease wheat production worldwide. We developed and validated breeder-friendly KASP markers for marker-assisted breeding to increase selection efficiency.

Abstract

Greenbug (Schizaphis graminum Rondani) and Hessian fly [Mayetiola destructor (Say)] are two major destructive insect pests of wheat (Triticum aestivum L.) throughout wheat production regions in the USA and worldwide. Greenbug and Hessian fly infestation can significantly reduce grain yield and quality. Breeding for resistance to these two pests using marker-assisted selection (MAS) is the most economical strategy to minimize losses. In this study, doubled haploid lines from the Synthetic W7984 × Opata M85 wheat reference population were used to construct linkage maps for the greenbug resistance gene Gb7 and the Hessian fly resistance gene H32 with genotyping-by-sequencing (GBS) and 90K array-based single nucleotide polymorphism (SNP) marker data. Flanking markers were closely linked to Gb7 and H32 and were located on chromosome 7DL and 3DL, respectively. Gb7-linked markers (synopGBS773 and synopGBS1141) and H32-linked markers (synopGBS901 and IWB65911) were converted into Kompetitive Allele Specific PCR (KASP) assays for MAS in wheat breeding. In addition, comparative mapping identified syntenic regions in Brachypodium distachyon, rice (Oryza sativa), and sorghum (Sorghum bicolor) for Gb7 and H32 that can be used for fine mapping and map-based cloning of the genes. The KASP markers developed in this study are the first set of SNPs tightly linked to Gb7 and H32 and will be very useful for MAS in wheat breeding programs and future genetic studies of greenbug and Hessian fly resistance.

     

An integrative AmpSeq platform for highly multiplexed marker-assisted pyramiding of grapevine powdery mildew resistance loci

Resistance breeding often requires the introgression and tracking of resistance loci from wild species into domesticated backgrounds, typically with the goal of pyramiding multiple resistance genes, to provide durable disease resistance to breeding selections and ultimately cultivars. While molecular markers are commonly used to facilitate these efforts, high genetic diversity and divergent marker technologies can complicate marker-assisted breeding strategies. Here, amplicon sequencing (AmpSeq) was used to integrate SNP markers with dominant presence/absence markers derived from genotyping-by-sequencing and other genotyping technologies, for the simultaneous tracking of five loci for resistance to grapevine powdery mildew. SNP haploblocks defined the loci for REN1, REN2 and REN3, which confer quantitative resistance phenotypes that are challenging to measure via field ratings of natural infections. Presence/absence markers for RUN1 and REN4 were validated to predict qualitative resistance phenotypes and corresponded with previous presence/absence fluorescent electrophoretic assays. Thus, 37 AmpSeq-derived markers were identified for the five loci, and markers for REN1, REN2, REN4 and RUN1 were used for multiplexed screening and selection within diverse breeding germplasm. Poor transferability of SNP markers indicated imperfect marker-trait association in some families. Together, AmpSeq SNP haploblocks and presence/absence markers provide a high-throughput, cost-effective tool to integrate divergent technologies for marker-assisted selection and genetic analysis of introgressed disease resistance loci in grapevine.     

Single nucleotide polymorphism in wheat chromosome region harboring Fhb1 for Fusarium head blight resistance

Fusarium head blight (FHB) is a destructive disease that reduces wheat grain yield and quality. To date, the quantitative trait locus on 3BS (Fhb1) from Sumai 3 has shown the largest effect on FHB resistance. Single nucleotide polymorphism (SNP) is the most common form of genetic variation and is suitable for high-throughput marker-assisted selection (MAS). We analyzed SNPs derived from 23 wheat expressed sequence tags (ESTs) that previously mapped near Fhb1 on chromosome 3BS. Using 71 Ning 7840/Clark BC7F7 recombinant inbred lines and the single-base extension method, we mapped seven SNP markers between Xgwm533 and Xgwm493, flanking markers for Fhb1. Five of the SNPs explained 45–54% of the phenotypic variation for FHB resistance. Haplotype analysis of 63 wheat accessions from eight countries based on SNPs in EST sequences, simple sequence repeats, and sequence tagged sites in the Fhb1 region identified four major groups: (1) US-Clark, (2) Asian, (3) US-Ernie, and (4) Chinese Spring. The Asian group consisted of Chinese and Japanese accessions that carry Fhb1 and could be differentiated from other groups by marker Xsnp3BS-11. All Sumai 3-related accessions formed a subgroup within the Asian group and could be sorted out by Xsnp3BS-8. The SNP markers identified in this study should be useful for MAS of Fhb1 and fine mapping to facilitate cloning of the Fhb1 resistance gene.     

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.     

Identification of a molecular marker tightly linked to bacterial wilt resistance in tomato by genome-wide SNP analysis

Key message

Genotyping of disease resistance to bacterial wilt in tomato by a genome-wide SNP analysis

Abstract

Bacterial wilt caused by Ralstonia pseudosolanacearum is one of the destructive diseases in tomato. The previous studies have identified Bwr-6 (chromosome 6) and Bwr-12 (chromosome 12) loci as the major quantitative trait loci (QTLs) contributing to resistance against bacterial wilt in tomato cultivar ‘Hawaii7996’. However, the genetic identities of two QTLs have not been uncovered yet. In this study, using whole-genome resequencing, we analyzed genome-wide single-nucleotide polymorphisms (SNPs) that can distinguish a resistant group, including seven tomato varieties resistant to bacterial wilt, from a susceptible group, including two susceptible to the same disease. In total, 5259 non-synonymous SNPs were found between the two groups. Among them, only 265 SNPs were located in the coding DNA sequences, and the majority of these SNPs were located on chromosomes 6 and 12. The genes that both carry SNP(s) and are near Bwr-6 and Bwr-12 were selected. In particular, four genes in chromosome 12 encode putative leucine-rich repeat (LRR) receptor-like proteins. SNPs within these four genes were used to develop SNP markers, and each SNP marker was validated by a high-resolution melting method. Consequently, one SNP marker, including a functional SNP in a gene, Solyc12g009690.1, could efficiently distinguish tomato varieties resistant to bacterial wilt from susceptible varieties. These results indicate that Solyc12g009690.1, the gene encoding a putative LRR receptor-like protein, might be tightly linked to Bwr-12, and the SNP marker developed in this study will be useful for selection of tomato cultivars resistant to bacterial wilt.

     

Genetic mapping of the wheat dimeric α-amylase inhibitor multi-gene family using allele-specific primers based on intergenomic SNPs

Single nucleotide polymorphisms (SNPs) identified in EST sequences can be used to map expressed genes. Though SNPs are useful markers for genetic mapping, SNP mapping of genes in common wheat is challenging because the genetic complement of wheat consists of three closely related genomes (designated A, B, and D), and most genes are present in triplicate sets. Mapping multi-gene family members is further complicated by the fact that it is difficult to distinguish SNP differences between the various paralogs from those between the different genomes. We have developed a PCR-based method for assigning wheat EST sequences to their proper genetic loci by first identifying and mapping SNPs that distinguish the three genomes. To develop this method, we focused on EST sequences encoding the dimeric α-amylase inhibitors (WDAI), The WDAI coding regions of hexaploid wheat were aligned. The sequences were classified into three groups based on nucleotide variations. Twenty-two SNPs were identified that distinguish the three groups. Group-specific primers based on these SNPs were designed to permit selective amplification of each group. The chromosomal location of each group was then determined using Group 3 ditelosomic lines of Chinese Spring. Groups 1 and 2 were assigned to chromosome locations 3DS and 3BS, respectively, whereas no sequence could be assigned to 3AS. A remarkable feature of this method is the ability to discriminate the location of homoeologous multigenes in the three genomes of wheat. This strategy can be useful for assigning unknown wheat EST sequences to specific chromosomes.     

Establishment of a high-efficiency SNP-based framework marker set for Arabidopsis

The major goal of this project was the establishment of a tool for rapid mapping of new mutations and genotyping in Arabidopsis consisting of at least 100 evenly spaced framework markers. We assembled a single nucleotide polymorphism (SNP)-based marker set consisting of 112 polymorphic sites with average spacing of 1.15 Mbp derived from an SNP database that we recently developed. This information was used to set up efficient SNP detection reactions based on multiplexed primer extension assays. The 112 Columbia (Col-0)/C24 framework markers were used to assemble 18 multiplexed SNaPshot assays with which up to eight separate loci can be genotyped in a single-tube/single-capillary format. In addition, for 110 framework markers matrix-assisted laser desorption/ionization time of flight (MALDI-ToF) assays have been established for high throughput analyses. We demonstrated the usefulness and the robustness of both procedures of this tool by genotyping 48 BC3F1 individuals created between the accessions Col-0 and C24. Subsets of 10-62 of the established markers discriminate between various combinations of the accessions Col-0, C24, Landsberg erecta (Ler), Cape Verdi Islands (Cvi) and Niederzenz (Nd). Using a subset of 17 evenly distributed and established SNP markers that are also polymorphic between Ler and Col-0, we were able to rapidly map a mutant gene (tbr1) to an interval of 2.3 Mbp in an Ler (tbr1) x Col-0 cross.     

SNP identification and marker assay development for high-throughput selection of soybean cyst nematode resistance

Background

Soybean cyst nematode (SCN) is the most economically devastating pathogen of soybean. Two resistance loci, Rhg1 and Rhg4 primarily contribute resistance to SCN race 3 in soybean. Peking and PI 88788 are the two major sources of SCN resistance with Peking requiring both Rhg1 and Rhg4 alleles and PI 88788 only the Rhg1 allele. Although simple sequence repeat (SSR) markers have been reported for both loci, they are linked markers and limited to be applied in breeding programs due to accuracy, throughput and cost of detection methods. The objectives of this study were to develop robust functional marker assays for high-throughput selection of SCN resistance and to differentiate the sources of resistance.

Results

Based on the genomic DNA sequences of 27 soybean lines with known SCN phenotypes, we have developed Kompetitive Allele Specific PCR (KASP) assays for two Single nucleotide polymorphisms (SNPs) from Glyma08g11490 for the selection of the Rhg4 resistance allele. Moreover, the genomic DNA of Glyma18g02590 at the Rhg1 locus from 11 soybean lines and cDNA of Forrest, Essex, Williams 82 and PI 88788 were fully sequenced. Pairwise sequence alignment revealed seven SNPs/insertion/deletions (InDels), five in the 6th exon and two in the last exon. Using the same 27 soybean lines, we identified one SNP that can be used to select the Rhg1 resistance allele and another SNP that can be employed to differentiate Peking and PI 88788-type resistance. These SNP markers have been validated and a strong correlation was observed between the SNP genotypes and reactions to SCN race 3 using a panel of 153 soybean lines, as well as a bi-parental population, F₅–derived recombinant inbred lines (RILs) from G00-3213 x LG04-6000.

Conclusions

Three functional SNP markers (two for Rhg1 locus and one for Rhg4 locus) were identified that could provide genotype information for the selection of SCN resistance and differentiate Peking from PI 88788 source for most germplasm lines. The robust KASP SNP marker assays were developed. In most contexts, use of one or two of these markers is sufficient for high-throughput marker-assisted selection of plants that will exhibit SCN resistance.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1531-3) contains supplementary material, which is available to authorized users.     

Use of VeraCode 384-plex assays for watermelon diversity analysis and integrated genetic map of watermelon with single nucleotide polymorphisms and simple sequence repeats

Watermelon (Citrullus lanatus var. lanatus) is one of the most important vegetable crops in the world. Molecular markers have become the tools of choice for resolving watermelon taxonomic relationships and evolution. Increased numbers of single nucleotide polymorphism (SNP) markers together with simple sequence repeat (SSR) markers would be useful for phylogenetic analyses of germplasm accessions and for linkage mapping for marker-assisted breeding with quantitative trait loci and single genes. We aimed to construct a genetic map based on SNPs (generated by Illumina Veracode multiplex assays for genotyping) and SSR markers and evaluate relationships inferred from SNP genotypes between 130 watermelon accessions collected throughout the world. We incorporated 282 markers (232 SNPs and 50 SSRs) into the linkage map. The genetic map consisted of 11 linkage groups spanning 924.72 cM with an average distance of 3.28 cM between markers. Because all of the SNP-containing sequences were assembled with the whole-genome sequence draft for watermelon, chromosome numbers could be readily assigned for all the linkage groups. We found that 134 SNPs were polymorphic in 130 watermelon accessions chosen for diversity studies. The current 384-plex SNP set is a powerful tool for characterizing genetic relatedness and for developing medium-resolution genetic maps.     

A high-throughput SNP marker system for parental polymorphism screening, and diversity analysis in common bean (Phaseolus vulgaris L.)

Single nucleotide polymorphism (SNP) detection has become a marker system of choice, because of the high abundance of source polymorphisms and the ease with which allele calls are automated. Various technologies exist for the evaluation of SNP loci and previously we validated two medium throughput technologies. In this study, our goal was to utilize a 768 feature, Illumina GoldenGate assay for common bean (Phaseolus vulgaris L.) developed from conserved legume gene sequences and to use the new technology for (1) the evaluation of parental polymorphisms in a mini-core set of common bean accessions and (2) the analysis of genetic diversity in the crop. A total of 736 SNPs were scored on 236 diverse common bean genotypes with the GoldenGate array. Missing data and heterozygosity levels were low and 94 % of the SNPs were scorable. With the evaluation of the parental polymorphism genotypes, we estimated the utility of the SNP markers in mapping for inter-genepool and intra-genepool populations, the latter being of lower polymorphism than the former. When we performed the diversity analysis with the diverse genotypes, we found Illumina GoldenGate SNPs to provide equivalent evaluations as previous gene-based SNP markers, but less fine-distinctions than with previous microsatellite marker analysis. We did find, however, that the gene-based SNPs in the GoldenGate array had some utility in race structure analysis despite the low polymorphism. Furthermore the SNPs detected high heterozygosity in wild accessions which was probably a reflection of ascertainment bias. The Illumina SNPs were shown to be effective in distinguishing between the genepools, and therefore were most useful in saturation of inter-genepool genetic maps. The implications of these results for breeding in common bean are discussed as well as the advantages and disadvantages of the GoldenGate system for SNP detection.     

Development of EST-based SNP and InDel markers and their utilization in tetraploid cotton genetic mapping

Background

Availability of molecular markers has proven to be an efficient tool in facilitating progress in plant breeding, which is particularly important in the case of less researched crops such as cotton. Considering the obvious advantages of single nucleotide polymorphisms (SNPs) and insertion-deletion polymorphisms (InDels), expressed sequence tags (ESTs) were analyzed in silico to identify SNPs and InDels in this study, aiming to develop more molecular markers in cotton.

Results

A total of 1,349 EST-based SNP and InDel markers were developed by comparing ESTs between Gossypium hirsutum and G. barbadense, mining G. hirsutum unigenes, and analyzing 3′ untranslated region (3′UTR) sequences. The marker polymorphisms were investigated using the two parents of the mapping population based on the single-strand conformation polymorphism (SSCP) analysis. Of all the markers, 137 (10.16%) were polymorphic, and revealed 142 loci. Linkage analysis using a BC₁ population mapped 133 loci on the 26 chromosomes. Statistical analysis of base variations in SNPs showed that base transitions accounted for 55.78% of the total base variations and gene ontology indicated that cotton genes varied greatly in harboring SNPs ranging from 1.00 to 24.00 SNPs per gene. Sanger sequencing of three randomly selected SNP markers revealed discrepancy between the in silico predicted sequences and the actual sequencing results.

Conclusions

In silico analysis is a double-edged blade to develop EST-SNP/InDel markers. On the one hand, the designed markers can be well used in tetraploid cotton genetic mapping. And it plays a certain role in revealing transition preference and SNP frequency of cotton genes. On the other hand, the developmental efficiency of markers and polymorphism of designed primers are comparatively low.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1046) contains supplementary material, which is available to authorized users.     

Accuracy of marker-assisted selection with single markers and marker haplotypes in cattle

A key question for the implementation of marker-assisted selection (MAS) using markers in linkage disequilibrium with quantitative trait loci (QTLs) is how many markers surrounding each QTL should be used to ensure the marker or marker haplotypes are in sufficient linkage disequilibrium (LD) with the QTL. In this paper we compare the accuracy of MAS using either single markers or marker haplotypes in an Angus cattle data set consisting of 9323 genome-wide single nucleotide polymorphisms (SNPs) genotyped in 379 Angus cattle. The extent of LD in the data set was such that the average marker-marker r2 was 0.2 at 200 kb. The accuracy of MAS increased as the number of markers in the haplotype surrounding the QTL increased, although only when the number of markers in the haplotype was 4 or greater did the accuracy exceed that achieved when the SNP in the highest LD with the QTL was used. A large number of phenotypic records (>1000) were required to accurately estimate the effects of the haplotypes.     

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.     

Development of single nucleotide polymorphism (SNP) markers for use in commercial maize (Zea mays L.) germplasm

The development of single nucleotide polymorphism (SNP) markers in maize offers the opportunity to utilize DNA markers in many new areas of population genetics, gene discovery, plant breeding and germplasm identification. However, the steps from sequencing and SNP discovery to SNP marker design and validation are lengthy and expensive. Access to a set of validated SNP markers is a significant advantage to maize researchers who wish to apply SNPs in scientific inquiry. We mined 1,088 loci sequenced across 60 public inbreds that have been used in maize breeding in North America and Europe. We then selected 640 SNPs using generalized marker design criteria that enable utilization with several SNP chemistries. While SNPs were found on average every 43 bases in 1,088 maize gene sequences, SNPs that were amenable to marker design were found on average every 623 bases; representing only 7% of the total SNPs discovered. We also describe the development of a 768 marker multiplex assay for use on the Illumina^® BeadArray? platform. SNP markers were mapped on the IBM2 intermated B73 × Mo17 high resolution genetic map using either the IBM2 segregating population, or segregation in multiple parent-progeny triplets. A high degree of colinearity was found with the genetic nested association map. For each SNP presented we give information on map location, polymorphism rates in different heterotic groups and performance on the Illumina^® platform.     

Single nucleotide polymorphisms for pig identification and parentage exclusion

Single nucleotide polymorphisms (SNPs) have become an important type of marker for commercial diagnostic and parentage genotyping applications as automated genotyping systems have been developed that yield accurate genotypes. Unfortunately, allele frequencies for public SNP markers in commercial pig populations have not been available. To fulfil this need, SNP markers previously mapped in the USMARC swine reference population were tested in a panel of 155 boars that were representative of US purebred Duroc, Hampshire, Landrace and Yorkshire populations. Multiplex assay groups of 5-7 SNP assays/group were designed and genotypes were determined using Sequenom's massarray system. Of 80 SNPs that were evaluated, 60 SNPs with minor allele frequencies >0.15 were selected for the final panel of markers. Overall identity power across breeds was 4.6 x 10(-23), but within-breed values ranged from 4.3 x 10(-14) (Hampshire) to 2.6 x 10(-22) (Yorkshire). Parentage exclusion probability with only one sampled parent was 0.9974 (all data) and ranged from 0.9594 (Hampshire) to 0.9963 (Yorkshire) within breeds. Sire exclusion probability when the dam's genotype was known was 0.99998 (all data) and ranged from 0.99868 (Hampshire) to 0.99997 (Yorkshire) within breeds. Power of exclusion was compared between the 60 SNP and 10 microsatellite markers. The parental exclusion probabilities for SNP and microsatellite marker panels were similar, but the SNP panel was much more sensitive for individual identification. This panel of SNP markers is theoretically sufficient for individual identification of any pig in the world and is publicly available.