Use Of Single Nucleotide Polymorphism Markers Research Articles

The use of SNP markers for cattle breed identification.

A potential application of single nucleotide polymorphisms (SNPs) in animal husbandry and production is identification of the animal breed. In this study, using chosen marker selection methods and genotypic data obtained with the use of Illumina Bovine SNP50 BeadChip for individuals belonging to ten cattle breeds, the reduced panels containing the most informative SNP markers were developed. The suitability of selected SNP panels for the effective and reliable assignment of the studied individuals to the breed of origin was checked by three allocation algorithms implemented in GeneClass 2. The studied breeds set included both Polish-native breeds under the genetic resources conservation programs and highly productive breeds with a global range. For all of the tested marker selection methods ("delta" and two FST-based variants), two separate methodological approaches of marker assortment were used and three marker panels were created with 96, 192, and 288 SNPs respectively, to determine the minimum number of markers required for effective differentiation of the studied breeds. Moreover, the usefulness of the most effective panels of markers to assess the population structure and genetic diversity of the analyzed breeds was examined. The conducted analyses showed the possibility of using SNP subsets from medium-density genotypic microarrays to distinguish breeds of cattle kept in Poland and to analyze their genetic structure.

Genetic diversity analysis and heterotic grouping of Indian white maize inbred lines using combining ability and SNP markers

AbstractMaize (Zea mays L.) is a versatile crop with high genetic yield potential and various uses. While yellow maize is primarily used for animal feed, white maize is preferred as a food by rural and tribal populations in India. However, white maize landraces suffer immensely in terms of low yield. Heterotic grouping is an excellent method to develop hybrids with increased yield potential in maize. The heterotic grouping of 78 white maize inbred lines was performed using combining ability and molecular diversity analysis. The lines were crossed with two testers, and the resulting hybrids were evaluated for grain yield at three locations representing different agroecological zones in India. The combining ability analysis based on specific combining ability effects on mean grain yield was used to classify the inbred lines into heterotic groups (HG‐A: 18 inbreds; HG‐B: 33 inbreds). Additionally, marker‐based clustering using 62,470 single nucleotide polymorphisms (SNPs) generated through genotyping by sequencing was also employed to determine the genetic relationships among these lines. The combining ability information was superimposed on marker‐based clustering data to obtain the heterotic grouping information, and two distinct HGs with 7 (HG‐1) and 18 inbred lines (HG‐2) were developed. The results of this study will contribute to the development of high‐performing white maize hybrids through the efficient utilization of clear‐cut and well‐defined HGs. Hence, the use of SNP markers along with the combining ability of inbred lines is a useful method for heterotic grouping and understanding of genetic relationships of white maize.

SNP Genotyping for Purity Assessment of a Forage Oat (Avena sativa L.) Variety from Colombia

Single nucleotide polymorphism (SNP) markers have multiple applications in plant breeding of small grains. They are used for the selection of divergent parents, the identification of genetic variants and marker-assisted selection. However, the use of SNPs in varietal purity assessment is under-reported, especially for multi-line varieties from the public sector. In the case of variety evaluation, these genetic markers are tools for maintaining varietal distinctness, uniformity and stability needed for cultivar release of multi-line or pure-line varieties of inbred crops. The objective of this research was to evaluate the purity and relationships of one original (AV-25) and two multi-line sub-populations (AV25-T and AV25-S) of the inbreeding species, oats (Avena sativa L.). Both sub-populations could be useful as forages in the central highland region of Colombia (>2000 masl), such as in the departments of Boyacá and Cundinamarca, even though they were derived from an original composite mixture widely used in the mountainsides of the southern department of Nariño named Avena 25. Representative single plant selections (SPS) from the two sub-populations were grown together with SPS harvests from off-type plants (early and late) and plants from the original AV25 composite mixture, to determine their genetic similarity. Plants were genotyped by DNA extraction of a plateful of 96 individual plant samples and SNPs were detected for an Illumina Infinium 6K Chip assay. The data were used for the analysis of genetic structure and population relationships. The grouping observed based on the genetic data indicated that AV25-T and AV25-S were homogeneous populations and somewhat divergent in their genetic profile compared to the original AV25-C mix. In addition, to the two commercial, certified oat varieties (Cajicá and Cayuse) were different from these. The early and late selections were probable contaminants and could be discarded. We concluded that the use of SNP markers is an appropriate tool for ensuring genetic purity of oat varieties.

Genome-wide SNP discovery and core marker sets for DNA barcoding and variety identification in commercial tomato cultivars

Single nucleotide polymorphisms (SNPs) have been widely used as a molecular marker in crop species with advances in next-generation sequencing technology. The use of SNP markers for variety identification is a cost-effective strategy to protect breeder’s intellectual property rights. This study was conducted to identify genome-wide SNPs and develop core marker sets for assessing genetic variations in commercial tomato cultivars. A total of 10,615 confident SNPs was generated from genotyping by sequencing for 48 F1 cultivars representing four market classes (large-fruited fresh market, cherry, grape, and rootstock). Of these, 288 SNPs across 12 chromosomes were selected to genotype additional 94 F1 cultivars using the Fluidigm assay and 224 SNPs showed reliable polymorphisms in 91 F1 cultivars. Both model-based and hierarchical clustering analyses with these markers found that the large-fruited fresh market cultivars were significantly distinct from the cherry and grape cultivars. In addition, the cherry and grape cultivars showed higher levels of genetic diversity relative to the large-fruited fresh market cultivars. The 224 SNP markers were also effective in differentiating all 139 F1 cultivars and Heinz 1706 (an inbred for the tomato reference genome). Of the five subsets, the 192 and 96 markers identified all of these tomato cultivars, respectively. Furthermore, the other three subsets of 48, 24, and 12 markers showed 80.0–93.6 % of identification rates. These results demonstrate that all five subsets of SNP markers will be useful in developing a high-throughput DNA barcoding system for variety identification in commercial tomato cultivars.

Use of SNP markers by KASP assay for MAS studies in sunflower againstPlasmopara halstedii

Downy mildew is a fungal disease caused by Plasmopara halstedii and leads to loss of yield up to 100% in sunflower. Disease control is performed mostly through chemical seed treatment and breeding. Due to the time consuming nature of conventional breeding, it is supported by biotechnological approaches. Marker-assisted selection (MAS) is a strategic approach in molecular breeding using molecular markers. Single nucleotide polymorphisms (SNPs) such as insertions, deletions, and base-pair substitutions are more advantageous than other molecular markers. The abundance and biallelic nature of SNPs in a genome provide flexibility in the choosing of SNPs at the desired loci. Competitive allele-specific PCR (KASP) is a genotyping technology for screening of trait-specific SNP markers. In this study, SNP markers (NSA002867, NSA006138; NS52, NSA000354; NSA002220, NSA002251) linked with the downy mildew resistance genes Plarg, Pl13, and Pl8, respectively, were analyzed via KASP in three parental crosses (RHA-419 x Colombi, RHA-419 x P64LC53, RHA-419 x Oliva) for Plarg, one parental cross (HA-R5 x P64LC53) for Pl13, one parental cross (P64LC53 x HA-89) for Pl8, and 140 F2 individuals. According to the allelic discrimination results, NSA002867 and NSA006138 markers were discriminative in all crosses for Plarg, NSA000354 marker was discriminative for Pl13, and NSA002220 and NSA002251 markers were discriminative for Pl8. This study has revealed the potential use of SNP markers in combination with KASP assay for MAS studies, particularly downy mildew resistance in sunflower.

Potential of combining morphometry and ancient DNA information to investigate grapevine domestication

The goal of this work was to explore the possibility (1) of carrying out both morphogeometric and archaeological DNA analyses on the same grape pips and (2) of comparing different molecular markers to reveal DNA variation, namely Simple Sequence Repeats (SSRs) and Single Nucleotide Polymorphisms (SNPs). We focused on waterlogged seeds originating from three Roman and one medieval archaeological sites in France. Our first results indicate that taking photographs of pips is not detrimental to the preservation of DNA, provided a specific protocol is respected. Regarding the genetic markers, obtaining reliable information in sufficient quantity proved very difficult using SSRs. SNPs have a much more interesting potential, providing greater success rates and reliability. Here in four archaeological pips we studied 842 SNPs, derived from known polymorphisms in several genes, including one gene related to sex. Phylogenies built using these genetic markers indicate that three pips from the Roman site of Gasquinoy are close to modern wild grapevines and/or the female group, while the only medieval pip from Colletiere is hermaphrodite and close to the modern cultivated group. Morphogeometrical results are in agreement with these findings. We conclude that the combined use of SNP markers and morphogeometry is promising for deciphering the intricate history of grapevine domestication.

Polymorphisms of heat shock protein 90 (Hsp90) in the sea cucumber Apostichopus japonicus and their association with heat-resistance

Heat shock protein 90 (Hsp90) functions as a molecular chaperone and plays an important role in the resistance of organisms to stress, particularly heat-stress. In our study, 12 exons and 11 introns of hsp90 were identified in the sea cucumber Apostichopus japonicus. Twenty-two single nucleotide polymorphisms (SNPs), including three non-synonymous mutations, were detected in the exons. Susceptible and resistant individuals were distinguished using a high-temperature (32 °C) challenge experiment. Three blocks with high linkage disequilibrium were detected among these SNPs. Five of the twenty-two SNPs were shown to be significantly associated with susceptibility/resistance to high temperature by correlation analysis (chi-square test, P < 0.05). To confirm the importance of these five SNPs, a heat-resistance strain (HRS) was selected through three generations. Using the common population as the control group, it was shown that the distributions of genotypes and alleles of SNP e10-1 and e11-6 were significantly different between the two groups (P < 0.05). SNP e10-1 was trimorphic, with three alleles (A, C and T) and five genotypes (AA, CC, AT, CT and AC). The allele frequency of SNP e2-3 was also significantly associated with this trait (P < 0.05). This is the first demonstration of SNPs related to heat-resistance in A. japonicus and supports the use of SNP markers in the selective breeding of sea cucumbers.

Confirmation of hybrid origin of Cyrtanthus based on the sequence analysis of internal transcribed spacer

The objectives of this study were to create interspecific hybrids between Cyrtanthus elatus and C. sanguineus and to confirm the hybrid origin of the progeny based on morphological characters and using molecular markers. The tip of the leaves, the shape and size of cells, and stomata distribution in abaxial and adaxial surface of leaf epidermis were analyzed by low temperature scanning electron microscope (LT-SEM). Molecular markers generated from random amplification polymorphic DNA (RAPD) and single nucleotide polymorphisms (SNPs) analysis of internal transcribed spacer 1 and 2 of rRNA gene (ITS 1, 2) were also analyzed to confirm hybrid status. Putative C. elatus×C. sanguineus hybrid A 3 resembled C. elatus and C. sanguineus ‘Pringle Bay’ (PB)בFred Meyer’ (FM) B 23 and was similar to C. sanguineus at flowering. However, the hybrid origin of hybrid A 3 could not be confirmed based on the morphological characteristics of flowers, the tip of the leaves, and cell structure and surface images in both abaxial and adaxial images of LT-SEM. Based on RAPD markers, putative hybrid A 3 and other putative hybrids that did not flower (A 4, A 5, A 13 and D 39, D 40, and D 42) can be considered as a hybrid based on the presence of both species-specific bands for parental taxa. SNP markers of ITS 1, 2 region also revealed that other putative hybrids of A and D that did not flower during the evaluation can be interspecific hybrids of C. elatus and C. sanguineus. Therefore, the use of SNP markers of ITS 1, 2 is considered a more accurate tool to characterize hybrid origin than morphological characteristics and RAPD markers.

Specific Detection of Bacillus Anthracis Using Ataqman® Mismatch Amplification Mutation Assay

Single nucleotide polymorphisms (SNPs) are increasingly recognized as important diagnostic markers for the detection and differentiation of Bacillus anthracis. The use of SNP markers for identifying B. anthracis DNA in environmental samples containing genetically similar bacteria requires the ability to amplify and detect DNA with single nucleotide specificity. We designed a TaqMan mismatch amplification mutation assay (TaqMAMA) around a SNP in the plcR gene of B. anthracis. The assay permits specific, low-level detection (25 fg DNA) of this B. anthracis-specific SNP, even in the presence of environmental DNA extracts containing a 20,000-fold excess of the alternate allele. We anticipate that the ability to selectively amplify and detect low copy number DNAs with single nucleotide specificity will represent a valuable tool in the arena of biodefense and microbial forensics.

The expected power of genome-wide linkage disequilibrium testing using single nucleotide polymorphism markers for detecting a low-frequency disease variant.

The expected power of genome-wide linkage disequilibrium (LD) testing for a low-frequency disease variant was examined using a simple genetic model in which the degree of LD between the disease variant and the adjacent single nucleotide polymorphism (SNP) marker decreases in proportion to the number of generations since the LD-generating event. In this study, the frequency of the SNP marker being in complete LD with a low-frequency disease variant at the LD-generating event was regarded as the random variable having the probability distribution expected from the neutral infinite sites model, which enables us to derive the formula for calculating the expected power of genome-wide LD testing without determining the allele frequency of the associated SNP marker. Such a treatment is essential for the evaluation of the power of LD testing, because the frequency of the associated marker allele is always unknown. The main results obtained are as follows: (1) genome-wide LD testing with a case-control design could identify a disease variant with a high penetrance, while a low-frequency disease variant showing a low penetrance is difficult to detect; (2) although the degree of LD increases as the number of markers increases, the power of LD testing does not necessarily increase after the significance level is adjusted by the Sidák correction or the Bonferroni correction based on the number of testings; (3) the use of SNP markers with only high-frequency minor alleles is more powerful for detecting LD even with a low-frequency disease variant than the use of SNP markers with both high- and low-frequency minor alleles. Thus, the study design of LD testing must be evaluated prior to the investigation. The present study will provide a guideline for determining the number of SNP markers and the range of SNP allele frequencies suitable for genome-wide LD testing.

The expected power of genome‐wide linkage disequilibrium testing using single nucleotide polymorphism markers for detecting a low‐frequency disease variant

The expected power of genome‐wide linkage disequilibrium (LD) testing for a low‐frequency disease variant was examined using a simple genetic model in which the degree of LD between the disease variant and the adjacent single nucleotide polymorphism (SNP) marker decreases in proportion to the number of generations since the LD‐generating event. In this study, the frequency of the SNP marker being in complete LD with a low‐frequency disease variant at the LD‐generating event was regarded as the random variable having the probability distribution expected from the neutral infinite sites model, which enables us to derive the formula for calculating the expected power of genome‐wide LD testing without determining the allele frequency of the associated SNP marker. Such a treatment is essential for the evaluation of the power of LD testing, because the frequency of the associated marker allele is always unknown. The main results obtained are as follows: (1) genome‐wide LD testing with a case‐control design could identify a disease variant with a high penetrance, while a low‐frequency disease variant showing a low penetrance is difficult to detect; (2) although the degree of LD increases as the number of markers increases, the power of LD testing does not necessarily increase after the significance level is adjusted by the Šidák correction or the Bonferroni correction based on the number of testings; (3) the use of SNP markers with only high‐frequency minor alleles is more powerful for detecting LD even with a low‐frequency disease variant than the use of SNP markers with both high‐ and low‐frequency minor alleles. Thus, the study design of LD testing must be evaluated prior to the investigation. The present study will provide a guideline for determining the number of SNP markers and the range of SNP allele frequencies suitable for genome‐wide LD testing.

Selection and use of SNP markers for animal identification and paternity analysis in U.S. beef cattle.

DNA marker technology represents a promising means for determining the genetic identity and kinship of an animal. Compared with other types of DNA markers, single nucleotide polymorphisms (SNPs) are attractive because they are abundant, genetically stable, and amenable to high-throughput automated analysis. In cattle, the challenge has been to identify a minimal set of SNPs with sufficient power for use in a variety of popular breeds and crossbred populations. This report describes a set of 32 highly informative SNP markers distributed among 18 autosomes and both sex chromosomes. Informativity of these SNPs in U.S. beef cattle populations was estimated from the distribution of allele and genotype frequencies in two panels: one consisting of 96 purebred sires representing 17 popular breeds, and another with 154 purebred American Angus from six herds in four Midwestern states. Based on frequency data from these panels, the estimated probability that two randomly selected, unrelated individuals will possess identical genotypes for all 32 loci was 2.0 x 10(-13) for multi-breed composite populations and 1.9 x 10(-10) for purebred Angus populations. The probability that a randomly chosen candidate sire will be excluded from paternity was estimated to be 99.9% and 99.4% for the same respective populations. The DNA immediately surrounding the 32 target SNPs was sequenced in the 96 sires of the multi-breed panel and found to contain an additional 183 polymorphic sites. Knowledge of these additional sites, together with the 32 target SNPs, allows the design of robust, accurate genotype assays on a variety of high-throughput SNP genotyping platforms.