Development Of Single Nucleotide Polymorphism Markers Research Articles

Transcriptome-wide comparison of sequence variation in divergent ecotypes of kokanee salmon

BackgroundHigh throughput next-generation sequencing technology has enabled the collection of genome-wide sequence data and revolutionized single nucleotide polymorphism (SNP) discovery in a broad range of species. When analyzed within a population genomics framework, SNP-based genotypic data may be used to investigate questions of evolutionary, ecological, and conservation significance in natural populations of non-model organisms. Kokanee salmon are recently diverged freshwater populations of sockeye salmon (Oncorhynchus nerka) that exhibit reproductive ecotypes (stream-spawning and shore-spawning) in lakes throughout western North America and northeast Asia. Current conservation and management strategies may treat these ecotypes as discrete stocks, however their recent divergence and low levels of gene flow make in-season genetic stock identification a challenge. The development of genome-wide SNP markers is an essential step towards fine-scale stock identification, and may enable a direct investigation of the genetic basis of ecotype divergence.ResultsWe used pooled cDNA samples from both ecotypes of kokanee to generate 750 million base pairs of transcriptome sequence data. These raw data were assembled into 11,074 high coverage contigs from which we identified 32,699 novel single nucleotide polymorphisms. A subset of these putative SNPs was validated using high-resolution melt analysis and Sanger resequencing to genotype independent samples of kokanee and anadromous sockeye salmon. We also identified a number of contigs that were composed entirely of reads from a single ecotype, which may indicate regions of differential gene expression between the two reproductive ecotypes. In addition, we found some evidence for greater pathogen load among the kokanee sampled in stream-spawning habitats, suggesting a possible evolutionary advantage to shore-spawning that warrants further study.ConclusionsThis study provides novel genomic resources to support population genetic and genomic studies of both kokanee and anadromous sockeye salmon, and has the potential to produce markers capable of fine-scale stock assessment. While this RNAseq approach was successful at identifying a large number of new SNP loci, we found that the frequency of alleles present in the pooled transcriptome data was not an accurate predictor of population allele frequencies.

SNP Markers and Their Impact on Plant Breeding

The use of molecular markers has revolutionized the pace and precision of plant genetic analysis which in turn facilitated the implementation of molecular breeding of crops. The last three decades have seen tremendous advances in the evolution of marker systems and the respective detection platforms. Markers based on single nucleotide polymorphisms (SNPs) have rapidly gained the center stage of molecular genetics during the recent years due to their abundance in the genomes and their amenability for high-throughput detection formats and platforms. Computational approaches dominate SNP discovery methods due to the ever-increasing sequence information in public databases; however, complex genomes pose special challenges in the identification of informative SNPs warranting alternative strategies in those crops. Many genotyping platforms and chemistries have become available making the use of SNPs even more attractive and efficient. This paper provides a review of historical and current efforts in the development, validation, and application of SNP markers in QTL/gene discovery and plant breeding by discussing key experimental strategies and cases exemplifying their impact.

SNP GENOTYPING IN TETRAPLOID CUT ROSES

Genetic analysis in tetraploid rose is complex: inheritance of genes could be disomic, keeping the two original diploid genomes separate but it could also be tetrasomic, permitting the four homologous chromosomes to pair and recombine during meiosis, and including the possibility of double reduction. Marker-assisted breeding (MAB) is particularly suited for traits such as disease resistance since disease resistance assays are laborious, time and money consuming, but we need to extend the number of markers for better coverage of the genome before being able to find associations and apply MAB. Our goal was to produce hundreds of single nucleotide polymorphism (SNP) markers to provide the coverage needed. Disease resistance, more specifically powdery mildew resistance is our primary target because chemical pest control will be increasingly limited. Molecular markers could provide a good tool to facilitate selection in an earlier stage of a breeding program. We investigated the inheritance of powdery mildew resistance in an F1 population originating from a cross between two tetraploid genotypes, P540 and P867, differing in powdery mildew resistance. SNP marker development started with Next Generation Sequencing (Illumina GAII) of cDNA obtained from RNA isolated from flowers at three developmental stages for each parent. We identified a large number of putative single nucleotide polymorphisms (SNPs) between and within both parents. SNPs that were expected to segregate as simplex × nulliplex and duplex × nulliplex markers were selected for genotyping the entire F1 population using the KASP platform (KBioscience). For a pilot study on a limited number of SNPs we defined specific selection criteria resulting in a set of 20 SNPs for testing on the population. It was found that selecting the SNPs according to the ratio of the number of reads from the Illumina sequencing data did not guarantee the appropriate segregation ratios in the progeny. Continued research will focus on identifying haplotypes and on QTL mapping of powdery mildew resistance. We will present results on the criteria used to select SNP markers, success rate, the realised segregation ratios in the offspring, the reconstruction of the homologous chromosomes (haplotypes) in the cross, and QTL mapping of powdery mildew resistance.

SNP identification and allelic-specific PCR markers development for TaGW2, a gene linked to wheat kernel weight

TaGW2, an orthologous gene of rice OsGW2, has been associated with kernel width and weight of bread wheat (Triticum aestivum). Difference in TaGW2 coding sequence was not found among different wheat varieties in previous researches. In this study, we found eight exons and seven introns in TaGW2 with a full-length cDNA sequence of 1,275 bp, which contains a conserved function domain and seven splice sites that shared homology with rice OsGW2. A single T-base insertion in the eighth exon of TaGW2 on chromosome 6A was detected in a large-kernel wheat variety, Lankaodali. This insertion mutation reduces the coding protein sequence from normal 424 amino acids (~47.2 kDa) to 328 amino acids (~37.1 kDa) by truncating 96 amino acids. The result was validated by identifying histidine-tagged TaGW2 proteins encoded by both alleles of the mutant and the wild types in SDS-PAGE. Allele-specific PCR markers were developed based on the single nucleotide polymorphism (SNP) site. The SNP markers were genotyped for an F(2) segregation population from the cross of Lankaodali × Chinese Spring. Seed traits of F(2:3) families were evaluated in three different environments. The association analysis indicated that F(2:3) families with the mutated TaGW2 allele significantly increased kernel width (KW) and thousand-kernel weight (TKW), and slightly improved kernel length (KL). Using the SNP markers, another two varieties harbored the mutated TaGW2 allele were successfully identified from 22 additional wheat varieties, and they both have large KW and TKW. Cloning and sequencing of the gene further confirmed the functions of the mutated allele of TaGW2 in the two large kernel varieties. The results suggested that TaGW2 may negatively regulate kernel size variation, which shares the same function as OsGW2 in rice. The successful development of SNP markers provides a useful tool for improving kernel yield in wheat.

DNA sequence variation and development of SNP markers in beech (Fagus sylvatica L.)

European beech (Fagus sylvatica L.) is one of the most important deciduous tree species in Central Europe. The potential of beech to adapt to climate change, higher temperatures, and less precipitation in the summer months is still unknown. Most studies in beech used microsatellite, AFLP (amplified fragment length polymorphism), or isozyme markers, which have only a restricted potential to analyze adaptation. Only few studies investigated genes probably involved in the adaptation to drought stress and bud phenology in beech. In this study, SNP (single nucleotide polymorphisms) markers were developed in order to analyze adaptation and their technical advantages compared to microsatellites and AFLPs were discussed. Partial sequences of ten candidate genes probably involved in drought stress and/or bud phenology were identified at the genomic level, and SNPs and indels (insertions/deletions) in coding and non-coding regions were analyzed. Plant material was sampled along a precipitation gradient in Germany. In total, 8,145 bp were sequenced and analyzed, 4,038 bp were located in exon and 4,107 bp in intron regions. 63 SNPs and 11 indels were detected, which are differently distributed over the studied gene regions. The nucleotide diversity ranged from 0 to 6.62 (π × 10−3) and is comparable to other tree species, whereas the mean nucleotide diversity (2.64) for F. sylvatica is comparatively low. These results will help to investigate the genetic basis of drought stress and bud burst and to conduct association mapping in natural populations. Furthermore, the detected SNPs can also be used for population genetic studies.

Rapid gene-based SNP and haplotype marker development in non-model eukaryotes using 3'UTR sequencing

BackgroundSweet cherry (Prunus avium L.), a non-model crop with narrow genetic diversity, is an important member of sub-family Amygdoloideae within Rosaceae. Compared to other important members like peach and apple, sweet cherry lacks in genetic and genomic information, impeding understanding of important biological processes and development of efficient breeding approaches. Availability of single nucleotide polymorphism (SNP)-based molecular markers can greatly benefit breeding efforts in such non-model species. RNA-seq approaches employing second generation sequencing platforms offer a unique avenue to rapidly identify gene-based SNPs. Additionally, haplotype markers can be rapidly generated from transcript-based SNPs since they have been found to be extremely utile in identification of genetic variants related to health, disease and response to environment as highlighted by the human HapMap project.ResultsRNA-seq was performed on two sweet cherry cultivars, Bing and Rainier using a 3' untranslated region (UTR) sequencing method yielding 43,396 assembled contigs. In order to test our approach of rapid identification of SNPs without any reference genome information, over 25% (10,100) of the contigs were screened for the SNPs. A total of 207 contigs from this set were identified to contain high quality SNPs. A set of 223 primer pairs were designed to amplify SNP containing regions from these contigs and high resolution melting (HRM) analysis was performed with eight important parental sweet cherry cultivars. Six of the parent cultivars were distantly related to Bing and Rainier, the cultivars used for initial SNP discovery. Further, HRM analysis was also performed on 13 seedlings derived from a cross between two of the parents. Our analysis resulted in the identification of 84 (38.7%) primer sets that demonstrated variation among the tested germplasm. Reassembly of the raw 3'UTR sequences using upgraded transcriptome assembly software yielded 34,620 contigs containing 2243 putative SNPs in 887 contigs after stringent filtering. Contigs with multiple SNPs were visually parsed to identify 685 putative haplotypes at 335 loci in 301 contigs.ConclusionsThis approach, which leverages the advantages of RNA-seq approaches, enabled rapid generation of gene-linked SNP and haplotype markers. The general approach presented in this study can be easily applied to other non-model eukaryotes irrespective of the ploidy level to identify gene-linked polymorphisms that are expected to facilitate efficient Gene Assisted Breeding (GAB), genotyping and population genetics studies. The identified SNP haplotypes reveal some of the allelic differences in the two sweet cherry cultivars analyzed. The identification of these SNP and haplotype markers is expected to significantly improve the genomic resources for sweet cherry and facilitate efficient GAB in this non-model crop.

Identification of a Seed Phospholipase D Null Allele in Rice (Oryza sativa L.) and Development of SNP Markers for Phospholipase D Deficiency

Rice (Oryza sativa L.) bran usage is severely limited by the rapid degradation of rice bran oil and rapid development of hydrolytic rancidity. Because phospholipase D (PLD) disintegrates phospholipid membranes of oil bodies to deteriorate bran quality, we isolated a PLD‐null rice mutant, ‘03‐s108’, whose PLD‐null phenotype is inherited as a single recessive trait. To develop a convenient DNA marker‐assisted selection method for the PLD‐null trait, we first performed a linkage analysis between the PLD‐null phenotype and simple sequence repeat (SSR) markers by using F2 progeny from 03‐s108 × ‘Koshihikari’ crosses, and we mapped the PLD‐null genotype to chromosome 1 near a putative PLD gene sequence, PLDα1. DNA sequencing of the 03‐s108 allele of PLDα1 revealed a transition mutation (G to A) in the third exon. This change resulted in a stop codon, indicating the presence of a nonsense mutation in the 03‐s108 allele. Using this DNA sequence information, we then developed methods of cleaved amplified polymorphic sequence (CAPS) and dot‐blot single‐nucleotide polymorphism (SNP) detection for this mutation, and we confirmed that the sequence polymorphism was predictive of the presence or absence of the seed PLD‐null phenotype. These CAPS and dot‐blot‐SNP methods are easy and cost‐effective means for identifying seed PLD‐null strains, to enable screening and breeding of PLD‐null rice varieties.

Transcriptome sequencing, and rapid development and application of SNP markers for the legume pod borer Maruca vitrata (Lepidoptera: Crambidae).

The legume pod borer, Maruca vitrata (Lepidoptera: Crambidae), is an insect pest species of crops grown by subsistence farmers in tropical regions of Africa. We present the de novo assembly of 3729 contigs from 454- and Sanger-derived sequencing reads for midgut, salivary, and whole adult tissues of this non-model species. Functional annotation predicted that 1320 M. vitrata protein coding genes are present, of which 631 have orthologs within the Bombyx mori gene model. A homology-based analysis assigned M. vitrata genes into a group of paralogs, but these were subsequently partitioned into putative orthologs following phylogenetic analyses. Following sequence quality filtering, a total of 1542 putative single nucleotide polymorphisms (SNPs) were predicted within M. vitrata contig assemblies. Seventy one of 1078 designed molecular genetic markers were used to screen M. vitrata samples from five collection sites in West Africa. Population substructure may be present with significant implications in the insect resistance management recommendations pertaining to the release of biological control agents or transgenic cowpea that express Bacillus thuringiensis crystal toxins. Mutation data derived from transcriptome sequencing is an expeditious and economical source for genetic markers that allow evaluation of ecological differentiation.

A method for selection of restriction enzymes for sdCAPS marker construction

With 2 figures Abstract Development of PCR-based markers for single-nucleotide polymorphism (SNP) detection is prerequisite for various genetic analyses. The use of restriction enzymes (REs) following PCR amplification is a common and relatively low cost method for SNP detection. Simple and cost-effective methodologies for SNP marker development that would enhance the use of SNP-based technologies are desirable. As an alternative analytical method for selection of REs for recognition of SNP motifs for marker development that does not require computer-based selection of REs is herein described. Given that only 12 REs are required for the detection of any SNP motif, the method described in this study is relatively inexpensive and technically simple.

Nucleotide diversity maps reveal variation in diversity among wheat genomes and chromosomes.

BackgroundA genome-wide assessment of nucleotide diversity in a polyploid species must minimize the inclusion of homoeologous sequences into diversity estimates and reliably allocate individual haplotypes into their respective genomes. The same requirements complicate the development and deployment of single nucleotide polymorphism (SNP) markers in polyploid species. We report here a strategy that satisfies these requirements and deploy it in the sequencing of genes in cultivated hexaploid wheat (Triticum aestivum, genomes AABBDD) and wild tetraploid wheat (Triticum turgidum ssp. dicoccoides, genomes AABB) from the putative site of wheat domestication in Turkey. Data are used to assess the distribution of diversity among and within wheat genomes and to develop a panel of SNP markers for polyploid wheat.ResultsNucleotide diversity was estimated in 2114 wheat genes and was similar between the A and B genomes and reduced in the D genome. Within a genome, diversity was diminished on some chromosomes. Low diversity was always accompanied by an excess of rare alleles. A total of 5,471 SNPs was discovered in 1791 wheat genes. Totals of 1,271, 1,218, and 2,203 SNPs were discovered in 488, 463, and 641 genes of wheat putative diploid ancestors, T. urartu, Aegilops speltoides, and Ae. tauschii, respectively. A public database containing genome-specific primers, SNPs, and other information was constructed. A total of 987 genes with nucleotide diversity estimated in one or more of the wheat genomes was placed on an Ae. tauschii genetic map, and the map was superimposed on wheat deletion-bin maps. The agreement between the maps was assessed.ConclusionsIn a young polyploid, exemplified by T. aestivum, ancestral species are the primary source of genetic diversity. Low effective recombination due to self-pollination and a genetic mechanism precluding homoeologous chromosome pairing during polyploid meiosis can lead to the loss of diversity from large chromosomal regions. The net effect of these factors in T. aestivum is large variation in diversity among genomes and chromosomes, which impacts the development of SNP markers and their practical utility. Accumulation of new mutations in older polyploid species, such as wild emmer, results in increased diversity and its more uniform distribution across the genome.

Development of SNP markers for individual identification and parentage test in a Japanese Black cattle population

This study describes the development of efficient single nucleotide polymorphism (SNP) markers for individual identification and parentage tests in a Japanese Black cattle population. An amplified fragment length polymorphism method was employed to detect informative candidate markers, and yielded 44 SNP markers from 220 primer combinations. 29 unlinked SNPs were finally selected as diagnostic markers. The allelic frequencies for each marker were estimated by using PCR-RFLP in the Japanese Black population. Based on the frequency data, the estimated identity power of these markers was 2.73 x 10(-12). Parentage exclusion probabilities, when both suspected parents' genotypes were known and when only one suspected parent was genotyped, were estimated as 0.96929 and 0.99693, respectively. This panel of SNP markers is theoretically sufficient for individual identification, and would also be a powerful tool for a parentage test in Japanese Black cattle. The markers could contribute to the management of the beef industry in Japan.

Development of gene-targeted SNP markers for genomic mapping in Pacific abalone Haliotis discus hannai Ino

Useful and novel DNA markers are needed for aquaculture genetics and breeding. In this study, we report the discovery and development of gene-targeted single nucleotide polymorphisms (SNPs) for genomic mapping in the Pacific abalone Haliotis discus hannai Ino. Single EST or EST-contigs from 66 genes that had positive BLASTx matches (E-value ≤ 1e-8) were used for polymerase chain reaction (PCR) amplification. PCR products from the two parents of one mapping family were directly sequenced, and 83 SNP loci were found from 17 genes. Allele-specific PCR (AS-PCR) was developed and optimized for genotyping of 11 SNP loci in 120 progeny of the mapping family. Nine of the loci conformed to the expected Mendelian ratio of 1:1 based on the χ(2) test (P > 0.05) and could potentially be used for linkage map construction. Our data also indicate that the sequencing of two parents may be a practical strategy for the discovery of informative SNPs for linkage mapping in a particular mapping population.

ConservedPrimers 2.0: A high-throughput pipeline for comparative genome referenced intron-flanking PCR primer design and its application in wheat SNP discovery

BackgroundIn some genomic applications it is necessary to design large numbers of PCR primers in exons flanking one or several introns on the basis of orthologous gene sequences in related species. The primer pairs designed by this target gene approach are called "intron-flanking primers" or because they are located in exonic sequences which are usually conserved between related species, "conserved primers". They are useful for large-scale single nucleotide polymorphism (SNP) discovery and marker development, especially in species, such as wheat, for which a large number of ESTs are available but for which genome sequences and intron/exon boundaries are not available. To date, no suitable high-throughput tool is available for this purpose.ResultsWe have developed, the ConservedPrimers 2.0 pipeline, for designing intron-flanking primers for large-scale SNP discovery and marker development, and demonstrated its utility in wheat. This tool uses non-redundant wheat EST sequences, such as wheat contigs and singleton ESTs, and related genomic sequences, such as those of rice, as inputs. It aligns the ESTs to the genomic sequences to identify unique colinear exon blocks and predicts intron lengths. Intron-flanking primers are then designed based on the intron/exon information using the Primer3 core program or BatchPrimer3. Finally, a tab-delimited file containing intron-flanking primer pair sequences and their primer properties is generated for primer ordering and their PCR applications. Using this tool, 1,922 bin-mapped wheat ESTs (31.8% of the 6,045 in total) were found to have unique colinear exon blocks suitable for primer design and 1,821 primer pairs were designed from these single- or low-copy genes for PCR amplification and SNP discovery. With these primers and subsequently designed genome-specific primers, a total of 1,527 loci were found to contain one or more genome-specific SNPs.ConclusionThe ConservedPrimers 2.0 pipeline for designing intron-flanking primers was developed and its utility demonstrated. The tool can be used for SNP discovery, genetic variation assays and marker development for any target genome that has abundant ESTs and a related reference genome that has been fully sequenced. The ConservedPrimers 2.0 pipeline has been implemented as a command-line tool as well as a web application. Both versions are freely available at .

Development and evaluation of single-nucleotide polymorphism markers in allotetraploid rapeseed (Brassica napus L.)

Single-nucleotide polymorphisms (SNPs) and insertion-deletions (INDELs) are currently the important classes of genetic markers for major crop species. In this study, methods for developing SNP markers in rapeseed (Brassica napus L.) and their in silico mapping and use for genotyping are demonstrated. For the development of SNP and INDEL markers, 181 fragments from 121 different gene sequences spanning 86 kb were examined. A combination of different selection methods (genome-specific amplification, hetero-duplex analysis and sequence analysis) allowed the detection of 18 singular fragments that showed a total of 87 SNPs and 6 INDELs between 6 different rapeseed varieties. The average frequency of sequence polymorphism was estimated to be one SNP every 247 bp and one INDEL every 3,583 bp. Most SNPs and INDELs were found in non-coding regions. Polymorphism information content values for SNP markers ranged between 0.02 and 0.50 in a set of 86 varieties. Using comparative genetics data for B. napus and Arabidopsis thaliana, an allocation of SNP markers to linkage groups in rapeseed was achieved: a unique location was determined for seven gene sequences; two and three possible locations were found for six and four sequences, respectively. The results demonstrate the usefulness of existing genomic resources for SNP discovery in rapeseed.

Development and mapping of gene-tagged SNP markers in laccases of maize (Zea maysL.)

Laccases, EC 1.10.3.2 or p-diphenol : dioxygen oxidoreductases, have been proposed to be involved in the oxidative polymerization of monolignols into lignins in plants. While 17 laccases have been identified in Arabidopsis, only five (ZmLac1–5) have so far been identified in maize. By a bioinformatic approach, 14 putative laccases were identified in maize. One putative laccase was identical to ZmLac1, while five were highly homologous to either ZmLac4 or ZmLac5. Sequence alignment of allelic sequences enabled the development of TaqMan single nucleotide polymorphism (SNP) markers for nine putative laccases. Four of these gene-tagged SNP markers were validated in a doubled-haploid mapping population of 140 individuals, mapping these loci to chromosomes 1, 2, 3 and 7, respectively.

Development of SNP markers and haplotype analysis of the candidate gene for rhg1, which confers resistance to soybean cyst nematode in soybean

Soybean cyst nematode (SCN; Heterodera glycines Ichinohe) is one of the most destructive pests in the cultivation of soybean (Glycine max (L.) Merr.) worldwide. Markers based on the SCN resistance gene will enable efficient marker-assisted selection (MAS). We sequenced the candidate gene rhg1 in six resistant and two susceptible soybean genotypes and identified 37 SNPs (single nucleotide polymorphisms) among the sequences, of which 11 were in the coding region. Seven of these 11 SNPs led to changes in the amino acid sequence of the gene. The amino acid sequence we obtained differs from the previously published one by a stretch of 26–27 amino acids. Six codominant allele-specific SNP markers based on agarose gel detection were developed and tested in 70 genotypes, among which occurred only nine different haplotypes. Two neutrality tests (Tajima’s D and Fu and Li’s F) were significant for the six SNP loci in the 70 genotypes, which is consistent with intensive directional selection. A strong LD pattern was detected among five SNPs except 2868T > C. Two SNPs (689C > A and 757C > T) formed one haplotype (689C-757C) that was perfectly associated with SCN resistance. The new allele-specific PCR markers located in the alleged sequence of the rhg1 candidate gene, combined with the microsatellite marker BACR-Satt309, will significantly improve the efficiency of MAS during the development of SCN-resistant cultivars.

SNP identification in crop plants

In many plants, single nucleotide polymorphism (SNP) markers are increasingly becoming the marker system of choice. However, for many crop plants there are surprisingly low numbers of validated SNP markers available although they are needed in large numbers for studies regarding genetic variation, linkage mapping, population structure analysis, association genetics, map-based gene isolation, and plant breeding. This review summarizes the current status of SNP marker development technologies for major crop plants. It will also provide an outlook into the future regarding possible SNP identification approaches in crop plants on the basis of current development in model systems such as Arabidopsis which will become available with the full sequencing of more plant genomes, genome resequencing, and in conjunction with the next-generation sequencing technologies.

Simultaneous mutation detection of three homoeologous genes in wheat by High Resolution Melting analysis and Mutation Surveyor®

BackgroundTILLING (Targeting Induced Local Lesions IN Genomes) is a powerful tool for reverse genetics, combining traditional chemical mutagenesis with high-throughput PCR-based mutation detection to discover induced mutations that alter protein function. The most popular mutation detection method for TILLING is a mismatch cleavage assay using the endonuclease CelI. For this method, locus-specific PCR is essential. Most wheat genes are present as three similar sequences with high homology in exons and low homology in introns. Locus-specific primers can usually be designed in introns. However, it is sometimes difficult to design locus-specific PCR primers in a conserved region with high homology among the three homoeologous genes, or in a gene lacking introns, or if information on introns is not available. Here we describe a mutation detection method which combines High Resolution Melting (HRM) analysis of mixed PCR amplicons containing three homoeologous gene fragments and sequence analysis using Mutation Surveyor® software, aimed at simultaneous detection of mutations in three homoeologous genes.ResultsWe demonstrate that High Resolution Melting (HRM) analysis can be used in mutation scans in mixed PCR amplicons containing three homoeologous gene fragments. Combining HRM scanning with sequence analysis using Mutation Surveyor® is sensitive enough to detect a single nucleotide mutation in the heterozygous state in a mixed PCR amplicon containing three homoeoloci. The method was tested and validated in an EMS (ethylmethane sulfonate)-treated wheat TILLING population, screening mutations in the carboxyl terminal domain of the Starch Synthase II (SSII) gene. Selected identified mutations of interest can be further analysed by cloning to confirm the mutation and determine the genomic origin of the mutation.ConclusionPolyploidy is common in plants. Conserved regions of a gene often represent functional domains and have high sequence similarity between homoeologous loci. The method described here is a useful alternative to locus-specific based methods for screening mutations in conserved functional domains of homoeologous genes. This method can also be used for SNP (single nucleotide polymorphism) marker development and eco-TILLING in polyploid species.

Genetic Diversity Analysis and Single-nucleotide Polymorphism Marker Development in Cultivated Bulb Onion Based on Expressed Sequence Tag–Simple Sequence Repeat Markers

Bulb onion (Allium cepa L.) is a globally significant crop, but the structure of genetic variation within and among populations is poorly understood. We broadly surveyed genetic variation in a cultivated onion germplasm using simple sequence repeat (SSR) markers and sequenced regions flanking expressed sequence tag (EST)-SSRs to develop single-nucleotide polymorphism (SNP) markers. Samples from 89 inbred and open-pollinated (OP) bulb onion populations of wide geographical adaptation and four related Allium L. accessions were genotyped with 56 EST-SSR and four genomic SSR markers. Multivariate analysis of genetic distances among populations resolved long-day, short-day, and Indian populations. EST-SSR markers frequently revealed two major alleles at high frequency in OP populations. The median proportion of single-locus polymorphic loci was 0.70 in OP and landrace populations compared with 0.43 in inbred lines. Resequencing of 24 marker amplicons revealed additional SNPs in 17 (68%) and five SNP assays were developed from these, suggesting that resequencing of EST markers can readily provide SNP markers for purity testing of inbreds and other applications in Allium genetics.

Bioinformatics Analysis of Distribution of Microsatellite Markers (SSRs) / Single Nucleotide Polymorphism (SNPs) in Expressed Transcripts of Prosopis Juliflora: Frequency and Distribution

The present paper aims to identify the distribution of Microsatellite markers (SSRs)/Single Nucleotide Polymorphism (SNPs) as resource tools for the analysis of interspecies hypervariability in Prosopis spp. Microsatellites are ubiquitously repeated extension of 1-5 bp motif extended throughout the genome. The dbEST division of Genbank contains 1467 ESTs of Prosopis juliflora which have been utilized for the development of genic microsatellite markers (SSRs). Locally installed assembling program was used for the cluster analysis of the EST. Analysis was performed on a Linux Cluster system. The analysis of the putative unigenes has been shown to have most abundant motif of A/T followed by dinucleotide AG/CT and trinucleotide repeat AAG/CTT. EST-derived SNPs are becoming the resources for the development of SNP markers. The relative rates of development of the high throughput computational methods for the detection of SNPs (Single Nucleotide Polymorphism) and small indels (insertion / deletion) has gained wide applications in the field of the molecular markers.