High-resolution melting analysis for SNP genotyping and mapping in tetraploid alfalfa (Medicago sativa L.)

Single nucleotide polymorphisms (SNPs) represent the most abundant type of genetic polymorphism in plant genomes. SNP markers are valuable tools for genetic analysis of complex traits of agronomic importance, linkage and association mapping, genome-wide selection, map-based cloning, and marker-assisted selection. Current challenges for SNP genotyping in polyploid outcrossing species include multiple alleles per loci and lack of high-throughput methods suitable for variant detection. In this study, we report on a high-resolution melting (HRM) analysis system for SNP genotyping and mapping in outcrossing tetraploid genotypes. The sensitivity and utility of this technology is demonstrated by identification of the parental genotypes and segregating progeny in six alfalfa populations based on unique melting curve profiles due to differences in allelic composition at one or multiple loci. HRM using a 384-well format is a fast, consistent, and efficient approach for SNP discovery and genotyping, useful in polyploid species with uncharacterized genomes. Possible applications of this method include variation discovery, analysis of candidate genes, genotyping for comparative and association mapping, and integration of genome-wide selection in breeding programs.     

Sequence-based ultra-dense genetic and physical maps reveal structural variations of allopolyploid cotton genomes

BackgroundSNPs are the most abundant polymorphism type, and have been explored in many crop genomic studies, including rice and maize. SNP discovery in allotetraploid cotton genomes has lagged behind that of other crops due to their complexity and polyploidy. In this study, genome-wide SNPs are detected systematically using next-generation sequencing and efficient SNP genotyping methods, and used to construct a linkage map and characterize the structural variations in polyploid cotton genomes.ResultsWe construct an ultra-dense inter-specific genetic map comprising 4,999,048 SNP loci distributed unevenly in 26 allotetraploid cotton linkage groups and covering 4,042 cM. The map is used to order tetraploid cotton genome scaffolds for accurate assembly of G. hirsutum acc. TM-1. Recombination rates and hotspots are identified across the cotton genome by comparing the assembled draft sequence and the genetic map. Using this map, genome rearrangements and centromeric regions are identified in tetraploid cotton by combining information from the publicly-available G. raimondii genome with fluorescent in situ hybridization analysis.ConclusionsWe report the genotype-by-sequencing method used to identify millions of SNPs between G. hirsutum and G. barbadense. We construct and use an ultra-dense SNP map to correct sequence mis-assemblies, merge scaffolds into pseudomolecules corresponding to chromosomes, detect genome rearrangements, and identify centromeric regions in allotetraploid cottons. We find that the centromeric retro-element sequence of tetraploid cotton derived from the D subgenome progenitor might have invaded the A subgenome centromeres after allotetrapolyploid formation. This study serves as a valuable genomic resource for genetic research and breeding of cotton.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0678-1) contains supplementary material, which is available to authorized users.     

FunciSNP: an R/bioconductor tool integrating functional non-coding data sets with genetic association studies to identify candidate regulatory SNPs

Single nucleotide polymorphisms (SNPs) are increasingly used to tag genetic loci associated with phenotypes such as risk of complex diseases. Technically, this is done genome-wide without prior restriction or knowledge of biological feasibility in scans referred to as genome-wide association studies (GWAS). Depending on the linkage disequilibrium (LD) structure at a particular locus, such tagSNPs may be surrogates for many thousands of other SNPs, and it is difficult to distinguish those that may play a functional role in the phenotype from those simply genetically linked. Because a large proportion of tagSNPs have been identified within non-coding regions of the genome, distinguishing functional from non-functional SNPs has been an even greater challenge. A strategy was recently proposed that prioritizes surrogate SNPs based on non-coding chromatin and epigenomic mapping techniques that have become feasible with the advent of massively parallel sequencing. Here, we introduce an R/Bioconductor software package that enables the identification of candidate functional SNPs by integrating information from tagSNP locations, lists of linked SNPs from the 1000 genomes project and locations of chromatin features which may have functional significance. Availability: FunciSNP is available from Bioconductor (bioconductor.org).     

MIG-seq is an effective method for high-throughput genotyping in wheat (Triticum spp.)

MIG-seq (Multiplexed inter-simple sequence repeats genotyping by sequencing) has been developed as a low cost genotyping technology, although the number of polymorphisms obtained is assumed to be minimal, resulting in the low application of this technique to analyses of agricultural plants. We applied MIG-seq to 12 plant species that include various crops and investigated the relationship between genome size and the number of bases that can be stably sequenced. The genome size and the number of loci, which can be sequenced by MIG-seq, are positively correlated. This is due to the linkage between genome size and the number of simple sequence repeats (SSRs) through the genome. The applicability of MIG-seq to population structure analysis, linkage mapping, and quantitative trait loci (QTL) analysis in wheat, which has a relatively large genome, was further evaluated. The results of population structure analysis for tetraploid wheat showed the differences among collection sites and subspecies, which agreed with previous findings. Additionally, in wheat biparental mapping populations, over 3,000 SNPs/indels with low deficiency were detected using MIG-seq, and the QTL analysis was able to detect recognized flowering-related genes. These results revealed the effectiveness of MIG-seq for genomic analysis of agricultural plants with large genomes, including wheat.     

Genomic heterogeneity in the density of noncoding single-nucleotide and microsatellite polymorphisms in Plasmodium falciparum

The density and distribution of single-nucleotide polymorphisms (SNPs) across the genome has important implications for linkage disequilibrium mapping and association studies, and the level of simple-sequence microsatellite polymorphisms has important implications for the use of oligonucleotide hybridization methods to genotype SNPs. To assess the density of these types of polymorphisms in P. falciparum, we sampled introns and noncoding DNA upstream and downstream of coding regions among a variety of geographically diverse parasites. Across 36,229 base pairs of noncoding sequence representing 41 genetic loci, a total of 307 polymorphisms including 248 polymorphic microsatellites and 39 SNPs were identified. We found a significant excess of microsatellite polymorphisms having a repeat unit length of one or two, compared to those with longer repeat lengths, as well as a nonrandom distribution of SNP polymorphisms. Almost half of the SNPs localized to only three of the 41 genetic loci sampled. Furthermore, we find significant differences in the frequency of polymorphisms across the two chromosomes (2 and 3) examined most extensively, with an excess of SNPs and a surplus of polymorphic microsatellites on chromosome 3 as compared to chromosome 2 (P=0.0001). Furthermore, at some individual genetic loci we also find a nonrandom distribution of polymorphisms between coding and flanking noncoding sequences, where completely monomorphic regions may flank highly polymorphic genes. These data, combined with our previous findings of nonrandom distribution of SNPs across chromosome 2, suggest that the Plasmodium falciparum genome may be a mosaic with regard to genetic diversity, containing chromosomal regions that are highly polymorphic interspersed with regions that are much less polymorphic.     

Applications of single nucleotide polymorphisms in crop genetics

The discovery of single nucleotide polymorphisms (SNPs) and insertions/deletions, which are the basis of most differences between alleles, has been simplified by recent developments in sequencing technology. SNP discovery in many crop species, such as corn and soybean, is relatively straightforward because of their high level of intraspecific nucleotide diversity, and the availability of many gene and expressed sequence tag (EST) sequences. For these species, direct readout of SNP haplotypes is possible. Haplotype-based analysis is more informative than analysis based on individual SNPs, and has more power in analyzing association with phenotypes. The elite germplasm of some crops may have been subjected to bottlenecks relatively recently, increasing the amount of linkage disequilibrium (LD) present and facilitating the association of SNP haplotypes at candidate gene loci with phenotypes. Whole-genome scans may help identify genome regions that are associated with interesting phenotypes if sufficient LD is present. Technological improvements make the use of SNP and indel markers attractive for high-throughput use in marker-assisted breeding, EST mapping and the integration of genetic and physical maps.     

Analysis of single nucleotide polymorphisms in three chromosomes of European sea bass Dicentrarchus labrax

Single nucleotide polymorphisms (SNPs) are believed to contain relevant information and have been therefore extensively used as genetic markers in population and conservation genetics, and molecular ecology studies. This study reports on the identification of potential SNPs in a diploid European sea bass Dicentrarchus labrax genome by using reference sequences from three assembled chromosomes and mapping all WGS datasets onto them (3× Sanger, 3× 454 and 20× SOLEXA). A total of 20,779 SNPs were identified over the 1469 gene loci and intergenic space analysed. Within chromosomes the occurrence of SNPs was the lowest in exons and higher in introns and intergenic regions, which may be explained by the fact, that coding regions are under strong selective pressure to maintain their biological function. The ratio of nonsynonymous to synonymous mutations was smaller than one for all the chromosomes, suggesting that most of deleterious nonsynonymous mutations were eliminated by negative selection. SNPs were not uniformly distributed over the chromosomes. Two chromosomes exhibited large regions with extremely low SNP density, which might represent homozygous regions in the diploid genome. The results of this study show how SNP detection can take profit from sequencing a single diploid individual, but also uncover the limits of such an approach. SNPs that have been identified will support marker development for genetic linkage mapping, population genetics and aquaculture related questions in general.     

Association and interaction of the IL4R,IL4, and IL13 loci with type 1 diabetes among Filipinos

In the search for genes involved in type 1 diabetes (T1D), other than the well-established risk alleles at the human leukocyte antigen loci, we have investigated the association and interaction of polymorphisms in genes involved in the IL4/IL13 pathway in a sample of 90 Filipino patients with T1D and 94 controls. Ten single-nucleotide polymorphisms (SNPs), including two promoter SNPs in the IL4R locus on chromosome 16p11, one promoter SNP in the IL4 locus on chromosome 5q31, and four SNPs--including two promoter SNPs--in the IL13 locus on chromosome 5q31 were examined for association, linkage disequilibrium, and interaction. We found that both individual SNPs (IL4R L389L; odds ratio [OR] 0.34; 95% confidence interval [CI] 0.17-0.67; P=.001) and specific haplotypes both in IL4R (OR 0.10; 95% CI 0-0.5; P=.001) and for the five linked IL4 and IL13 SNPs (OR 3.47; P=.004) were strongly associated with susceptibility to T1D. Since IL4 and IL13 both serve as ligands for a receptor composed, in part, of the IL4R alpha chain, we looked for potential epistasis between polymorphisms in the IL4R locus on chromosome 16p11 and the five SNPs in the IL4 and IL13 loci on chromosome 5q31 and found, through use of a logistic-regression model, significant gene-gene interactions (P=.045, corrected for multiple comparisons by permutation analysis). Our data suggest that the risk for T1D is determined, in part, by polymorphisms within the IL4R locus, including promoter and coding-sequence variants, and by specific combinations of genotypes at the IL4R and the IL4 and IL13 loci.     

Linkage mapping of gene-associated SNPs to pig chromosome 11

Single nucleotide polymorphisms (SNPs) were discovered in porcine expressed sequence tags (ESTs) orthologous to genes from human chromosome 13 (HSA13) and predicted to be located on pig chromosome 11 (SSC11). The SNPs were identified as sequence variants in clusters of EST sequences from pig cDNA libraries constructed in the Sino-Danish pig genome project. In total, 312 human gene sequences from HSA13 were used for similarity searches in our pig EST database. Pig ESTs showing significant similarity with HSA13 genes were clustered and candidate SNPs were identified. Allele frequencies for 26 SNPs were estimated in a group of 80 unrelated pigs from Danish commercial pig breeds: Duroc, Hampshire, Landrace and Large White. Eighteen of the 26 SNPs genotyped in the PiGMaP Reference Families were mapped by linkage analysis to SSC11. The EST-based SNPs published here are new genetic markers useful for linkage and association studies in commercial and experimental pig populations. This study represents the first gene-associated SNP linkage map of pig chromosome 11 and adds new comparative mapping information between SSC11 and HSA13. Furthermore, our data facilitate future studies aimed at the identification of interesting regions on pig chromosome 11, positional cloning and fine mapping of quantitative trait loci in pig.     

Reciprocal mouse and human limb phenotypes caused by gain- and loss-of-function mutations affecting Lmbr1

An intensive linkage map of the yellow fever mosquito, Aedes aegypti, was constructed using single-strand conformation polymorphism (SSCP) analysis of cDNA markers to identify single nucleotide polymorphisms (SNPs). A total of 94 A. aegypti cDNAs were downloaded from GenBank and primers were designed to amplify fragments <500 bp in size. These primer pairs amplified 94 loci, 57 (61%) of which segregated in a single F(1) intercross family among 83 F(2) progeny. This allowed us to produce a dense linkage map of one marker every 2 cM distributed over a total length of 134 cM. Many A. aegypti cDNAs were highly similar to genes in the Drosophila melanogaster genome project. Comparative linkage analysis revealed areas of synteny between the two species. SNP polymorphisms are abundant in A. aegypti genes and should prove useful in both population genetics and mapping studies.     

Genetic mapping and QTL analysis of fiber-related traits in cotton (<Emphasis Type="Italic">Gossypium</Emphasis>)

Cotton, the leading natural fiber crop, is largely produced by two primary cultivated allotetraploid species known as Upland or American cotton (Gossypium hirsutum L.) and Pima or Egyptian cotton (G. barbadense L.). The allotetraploid species diverged from each other and from their diploid progenitors (A or D genome) through selection and domestication after polyploidization. To analyze cotton AD genomes and dissect agronomic traits, we have developed a genetic map in an F₂ population derived from interspecific hybrids between G. hirsutum L. cv. Acala-44 and G. barbadense L. cv. Pima S-7. A total of 392 genetic loci, including 333 amplified fragment length polymorphisms (AFLPs), 47 simple sequence repeats (SSRs), and 12 restriction fragment length polymorphisms (RFLPs), were mapped in 42 linkage groups, which span 3,287 cM and cover approximately 70% of the genome. Using chromosomal aneuploid interspecific hybrids and a set of 29 RFLP and SSR framework markers, we assigned 19 linkage groups involving 223 loci to 12 chromosomes. Comparing four pairs of homoeologous chromosomes, we found that with one exception linkage distances in the A-subgenome chromosomes were larger than those in their D-subgenome homoeologues, reflecting higher recombination frequencies and/or larger chromosomes in the A subgenome. Segregation distortion was observed in 30 out of 392 loci mapped in cotton. Moreover, approximately 29% of the RFLPs behaved as dominant loci, which may result from rapid genomic changes. The cotton genetic map was used for quantitative trait loci (QTL) analysis using composite interval mapping and permutation tests. We detected seven QTLs for six fiber-related traits; five of these were distributed among A-subgenome chromosomes, the genome donor of fiber traits. The detection of QTLs in both the A subgenome in this study and the D subgenome in a previous study suggests that fiber-related traits are controlled by the genes in homoeologous genomes, which are subjected to selection and domestication. Some chromosomes contain clusters of QTLs and presumably contribute to the large amount of phenotypic variation that is present for fiber-related traits.Communicated by J. Dvorak     

Evaluation of linkage disequilibrium and its effect on non-parametric multipoint linkage analysis using two high density single-nucleotide polymorphism mapping panels

Genotype data from the Illumina Linkage III SNP panel (n = 4,720 SNPs) and the Affymetrix 10 k mapping array (n = 11,120 SNPs) were used to test the effects of linkage disequilibrium (LD) between SNPs in a linkage analysis in the Collaborative Study on the Genetics of Alcoholism pedigree collection (143 pedigrees; 1,614 individuals). The average r2 between adjacent markers across the genetic map was 0.099 +/- 0.003 in the Illumina III panel and 0.17 +/- 0.003 in the Affymetrix 10 k array. In order to determine the effect of LD between marker loci in a nonparametric multipoint linkage analysis, markers in strong LD with another marker (r2 > 0.40) were removed (n = 471 loci in the Illumina panel; n = 1,804 loci in the Affymetrix panel) and the linkage analysis results were compared to the results using the entire marker sets. In all analyses using the ALDX1 phenotype, 8 linkage regions on 5 chromosomes (2, 7, 10, 11, X) were detected (peak markers p < 0.01), and the Illumina panel detected an additional region on chromosome 6. Analysis of the same pedigree set and ALDX1 phenotype using short tandem repeat markers (STRs) resulted in 3 linkage regions on 3 chromosomes (peak markers p < 0.01). These results suggest that in this pedigree set, LD between loci with spacing similar to the SNP panels tested may not significantly affect the overall detection of linkage regions in a genome scan. Moreover, since the data quality and information content are greatly improved in the SNP panels over STR genotyping methods, new linkage regions may be identified due to higher information content and data quality in a dense SNP linkage panel.     

Cot-based sampling of genomes for polymorphic low-copy DNA

DNA polymorphisms are powerful tools for many evolutionary and genomic studies in plants including molecular breeding. Single nucleotide polymorphisms (SNPs) are the most elemental DNA marker for genomic studies, but even with advances in DNA sequencing technology, SNP discovery remains costly and computationally demanding, especially in large genomes that are rich in repetitive DNA such as those of many plants. Here we report a method using DNA renaturation kinetics (Cot techniques), sequencing, and BLAST-based screening to identify low-copy, non-coding DNA sequences that were subsequently found to be relatively rich in polymorphisms. A total of of 63 such fragments isolated from a diploid D genome cotton species (Gossypium raimondii) revealed a higher frequency of polymorphisms than that observed for cotton expressed sequence tags or hypomethylated (PstI-susceptible) genomic DNA. While microsatellite-derived loci show still higher polymorphism rates, they often fall in repetitive elements and their sequence analysis is often complicated by alignment difficulties. The potential applications of Cot-filtered noncoding (CFNC) DNA in development of DNA markers are discussed.     

Comparative LD mapping using single SNPs and haplotypes identifies QTL for plant height and biomass as secondary traits of drought tolerance in maize

Drought often delays developmental events so that plant height and above-ground biomass are reduced, resulting in yield loss due to inadequate photosynthate. In this study, plant height and biomass measured by the Normalized Difference Vegetation Index (NDVI) were used as criteria for drought tolerance. A total of 305 lines representing temperate, tropical and subtropical maize germplasm were genotyped using two single nucleotide polymorphism (SNP) chips each containing 1536 markers, from which 2052 informative SNPs and 386 haplotypes each constructed with two or more SNPs were used for linkage disequilibrium (LD) or association mapping. Single SNP- and haplotype-based LD mapping identified two significant SNPs and three haplotype loci [a total of four quantitative trait loci (QTL)] for plant height under well-watered and water-stressed conditions. For biomass, 32 SNPs and 12 haplotype loci (30 QTL) were identified using NDVIs measured at seven stages under the two water regimes. Some significant SNP and haplotype loci for NDVI were shared by different stages. Comparing significant loci identified by single SNP- and haplotype-based LD mapping, we found that six out of the 14 chromosomal regions defined by haplotype loci each included at least one significant SNP for the same trait. Significant SNP haplotype loci explained much higher phenotypic variation than individual SNPs. Moreover, we found that two significant SNPs (two QTL) and one haplotype locus were shared by plant height and NDVI. The results indicate the power of comparative LD mapping using single SNPs and SNP haplotypes with QTL shared by plant height and biomass as secondary traits for drought tolerance in maize.     

Development and evaluation of single-nucleotide polymorphism markers in allotetraploid rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> 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.     

Genome-Wide Association Studies Using Single Nucleotide Polymorphism Markers Developed by Re-Sequencing of the Genomes of Cultivated Tomato

With the aim of understanding relationship between genetic and phenotypic variations in cultivated tomato, single nucleotide polymorphism (SNP) markers covering the whole genome of cultivated tomato were developed and genome-wide association studies (GWAS) were performed. The whole genomes of six tomato lines were sequenced with the ABI-5500xl SOLiD sequencer. Sequence reads covering ∼13.7× of the genome for each line were obtained, and mapped onto tomato reference genomes (SL2.40) to detect ∼1.5 million SNP candidates. Of the identified SNPs, 1.5% were considered to confer gene functions. In the subsequent Illumina GoldenGate assay for 1536 SNPs, 1293 SNPs were successfully genotyped, and 1248 showed polymorphisms among 663 tomato accessions. The whole-genome linkage disequilibrium (LD) analysis detected highly biased LD decays between euchromatic (58 kb) and heterochromatic regions (13.8 Mb). Subsequent GWAS identified SNPs that were significantly associated with agronomical traits, with SNP loci located near genes that were previously reported as candidates for these traits. This study demonstrates that attractive loci can be identified by performing GWAS with a large number of SNPs obtained from re-sequencing analysis.