Heterozygous Single Nucleotide Polymorphisms Research Articles

Mining of genic SNPs and diversity evaluation of landraces in loquat

With the development of next-generation sequencing technologies, the exploitation of single nucleotide polymorphism (SNP) markers from transcriptome sequences has gained more attention and this kind of marker is widely utilized in plant genetic research. In this study, genic SNP markers derived from transcriptome sequences were identified from 26 loquat cultivars. A total length of 4183bp using 9 sets of primers was sequenced and used for alignments. From this, 103 SNPs, with a frequency of one SNP per 41bp, were found, which were comprised of 55 transitions, 37 transversions, 8 InDels and 3 other types. Among these SNPs, the percentage of transitions and transversions in A/G, C/T, G/T, A/T, G/C and A/C were 31.1% (32), 22.3% (23), 13.6% (14), 11.7% (12), 6.8% (7) and 3.9% (4), respectively. Of these SNP genotypes, 84 were detected as homozygotes in the 26 loquat cultivars, and 8 heterozygous SNPs were found in some cultivars. Cluster analysis of the 26 loquat cultivars was also carried out using the SNPs data, and a phylogenetic tree constructed showed that all the cultivars were separated well, and clustered into several groups which basically corresponds to the genetic background of them. The newly identified genic SNPs in this study will provide powerful tools in molecular studies for cultivar identification, genetic diversity analyses and marker-assisted selection breeding in loquat.

CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting.

CRISPR/Cas9-based therapeutics hold the possibility for permanent treatment of genetic disease. The potency and specificity of this system has been used to target dominantly inherited conditions caused by heterozygous missense mutations through inclusion of the mutated base in the short-guide RNA (sgRNA) sequence. This research evaluates a novel approach for targeting heterozygous single-nucleotide polymorphisms (SNPs) using CRISPR/Cas9. We determined that a mutation within KRT12, which causes Meesmann's epithelial corneal dystrophy (MECD), leads to the occurrence of a novel protospacer adjacent motif (PAM). We designed an sgRNA complementary to the sequence adjacent to this SNP-derived PAM and evaluated its potency and allele specificity both in vitro and in vivo. This sgRNA was found to be highly effective at reducing the expression of mutant KRT12 mRNA and protein in vitro. To assess its activity in vivo we injected a combined Cas9/sgRNA expression construct into the corneal stroma of a humanized MECD mouse model. Sequence analysis of corneal genomic DNA revealed non-homologous end-joining repair resulting in frame-shifting deletions within the mutant KRT12 allele. This study is the first to demonstrate in vivo gene editing of a heterozygous disease-causing SNP that results in a novel PAM, further highlighting the potential for CRISPR/Cas9-based therapeutics.

A Rapid, High-Quality, Cost-Effective, Comprehensive and Expandable Targeted Next-Generation Sequencing Assay for Inherited Heart Diseases.

Thousands of mutations across >50 genes have been implicated in inherited cardiomyopathies. However, options for sequencing this rapidly evolving gene set are limited because many sequencing services and off-the-shelf kits suffer from slow turnaround, inefficient capture of genomic DNA, and high cost. Furthermore, customization of these assays to cover emerging targets that suit individual needs is often expensive and time consuming. We sought to develop a custom high throughput, clinical-grade next-generation sequencing assay for detecting cardiac disease gene mutations with improved accuracy, flexibility, turnaround, and cost. We used double-stranded probes (complementary long padlock probes), an inexpensive and customizable capture technology, to efficiently capture and amplify the entire coding region and flanking intronic and regulatory sequences of 88 genes and 40 microRNAs associated with inherited cardiomyopathies, congenital heart disease, and cardiac development. Multiplexing 11 samples per sequencing run resulted in a mean base pair coverage of 420, of which 97% had >20× coverage and >99% were concordant with known heterozygous single nucleotide polymorphisms. The assay correctly detected germline variants in 24 individuals and revealed several polymorphic regions in miR-499. Total run time was 3 days at an approximate cost of $100 per sample. Accurate, high-throughput detection of mutations across numerous cardiac genes is achievable with complementary long padlock probe technology. Moreover, this format allows facile insertion of additional probes as more cardiomyopathy and congenital heart disease genes are discovered, giving researchers a powerful new tool for DNA mutation detection and discovery.

Analyzing allele specific RNA expression using mixture models

BackgroundMeasuring allele-specific RNA expression provides valuable insights into cis-acting genetic and epigenetic regulation of gene expression. Widespread adoption of high-throughput sequencing technologies for studying RNA expression (RNA-Seq) permits measurement of allelic RNA expression imbalance (AEI) at heterozygous single nucleotide polymorphisms (SNPs) across the entire transcriptome, and this approach has become especially popular with the emergence of large databases, such as GTEx. However, the existing binomial-type methods used to model allelic expression from RNA-seq assume a strong negative correlation between reference and variant allele reads, which may not be reasonable biologically.ResultsHere we propose a new strategy for AEI analysis using RNA-seq data. Under the null hypothesis of no AEI, a group of SNPs (possibly across multiple genes) is considered comparable if their respective total sums of the allelic reads are of similar magnitude. Within each group of “comparable” SNPs, we identify SNPs with AEI signal by fitting a mixture of folded Skellam distributions to the absolute values of read differences. By applying this methodology to RNA-Seq data from human autopsy brain tissues, we identified numerous instances of moderate to strong imbalanced allelic RNA expression at heterozygous SNPs. Findings with SLC1A3 mRNA exhibiting known expression differences are discussed as examples.ConclusionThe folded Skellam mixture model searches for SNPs with significant difference between reference and variant allele reads (adjusted for different library sizes), using information from a group of “comparable” SNPs across multiple genes. This model is particularly suitable for performing AEI analysis on genes with few heterozygous SNPs available from RNA-seq, and it can fit over-dispersed read counts without specifying the direction of the correlation between reference and variant alleles.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1749-0) contains supplementary material, which is available to authorized users.

Abstract 4865: PhyloSpan: using multi-mutation reads to resolve subclonal architectures from heterogeneous tumor samples

Abstract We have developed a new method that uses high-throughput reads that span multiple somatic point mutations to reconstruct multiple, genetically diverse subclonal populations from one or more heterogeneous tumor samples. Subclonal reconstruction algorithms attempt to infer the prevalence and genotype of multiple, genetically-related subclonal populations using the variant allele frequency (VAF) of somatic variants. To date, these algorithms exclusively use data on individual somatic mutations. This restriction greatly reduces their ability to fully resolve phylogenic ambiguities. In some cases, it is possible to determine the mutation status of >1 mutation in a single cell, for example, when single reads cover multiple single nucleotide variants (SNVs). This type of information can add considerable power to the phylogenetic reconstruction of the tumor subclonal population. We have developed the PhyloSpan algorithm which attempts to infer the states of multiple SNVs in single cells, and then exploits that information in subclonal reconstruction. Our algorithm starts with phasing somatic SNVs by looking for reads / read-pairs that cover both a somatic mutation and germline heterozygous single nucleotide polymorphism (SNP). These germline SNPs are often available through profiling of normal tissue. PhyloSpan then identifies SNVs that are on the same chromosome and close enough to be covered by a single read or paired reads. These pairs of mutations provide more phylogenetic certainty than can be found by looking at mutations independently. For example, if those SNVs are found in the same evolutionary branch, then we expect to see some reads containing both mutations. If however, the SNVs are an separate branches then no reads should show both SNVs. PhyloSpan integrates this phylogenetic information, along with information about the VAF of each somatic SNV in order to perform subclonal reconstruction. Incorporating these various types of information requires a rigorous statistical approach, and so we have developed a Bayesian non-parametric tree-based clustering algorithm. This algorithm not only infers the number of subclonal populations and their genotype but also provides a measure of uncertainty about this inference, enabling users to determine which parts of the subclonal reconstruction are certain and which parts remain ambiguous. While the number of SNVs a short-read length distance away from another SNV is small, a handful of such pairs are all that is needed to eliminate a substantial amount of ambiguity in subclonal reconstruction. Furthermore, long read technologies, such as PacBio, can be used to supplement short reads. Our approach generalizes to permit the integration of single-cell sequencing with bulk tumor sequencing. We will present results applying our algorithm to whole genome sequencing data showing the added value of considering multiple SNVs compared to independent SNVs. Citation Format: Amit G. Deshwar, Levi Boyles, Jeff Wintersinger, Paul C. Boutros, Yee Whye Teh, Quaid Morris. PhyloSpan: using multi-mutation reads to resolve subclonal architectures from heterogeneous tumor samples. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4865. doi:10.1158/1538-7445.AM2015-4865

AllelicImbalance: an R/bioconductor package for detecting, managing, and visualizing allele expression imbalance data from RNA sequencing.

BackgroundOne aspect in which RNA sequencing is more valuable than microarray-based methods is the ability to examine the allelic imbalance of the expression of a gene. This process is often a complex task that entails quality control, alignment, and the counting of reads over heterozygous single-nucleotide polymorphisms. Allelic imbalance analysis is subject to technical biases, due to differences in the sequences of the measured alleles. Flexible bioinformatics tools are needed to ease the workflow while retaining as much RNA sequencing information as possible throughout the analysis to detect and address the possible biases.ResultsWe present AllelicImblance, a software program that is designed to detect, manage, and visualize allelic imbalances comprehensively. The purpose of this software is to allow users to pose genetic questions in any RNA sequencing experiment quickly, enhancing the general utility of RNA sequencing. The visualization features can reveal notable, non-trivial allelic imbalance behavior over specific regions, such as exons.ConclusionsThe software provides a complete framework to perform allelic imbalance analyses of aligned RNA sequencing data, from detection to visualization, within the robust and versatile management class, ASEset.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0620-2) contains supplementary material, which is available to authorized users.

Multilocus heterozygosity and coronary heart disease: nested case-control studies in men and women.

BackgroundGeneralized allelic heterozygosity has been proposed to improve reproductive fitness and has been associated with higher blood pressure, but its association with chronic disease is not well characterized.MethodsUsing the Affymetrix Genome-Wide Human 6.0 array, we performed whole genome scans in parallel case-control studies of coronary heart disease (CHD) nested in the Health Professionals Follow-up Study and Nurses’ Health Study. We examined ~700,000 single nucleotide polymorphisms (SNPs) in 435 men with incident CHD and 878 matched controls and 435 women with incident CHD with 931 matched controls. We examined the relationship of genome-wide heterozygosity with risk of incident of CHD and with baseline levels of cardiovascular risk factors.ResultsIn both cohorts, approximately 227650 (SD 2000) SNPs were heterozygous. The number of heterozygous SNPs was not related to risk of CHD in either men or women (adjusted odds ratios per 2000 heterozygous SNPs 1.01 [95% confidence interval, 0.91-1.13] in women and 0.94 [0.84-1.06] in men). We also found no consistent associations of genome-wide heterozygosity with levels of lipids, inflammatory markers, adhesion molecules, homocysteine, adiponectin, or body-mass index.ConclusionsIn these parallel nested case-control studies, we found no relationship of multilocus heterozygosity with risk of CHD or its major risk factors. Studies in other populations are needed to rule out associations with lower levels of heterozygosity.

Genome-wide identification of SSR and SNP markers from the non-heading Chinese cabbage for comparative genomic analyses.

BackgroundNon-heading Chinese cabbage (NHCC), belonging to Brassica, is an important leaf vegetable in Asia. Although genetic analyses have been performed through conventional selection and breeding efforts, the domestication history of NHCC and the genetics underlying its morphological diversity remain unclear. Thus, the reliable molecular markers representative of the whole genome are required for molecular-assisted selection in NHCC.ResultsA total of 20,836 simple sequence repeats (SSRs) were detected in NHCC, containing repeat types from mononucleotide to nonanucleotide. The average density was 62.93 SSRs/Mb. In gene regions, 5,435 SSRs were identified in 4,569 genes. A total of 5,008 primer pairs were designed, and 74 were randomly selected for validation. Among these, 60 (81.08%) were polymorphic in 18 Cruciferae. The number of polymorphic bands ranged from two to five, with an average of 2.70 for each primer. The average values of the polymorphism information content, observed heterozygosity, Hardy-Weinberg equilibrium, and Shannon’s information index were 0.2970, 0.4136, 0.5706, and 0.5885, respectively. Four clusters were classified according to the unweighted pair-group method with arithmetic average cluster analysis of 18 genotypes. In addition, a total of 1,228,979 single nucleotide polymorphisms (SNPs) were identified in the NHCC through a comparison with the genome of Chinese cabbage, and the average SNP density in the whole genome was 4.33/Kb. The number of SNPs ranged from 341,939 to 591,586 in the 10 accessions, and the average heterozygous SNPs ratio was ~42.53%. All analyses showed these markers were high quality and reliable. Therefore, they could be used in the construction of a linkage map and for genetic diversity studies for NHCC in future.ConclusionsThis is the first systematic and comprehensive analysis and identification of SSRs in NHCC and 17 species. The development of a large number of SNP and SSR markers was successfully achieved for NHCC. These novel markers are valuable for constructing genetic linkage maps, comparative genome analysis, quantitative trait locus (QTL) mapping, genome-wide association studies, and marker-assisted selection in NHCC breeding system research.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1534-0) contains supplementary material, which is available to authorized users.

Embryo genome profiling by single-cell sequencing for preimplantation genetic diagnosis in a β-thalassemia family.

The embryonic genome, including genotypes and haplotypes, contains all the information for preimplantation genetic diagnosis, representing great potential for mendelian disorder carriers to conceive healthy babies. We developed a strategy to obtain the full embryonic genome for a β-thalassemia-carrier couple to have a healthy second baby. We carried out sequencing for single blastomere cells and the family trio and further developed the analysis pipeline, including recovery of the missing alleles, removal of the majority of errors, and phasing of the embryonic genome. The final accuracy for homozygous and heterozygous single-nucleotide polymorphisms reached 99.62% and 98.39%, respectively. The aneuploidies of embryos were detected as well. Based on the comprehensive embryonic genome, we effectively performed whole-genome mendelian disorder diagnosis and human leukocyte antigen matching tests. This retrospective study in a β-thalassemia family demonstrates a method for embryo genome recovery through single-cell sequencing, which permits detection of genetic variations in preimplantation genetic diagnosis. It shows the potential of single-cell sequencing technology in preimplantation genetic diagnosis clinical practices.

Introgression browser: high-throughput whole-genome SNP visualization.

Breeding by introgressive hybridization is a pivotal strategy to broaden the genetic basis of crops. Usually, the desired traits are monitored in consecutive crossing generations by marker-assisted selection, but their analyses fail in chromosome regions where crossover recombinants are rare or not viable. Here, we present the Introgression Browser (iBrowser), a bioinformatics tool aimed at visualizing introgressions at nucleotide or SNP (Single Nucleotide Polymorphisms) accuracy. The software selects homozygous SNPs from Variant Call Format (VCF) information and filters out heterozygous SNPs, multi-nucleotide polymorphisms (MNPs) and insertion-deletions (InDels). For data analysis iBrowser makes use of sliding windows, but if needed it can generate any desired fragmentation pattern through General Feature Format (GFF) information. In an example of tomato (Solanum lycopersicum) accessions we visualize SNP patterns and elucidate both position and boundaries of the introgressions. We also show that our tool is capable of identifying alien DNA in a panel of the closely related S. pimpinellifolium by examining phylogenetic relationships of the introgressed segments in tomato. In a third example, we demonstrate the power of the iBrowser in a panel of 597 Arabidopsis accessions, detecting the boundaries of a SNP-free region around a polymorphic 1.17 Mbp inverted segment on the short arm of chromosome 4. The architecture and functionality of iBrowser makes the software appropriate for a broad set of analyses including SNP mining, genome structure analysis, and pedigree analysis. Its functionality, together with the capability to process large data sets and efficient visualization of sequence variation, makes iBrowser a valuable breeding tool.

Variant rs1801157 in the 3'UTR of SDF-1ß does not explain variability of healthy-donor G-CSF responsiveness.

The genetics responsible for the inter-individually variable G-CSF responsiveness remain elusive. A single nucleotide polymorphism (SNP) in the 3’UTR of CXCL12, rs1801157, was implicated in X4-tropic HiV susceptibility and later, in two small studies, in G-CSR responsiveness in patients and donors. The position of the SNP in the 3’UTR together with in-silico predictions suggested differential binding of micro-RNA941 as an underlying mechanism. In a cohort of 515 healthy stem cell donors we attempted to reproduce the correlation of the CXCL12 3’UTR SNP and mobilization responses and tested the role of miR941 in this context. The SNP was distributed with the expected frequency. Mobilization efficiency for CD34+ cells in WT, heterozygous and homozygous SNP individuals was indistinguishable, even after controlling for gender. miR941 expression in non-hematopoietic bone marrow cells was undetectable and miR941 did not interact with the 3’ UTR of CXCL12. Proposed effects of the SNP rs1801157 on G-CSF responsiveness cannot be confirmed in a larger cohort.

WhatsHap: Weighted Haplotype Assembly for Future-Generation Sequencing Reads.

The human genome is diploid, which requires assigning heterozygous single nucleotide polymorphisms (SNPs) to the two copies of the genome. The resulting haplotypes, lists of SNPs belonging to each copy, are crucial for downstream analyses in population genetics. Currently, statistical approaches, which are oblivious to direct read information, constitute the state-of-the-art. Haplotype assembly, which addresses phasing directly from sequencing reads, suffers from the fact that sequencing reads of the current generation are too short to serve the purposes of genome-wide phasing. While future-technology sequencing reads will contain sufficient amounts of SNPs per read for phasing, they are also likely to suffer from higher sequencing error rates. Currently, no haplotype assembly approaches exist that allow for taking both increasing read length and sequencing error information into account. Here, we suggest WhatsHap, the first approach that yields provably optimal solutions to the weighted minimum error correction problem in runtime linear in the number of SNPs. WhatsHap is a fixed parameter tractable (FPT) approach with coverage as the parameter. We demonstrate that WhatsHap can handle datasets of coverage up to 20×, and that 15× are generally enough for reliably phasing long reads, even at significantly elevated sequencing error rates. We also find that the switch and flip error rates of the haplotypes we output are favorable when comparing them with state-of-the-art statistical phasers.

Limiting Dilution Bisulfite Pyrosequencing®: A Method for Methylation Analysis of Individual DNA Molecules in a Single or a Few Cells.

Bisulfite-based methods for DNA methylation analysis of small amounts of DNA from a limited number of cells are technologically challenging. Degradation of genomic DNA by bisulfite treatment, contamination with foreign DNA, and biases in the amplification of individual DNA molecules can generate results, which are not representative of the starting sample. Limiting dilution (LD) bisulfite Pyrosequencing(®) (BSP) is a relatively simple technique to circumvent these problems. The bisulfite-treated DNA of a single or a few cells is diluted to an extent, that only a single DNA target molecule is present in the reaction. Then each individual DNA molecule in the starting sample is separately amplified and analyzed by Pyrosequencing. This allows the detection of rare alleles that are easily masked when pools of DNA target molecules are analyzed. Amplicons containing a heterozygous single nucleotide polymorphism (SNP) allow one to delineate the parental origin of the recovered molecules in addition to their methylation status. The number of cells (DNA target molecules) in the starting sample determines the dilution level and the number of reactions that have to be performed. LD-BSP allows methylation analysis of small cell pools (i.e., 5-10 microdissected cells) and even individual cells. The primers and PCR conditions described here have been successfully employed to analyze the methylation status of up to eight target genes in individual 2-16 cell embryos, germinal vesicle (GV) oocytes, and haploid sperms.

SNP-Based Quantification of Allele-Specific DNA Methylation Patterns by Pyrosequencing®.

The analysis of allele-specific DNA methylation patterns has recently attracted much interest as loci of allele-specific DNA methylation overlap with known risk loci for complex diseases and the analysis might contribute to the fine-mapping and interpretation of non-coding genetic variants associated with complex diseases and improve the understanding between genotype and phenotype. In the presented protocol, we present a method for the analysis of DNA methylation patterns on both alleles separately using heterozygous Single Nucleotide Polymorphisms (SNPs) as anchor for allele-specific PCR amplification followed by analysis of the allele-specific DNA methylation patterns by Pyrosequencing(®). Pyrosequencing is an easy-to-handle, quantitative real-time sequencing method that is frequently used for genotyping as well as for the analysis of DNA methylation patterns. The protocol consists of three major steps: (1) identification of individuals heterozygous for a SNP in a region of interest using Pyrosequencing; (2) analysis of the DNA methylation patterns surrounding the SNP on bisulfite-treated DNA to identify regions of potential allele-specific DNA methylation; and (3) the analysis of the DNA methylation patterns associated with each of the two alleles, which are individually amplified using allele-specific PCR. The enrichment of the targeted allele is re-enforced by modification of the allele-specific primers at the allele-discriminating base with Locked Nucleic Acids (LNA). For the proof-of-principle of the developed approach, we provide assay details for three imprinted genes (IGF2, IGF2R, and PEG3) within this chapter. The mean of the DNA methylation patterns derived from the individual alleles corresponds well to the overall DNA methylation patterns and the developed approach proved more reliable compared to other protocols for allele-specific DNA methylation analysis.

Genome-wide Single Nucleotide Polymorphism-based Assay for Phylogenetic Relationship of the Flammulina velutipes

Genome-wide reanalyzed data of 25 Flammulina strains were compared against the reference genome (KACC42780) to establish a genome-wide single nucleotide polymorphism (SNP). The rate of mapping differences between the strains reflected in the strain variation in its result. Genome-wide SNPs distribution divided into types of homozygous SNP and heterozygous SNP moreover all of the strains demonstrated a wide variation in all of the regions. In the further study of topological relationship between the collected strains, phylogenetic tree was separated into 3 major groups. Group I contained F. velutipes var. related strains of ASI 4062, 4148, 4195. Group 2 contained strains that are different species of ASI 4188 F. elastica, ASI 4190 F. fennae, and ASI 4194 F. rossica. The other 19 strains F. velutipes were classified as a single group. However, further experiment to discriminate its genetic relationship between the white group and brown group did not verify its validity. The inferred tree exhibited a phylogenetic relationship between Korea white fruitbody forming strains of ASI 4210, 4166, 4178 and Japan white fruitbody forming strains of ASI 4209, 4167 confirmed to be genetically closely related.

Low-density oligonucleotide microarrays - A major step in Wilson's disease diagnosis.

Low-density oligonucleotide microarrays - A major step in Wilson's disease diagnosis.

HapTree-X: An Integrative Bayesian Framework for Haplotype Reconstruction from Transcriptome and Genome Sequencing Data.

By running standard genotype calling tools, it is possible to accurately identify the number of “wild type” and “mutant” alleles for each single-nucleotide polymorphism (SNP) site. However, in the case of two heterozygous SNP sites, genotype calling tools cannot determine whether “mutant” alleles from different SNP loci are on the same chromosome or on different homologous chromosomes (i.e. compound heterozygote).

Subtle changes in Plasmodium falciparum infection complexity following enhanced intervention in Malawi

With support from the Global Fund, the United States President's Malaria Initiative (PMI) and other cooperating partners, Malawi is implementing a comprehensive malaria control programme involving indoor residual spraying in targeted districts, universal coverage with insecticide-treated bed nets, use of rapid diagnostic tests to confirm the clinical diagnosis of malaria and use of the highly effective artemisinin-based combination therapy, artemether-lumefantrine (AL), as the first-line treatment for malaria. We genotyped 24 genome-wide single nucleotide polymorphisms (SNPs) in Plasmodium falciparum infections (n=316) sampled from a single location in Malawi before (2006 and 2007) and after enhanced intervention (2008 and 2012). The SNP data generated were used to examine temporal changes in the proportion of multiple-genotype infections (MIs), mean number of heterozygous SNPs within MIs, parasite genetic diversity (expected heterozygosity and genotypic richness), multilocus linkage disequilibrium and effective population size (Ne). While the proportion of MIs, expected heterozygosity, genotypic richness, multilocus linkage disequilibrium and Ne were unchanged over time, the mean number (±standard deviation) of heterozygous SNPs within MIs decreased significantly (p=0.01) from 9(±1) in 2006 to 7(±1) in 2012. These findings indicate that the genetic diversity of P. falciparum malaria parasites in this area remains high, suggesting that only subtle gains, if any, have been made in reducing malaria transmission. Continued surveillance is required to evaluate the impact of malaria control interventions in this area and the rest of Malawi, and to better target control interventions.

Hundreds of SNPs vs. dozens of SSRs: which dataset better characterizes natural clonal lineages in a self-fertilizing fish?

For more than two decades, mitochondrial DNA sequences and simple sequence repeats (SSRs, or microsatellite loci) have served as gold standards in population genetics. More recently, next generation sequencing (NGS) has enabled researchers to address biological questions that can benefit from hundreds or even thousands of nuclear single-nucleotide polymorphisms (SNPs) generated by restriction-site associated DNA sequencing (RAD-seq). Here we compare the performance of SSR and RAD-seq SNP methods to characterize clonal patterns in a self-fertilizing and highly inbred killifish, Kryptolebias marmoratus (mangrove rivulus) in Florida. RAD-seq analyses conducted on 18 inbred lineages of mangrove rivulus obtained from western Florida and a distant location in eastern Florida unveiled 481 polymorphic RAD loci of which 129 were homozygous within individuals and 352 loci were heterozygous in at least one individual. An initial UPGMA phenogram was constructed, based on 32 microsatellite loci, and used as a benchmark for comparisons with SNP-based phenograms, using a number of different criteria for SNP selection. A phenogram produced by the homozygous SNPs was in excellent agreement with the one generated from 32 microsatellite loci. However, heterozygous SNP data and RAD loci with more than one polymorphic site contributed more noise than usable signal and were unable to resolve clades consistently. This is likely due to errors in identifying homologous loci in the absence of a reference genome. In summary, although the RAD data were powerful in distinguishing the clonal lineages identified by SSR analyses, they also carried considerable phylogenetic noise. Our results suggest that RAD-seq methods should be used with caution for inferring fine population structure, and that stringent quality controls are necessary to reduce false phylogenetic signals.

Genotyping by sequencing transcriptomes in an evolutionary pre-breeding durum wheat population

The genetic diversity in durum wheat, Triticum turgidum durum, has been strongly reduced since the domestication of the wild Triticum turgidum dicoccoides. Monitoring durum wheat composite crosses incorporating related tetraploid taxa, such as wild and domesticated emmer wheat, is a suitable evolutionary pre-breeding method. Transcriptome sequencing paves the way for a genomic survey of single nucleotide polymorphisms (SNPs) segregating in such populations, offering the possibility of genotyping by sequencing to use these resources in genome-wide association studies (GWAS) and genomic selection (GS) programs. Evolutionary Pre-breeding pOpulation (EPO) is an evolutionary durum wheat pre-breeding population. Sequencing the transcriptome of 179 durum wheat lines (175 from EPO) led to the detection of 103,262 SNPs on two reference transcriptomes: one from the International Wheat Genome Sequencing Consortium and one assembled de novo on durum wheat. Using strict filtering to remove dubious heterozygous SNPs, EPO genetic diversity was eventually described with 76,188 high-confidence SNPs. The percentage of missing genotyping data depended on the expression level, and 88 individuals out of 175 were genotyped per SNP on average. Using the 3B pseudo-molecule of bread wheat, the transcription and diversity levels were shown to be higher in distal regions than in proximal regions, but SNPs were available throughout the chromosomes. Assuming good synteny with Hordeum, the trend was similar on the 14 chromosomes of the durum wheat genome. EPO hosts a high level of diversity, has a number of SNPs in low linkage disequilibrium (<40 Mb) and would be well suited for GWAS and GS programs.