Variable Number Of Repeats Research Articles

Atlas of telomeric repeat diversity in Arabidopsis thaliana

BackgroundTelomeric repeat arrays at the ends of chromosomes are highly dynamic in composition, but their repetitive nature and technological limitations have made it difficult to assess their true variation in genome diversity surveys.ResultsWe have comprehensively characterized the sequence variation immediately adjacent to the canonical telomeric repeat arrays at the very ends of chromosomes in 74 genetically diverse Arabidopsis thaliana accessions. We first describe several types of distinct telomeric repeat units and then identify evolutionary processes such as local homogenization and higher-order repeat formation that shape diversity of chromosome ends. By comparing largely isogenic samples, we also determine repeat number variation of the degenerate and variant telomeric repeat array at both the germline and somatic levels. Finally, our analysis of haplotype structure uncovers chromosome end-specific patterns in the distribution of variant telomeric repeats, and their linkage to the more proximal non-coding region.ConclusionsOur findings illustrate the spectrum of telomeric repeat variation at multiple levels in A. thaliana—in germline and soma, across all chromosome ends, and across genetic groups—thereby expanding our knowledge of the evolution of chromosome ends.

In silico and experimental analysis of the repeated domains in BvaP, a protein important for GBS vaginal colonization.

Streptococcus agalactiae (group B strep, GBS) infections in neonates are often fatal and strongly associated with maternal GBS vaginal colonization. Previously, we highlighted the importance of a formerly uncharacterized protein, BvaP, in GBS vaginal colonization. BvaP is highly conserved across GBS and is made up of repeated domains, with a variable number of repeats between strains. Here, we evaluate the prevalence of BvaP repeated domains and their relevance in phenotypes previously associated with vaginal colonization. Using in silico analysis, we found that the number of repeats in the BvaP protein does not generally appear to be associated with serotype, isolation site, or host. Using BvaP truncations in GBS strain A909, we determined that a smaller number of repeats was correlated with decreased bacterial chain length, but adherence to vaginal epithelial cells was complemented using BvaP containing one, two, three, or five repeats. Future research will be geared toward understanding the host immune response to BvaP in vivo and whether vaginal carriage or host response is dependent on the BvaP repeated domains.

Microsatellite Genome-Wide Database Development for the Commercial Blackhead Seabream (Acanthopagrus schlegelii)

Simple sequence repeats (SSRs), the markers with the highest polymorphism and co-dominance degrees, offer a crucial genetic research resource. Limited SSR markers in blackhead seabream have been reported. The availability of the blackhead seabream genome assembly provided the opportunity to carry out genome-wide identification for all microsatellite markers, and bioinformatic analyses open the way for developing a microsatellite genome-wide database in blackhead seabream. In this study, a total of 412,381 SSRs were identified in the 688.08 Mb genome by Krait software. Whole-genome sequences (10×) of 42 samples were aligned against the reference genome and genotyped using the HipSTR tools by comparing and counting repeat number variation across the SSR loci. A total of 156,086 SSRs with a 2-4 bp repeat were genotyped by HipSTR tools, which accounted for 55.78% of the 2-4 bp SSRs in the reference genome. High accuracy of genotyping was observed by comparing HipSTR tools and PCR amplification. A set of 109,131 loci with a number of alleles ≥ 3 and with a number of genotyped individuals ≥ 6 were reserved to constitute the polymorphic SSR database. Fifty-one polymorphic SSR loci were identified through PCR amplification. This strategy to develop polymorphic SSR markers not only obtained a large set of polymorphic SSRs but also eliminated the need for laborious experimental screening. SSR markers developed in this study may facilitate blackhead seabream research, which lays a certain foundation for further gene tagging and genetic linkage analysis, such as marker-assisted selection, genetic mapping, as well as comparative genomic analysis.

Genome-wide distribution of histone trimethylation reveals a global impact of bisphenol A on telomeric binding proteins and histone acetyltransferase factors: a pilot study with human and in vitro data

ObjectiveTo assess the genetic and epigenetic effects promoted by Bisphenol A (BPA) exposure in adolescent males from the Spanish INMA-Granada birth cohort, and in human cells.MethodsDNA methylation was analysed using MEDIP. Repeat number variation in genomic DNA was evaluated, along with the analysis of H3K4me3 by using chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq). Analyses were performed with material extracted from whole blood of the adolescents, complemented by in vitro assessments of human (HeLa) cells exposed to 10 nM BPA, specifically, immunofluorescence evaluation of protein levels, gene expression analysis and ChIP‒qPCR analysis.ResultsAdolescents in the high urinary BPA levels group presented a higher level of Satellite A (SATA) repetitive region copy numbers compared to those in the low BPA group and a tendency towards increase in telomere length. We also observed decreased DNA methylation at the promoters of the imprinted genes H19, KCNQ1, and IGF2; at LINE1 retroelements; and at the ARID2, EGFR and ESRRA and TERT genes. Genome-wide sequencing revealed increased H3K4me3 occupancy at the promoters of genes encoding histone acetyltransferases, telomeric DNA binding factors and DNA repair genes. Results were supported in HeLa cells exposed to 10 nM BPA in vitro. In accordance with the data obtained in blood samples, we observed higher H3K4me3 occupancy and lower DNA methylation at some specific targets in HeLa cells. In exposed cells, changes in the expression of genes encoding DNA repair factors (ATM, ARID2, TRP53) were observed, and increased expression of several genes encoding telomeric DNA binding factors (SMG7, TERT, TEN1, UPF1, ZBTB48) were also found. Furthermore, an increase in ESR1/ERa was observed in the nuclei of HeLa cells along with increased binding of ESR1 to KAT5, KMT2E and TERF2IP promoters and decreased ESR1 binding at the RARA promoter. The DNA damage marker p53/TP53 was also increased.ConclusionIn this pilot study, genome-wide analysis of histone trimethylation in adolescent males exposed to BPA revealed a global impact on the expression of genes encoding telomeric binding proteins and histone acetyltransferase factors with similar results in HeLa cells. Nevertheless, larger studies should confirm our findings.

The long and the short of Periscope Proteins

Bacteria sense, interact with, and modify their environmental niche by deploying a molecular ensemble at the cell surface. The changeability of this exposed interface, combined with extreme changes in the functional repertoire associated with lifestyle switches from planktonic to adherent and biofilm states necessitate dynamic variability. Dynamic surface changes include chemical modifications to the cell wall; export of diverse extracellular biofilm components; and modulation of expression of cell surface proteins for adhesion, co-aggregation and virulence. Local enrichment for highly repetitive proteins with high tandem repeat identity has been an enigmatic phenomenon observed in diverse bacterial species. Preliminary observations over decades of research suggested these repeat regions were hypervariable, as highly related strains appeared to express homologues with diverse molecular mass. Long-read sequencing data have been interrogated to reveal variation in repeat number; in combination with structural, biophysical and molecular dynamics approaches, the Periscope Protein class has been defined for cell surface attached proteins that dynamically expand and contract tandem repeat tracts at the population level. Here, I review the diverse high-stability protein folds and coherent interdomain linkages culminating in the formation of highly anisotropic linear repeat arrays, so-called rod-like protein 'stalks', supporting roles in bacterial adhesion, biofilm formation, cell surface spatial competition, and immune system modulation. An understanding of the functional impacts of dynamic changes in repeat arrays and broader characterisation of the unusual protein folds underpinning this variability will help with the design of immunisation strategies, and contribute to synthetic biology approaches including protein engineering and microbial consortia construction.

Calculation and implementation of sample-wide stochastic thresholds for forensic genetic analysis of STRs and SNPs for massively parallel sequencing platforms

Capillary electrophoresis (CE) analysis of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) use a stochastic threshold to consider the possibility of missing alleles (dropouts) or detecting additional alleles (drop-ins). In CE, this threshold may be approximately 200 RFU, and peak heights are assessed relative to this threshold. In next generation sequencing (NGS), also known as massively parallel sequencing (MPS), STRs are identified by their sequence, and specific alleles are identified by their repeat number and intra-allelic variation. Abundance is approximated by the number of sequence reads for each allele. The total number of reads generated for each marker in a sample depends on factors such as the numbers of samples pooled for sequencing, the number of markers in the assay, the integrity and quantity of the input DNA sample, and the inter-locus balance of the assay. For multiplexes that contain both autosomal and sex-linked markers, the biological sex of the sample also influences total reads per locus. To normalize these variables and better establish a robust stochastic threshold, a sample-wide metric is proposed for estimating the possibility of dropouts or drop-ins based on the variance of the inter-locus balance of the markers across a sample. The intuition is that samples with variable allele balance globally are more likely to have noisier data and therefore require more stringent read count thresholds. This method is robust to sequencing multiplexity, biological sex and manufacturing lot variation.

Identification of a CCG-Enriched Expanded Allele in Patients with Myotonic Dystrophy Type 1 Using Amplification-Free Long-Read Sequencing

Myotonic dystrophy type 1 (DM1) exhibits highly heterogeneous clinical manifestations caused by an unstable CTG repeat expansion reaching up to 4000 CTG. The clinical variability depends on CTG repeat number, CNG repeat interruptions, and somatic mosaicism. Currently, none of these factors are simultaneously and accurately determined due to the limitations of gold standard methods used in clinical and research laboratories. An amplicon method for targeting the DMPK locus using single-molecule real-time sequencing was recently developed to accurately analyze expanded alleles. However, amplicon-based sequencing still depends on PCR, and the inherent bias toward preferential amplification of smaller repeats can be problematic in DM1. Thus, an amplification-free long-read sequencing method was developed by using CRISPR/Cas9 technology in DM1. This method was used to sequence the DMPK locus in patients with CTG repeat expansion ranging from 130 to >1000 CTG. We showed that elimination of PCR amplification improves the accuracy of measurement of inherited repeat number and somatic repeat variations, two key factors in DM1 severity and age at onset. For the first time, an expansion composed of >85% CCG repeats was identified by using this innovative method in a DM1 family with an atypical clinical profile. No-amplification targeted sequencing represents a promising method that can overcome research and diagnosis shortcomings, with translational implications for clinical and genetic counseling in DM1.

Molecular Typing of Methicillin Resistant Staphylococcus aureus using Coa Gene Polymerase Chain Reaction-Restriction Fragment Length Polymorphism: A Cross-sectional Study

Introduction: Methicillin Resistant Staphylococcus aureus (MRSA) are the most important multidrug resistant pathogen of humans causing a wide array of infections. Polymorphic Coagulase gene (coa) could be targeted for specific typing of Staphylococcus aureus isolates. ‘Possible source’ can be identified and discriminated rapidly for control and prevention of Staphylococcus aureus infections especially in case of suspected outbreaks. Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) have high discriminatory power and enhanced reproducibility. Mutation and variation in number of short sequence repeats lead to distinct amplification of this region of coa gene by PCR. Deoxyribonucleic Acid (DNA) fragments of different sizes obtained by digestion with specific restriction enzymes like Arthrobacter luteus I (AluI) help to discriminate various types based on the patterns produced as a result of PCR-RFLP. Aim: To determine molecular typing of MRSA by coa gene PCR-RFLP. Materials and Methods: A cross-sectional study was done in the Department of Microbiology involving 150 clinical isolates of Staphylococcus aureus obtained from various clinical specimens. Genotypic identification of MRSA was done by detecting coa gene and mecA gene by PCR. Molecular typing of MRSA was done by coa gene PCR-RFLP. Results: Coa gene PCR identified six Genotypic codes (Code I to Code VI) ranging from 300 bp-800 bp size. Restriction digestion of the amplicons of the coa gene by PCR-RFLP using the enzyme AluI provided 17 unique restriction patterns among MRSA isolates in toto. The predominant coa gene PCR Genotype code was “Type IV” which was 600 bp size and predominant RFLP fragment for Genotype code “Type IV” was RFLP Pattern ‘b’ (500 bp, 240 bp and 140 bp fragments) among the study population. Conclusion: Genotype code ‘IV’ and RFLP pattern ‘b’ was found to be predominant coa gene type among MRSA in this study

Pervasive cis effects of variation in copy number of large tandem repeats on local DNA methylation and gene expression

Variable number tandem repeats (VNTRs) are composed of large tandemly repeated motifs, many of which are highly polymorphic in copy number. However, because of their large size and repetitive nature, they remain poorly studied. To investigate the regulatory potential of VNTRs, we used read-depth data from Illumina whole-genome sequencing to perform association analysis between copy number of ∼70,000 VNTRs (motif size ≥ 10bp) with both gene expression (404 samples in 48 tissues) and DNA methylation (235 samples in peripheral blood), identifying thousands of VNTRs that are associated with local gene expression (eVNTRs) and DNA methylation levels (mVNTRs). Using an independent cohort, we validated 73%-80% of signals observed in the two discovery cohorts, while allelic analysis of VNTR length and CpG methylation in 30 Oxford Nanopore genomes gave additional support for mVNTR loci, thus providing robust evidence to support that these represent genuine associations. Further, conditional analysis indicated that many eVNTRs and mVNTRs act as QTLs independently of other local variation. We also observed strong enrichments of eVNTRs and mVNTRs for regulatory features such as enhancers and promoters. Using the Human Genome Diversity Panel, we define sets of VNTRs that show highly divergent copy numbers among human populations and show that these are enriched for regulatory effects and preferentially associate with genes that have been linked with human phenotypes through GWASs. Our study provides strong evidence supporting functional variation at thousands of VNTRs and defines candidate sets of VNTRs, copy number variation of which potentially plays a role in numerous human phenotypes.

The Medical Triazole Voriconazole Can Select for Tandem Repeat Variations in Azole-Resistant Aspergillus Fumigatus Harboring TR34/L98H Via Asexual Reproduction.

Azole-resistant Aspergillus fumigatus isolates recovered at high frequency from patients, harbor mutations that are associated with variation of promoter length in the cyp51A gene. Following the discovery of the TR34/L98H genotype, new variations in tandem repeat (TR) length and number of repeats were identified, as well as additional single nucleotide polymorphisms (SNPs) in the cyp51A gene, indicating that the diversity of resistance mutations in A. fumigatus is likely to continue to increase. Investigating the development routes of TR variants is critical to be able to design preventive interventions. In this study, we tested the potential effects of azole exposure on the selection of TR variations, while allowing haploid A. fumigatus to undergo asexual reproduction. Through experimental evolution involving voriconazole (VOR) exposure, an isolate harboring TR343/L98H evolved from a clinical TR34/L98H ancestor isolate, confirmed by whole genome sequencing. TR343/L98H was associated with increased cyp51A expression and high VOR and posaconazole MICs, although additional acquired SNPs could also have contributed to the highly azole-resistant phenotype. Exposure to medical azoles was found to select for TR343, thus supporting the possibility of in-host selection of TR34 variants.

Balancing selection and high genetic diversity of Plasmodium vivax circumsporozoite central region in parasites from Brazilian Amazon and Rio de Janeiro Atlantic Forest.

Circumsporozoite protein (CSP) is the primary pre-erythrocytic vaccine target in Plasmodium species. Knowledge about their genetic diversity can help predict vaccine efficacy and the spread of novel parasite variants. Thus, we investigated pvcsp gene polymorphisms in 219 isolates (136 from Brazilian Amazon [BA], 71 from Rio de Janeiro Atlantic Forest [AF], and 12 from non-Brazilian countries [NB]). Forty-eight polymorphic sites were detected, 46 in the central repeat region (CR), and two in the C-terminal region. Also, the CR presents InDels and a variable number of repeats. All samples correspond to the VK210 variant, and 24 VK210 subtypes based on CR. Nucleotide diversity (π = 0.0135) generated a significant number of haplotypes (168) with low genetic differentiation between the Brazilian regions (Fst = 0.208). The haplotype network revealed similar distances among the BA and AF regions. The linkage disequilibrium indicates that recombination does not seem to be acting in diversity, reinforcing natural selection’s role in accelerating adaptive evolution. The high diversity (low Fst) and polymorphism frequencies could be indicators of balancing selection. Although malaria in BA and AF have distinct vector species and different host immune pressures, consistent genetic signature was found in two regions. The immunodominant B-cell epitope mapped in the CR varies from seven to 19 repeats. The CR T-cell epitope is conserved only in 39 samples. Concerning to C-terminal region, the Th2R epitope presented nonsynonymous SNP only in 6% of Brazilian samples, and the Th3R epitope remained conserved in all studied regions. We conclude that, although the uneven distribution of alleles may jeopardize the deployment of vaccines directed to a specific variable locus, a unique vaccine formulation could protect populations in all Brazilian regions.

The complete mitochondrial genome of Talpa aquitania (Talpidae; Insectivora), a mole species endemic to northern Spain and southern France.

The complete mitogenome sequence of Talpa aquitania, a recently described Talpa species, was assembled using whole-genome sequencing data. It varies in length from 16,776 to 16,846 bp, contains 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA genes, one origin of L-strand replication, and a control region. In the control region, which varied from 1320 to 1390 bp, we identified the extended termination-associated sequence (ETAS-1 and ETAS-2) and the conserved sequence blocks (CSB-1, 2, 3, B, C, D, E, F). In addition, this region includes a 10 bp tandem repeat DNA sequence, with a variable number of repeats that suggest the existence of heteroplasmy. Phylogeny reconstructions based on Maximum Likelihood, Neighbor-joining and Bayesian inference analyses yielded phylogenies with similar topologies demonstrating that T. aquitania and T. occidentalis are sister species.

Small size apolipoprotein(a) isoforms enhance inflammatory and proteolytic potential of collagen-primed monocytes

BackgroundAtherosclerosis is an inflammatory process involving activation of monocytes recruited by various chemoattractant factors, among which lipoprotein(a) and its specific apolipoprotein apo(a). Lp(a) contains a specific apolipoprotein apo(a) which size is determined by a variable number of repeats of a specific structural domain, the kringle IV type 2 (IV-2). Lp(a) plasma concentration and apo(a) size is inversely correlated, and smaller apo(a) are major risk factors for coronary heart disease.Design and methodsThe aim of this study was to evaluate the effect of recombinant apo(a) isoforms (containing 10, 18 or 34 kringles) on monocytes interacting with type I collagen.ResultsApo(a) isoforms stimulated reactive oxygen species (ROS) and matrix metalloproteinase-9 (MMP-9) production by monocytes, and not modified monocytes adhesion on type I collagen. This effect was specific of apo(a) since no effect was observed in the presence of plasminogen and was inversely related to apo(a) size. The lysine analogue 6-aminohexanoic acid which blocks the lysine binding sites (LBS), and carboxypeptidase B (CpB) which cleaves carboxy-terminal lysine residues, abolished apo(a)-induced ROS and MMP-9 production, highlighting an effect mediated by apo(a) lysing-binding sites.ConclusionsThese results indicate that activation of collagen-primed monocytes stimulated with apo(a) is a Kringle number-dependent effect and reinforce the hypothesis of a role for small size apo(a) isoforms in atherothrombosis.

Genome-wide investigation of microsatellite polymorphism in coding region of the giant panda (Ailuropoda melanoleuca) genome: a resource for study of phenotype diversity and abnormal traits

Repeat number variations of coding simple sequence repeats (coding SSRs) generate phenotype diversity for environmental adaptation and rapid adaptive evolution. Despite the positive potential of coding SSR mutations, the repeat variations have also been shown to cause hereditary disorders and severe diseases. However, there have been few reports on the analysis of SSR polymorphism in genome-wide coding regions. We identified 1016 coding SSRs, of which 283 SSRs (27.9%) were polymorphic with 2 to 8 alleles, using 33 re-sequenced giant panda genomes. The 283 polymorphic coding SSRs were mapped on 260 genes, which were assigned into 547 GO terms. Functional analysis of 260 genes showed that four significantly enriched pathways: thyroid hormone signaling pathway, pancreatic secretion, gastric acid secretion, and thyroid hormone synthesis, were relevant to digestion and metabolism. The comparison analysis of gene functions exhibited positive regulation of transcription, DNA-templated term (GO:0045893), the poly(A) RNA-binding term (GO:0008270), and zinc ion–binding term (GO:0044822) which were only significantly enriched in genes containing polymorphic coding SSRs. This indicates coding SSR polymorphism may be involved in binding and regulation functions that can affect the expression of their downstream genes and contribute to diverse phenotypes. Our results provide a solid foundation for further study regarding the effect of coding SSR polymorphism on the adaptation, adaptive evolution, and abnormal traits of the giant panda.

Evolution of salivary glue genes in Drosophila species

BackgroundAt the very end of the larval stage Drosophila expectorate a glue secreted by their salivary glands to attach themselves to a substrate while pupariating. The glue is a mixture of apparently unrelated proteins, some of which are highly glycosylated and possess internal repeats. Because species adhere to distinct substrates (i.e. leaves, wood, rotten fruits), glue genes are expected to evolve rapidly.ResultsWe used available genome sequences and PCR-sequencing of regions of interest to investigate the glue genes in 20 Drosophila species. We discovered a new gene in addition to the seven glue genes annotated in D. melanogaster. We also identified a phase 1 intron at a conserved position present in five of the eight glue genes of D. melanogaster, suggesting a common origin for those glue genes. A slightly significant rate of gene turnover was inferred. Both the number of repeats and the repeat sequence were found to diverge rapidly, even between closely related species. We also detected high repeat number variation at the intrapopulation level in D. melanogaster.ConclusionMost conspicuous signs of accelerated evolution are found in the repeat regions of several glue genes.

Caution for simple sequence repeat number variation in the mitochondrial DNA D-loop to determine cancer-specific variants.

The mitochondrial DNA (mtDNA) displacement loop (D-loop) is often altered in various cancer types, including with regard to simple sequence repeat number variation (SSRNV), which includes the C-tract and CA-tract. However, because of mitochondrial heteroplasmy and slippage errors by the Taq DNA polymerase used in polymerase chain reaction (PCR) analysis, it is difficult to precisely evaluate mtDNA D-loop SSRNV experimentally. In this study, to precisely determine cancer-specific variants in mtDNA SSRNV, various microscopic portions of cancerous tissues and normal control tissues were obtained from a patient with breast cancer, followed by laser-capture microdissection of formalin-fixed paraffin-embedded specimens. Regions containing (CA)7 repeats (positions 514-523) and (C)8 repeats (positions 303-315) of the mitochondria DNA D-loop were amplified and sequenced. Variant signals of mtDNA SSRs of (CA)7 and (C)8 were observed in normal and cancerous tissues, with the content of minor alleles (CA)6 and (C)7/(C)9 differing among samples. These results were confirmed by PCR using various primers and proofreading DNA polymerases. PCR of genomic SSRs of (CA)7 in the NAALD2 gene and (C)8 in the BMP6 gene showed a simple repeat in all samples that was different from the observed mtDNA SSRNV. The present study suggests a reliable procedure for determining cancer-specific variants in mtDNA SSRNV: Using a proofreading DNA polymerase for PCR, the background of slippage by PCR is determined by PCR of the same genomic sequence as the target. Due to the varied heteroplasmy level of mtDNA SSRNV among normal tissues, the second background of polymorphic variations should be determined by several normal tissue DNA as PCR templates. Finally, the cancer-specific variant, including its variation frequency, is determined by subtracting the two background signals from the variant signals in cancer. However, care must be taken, as normal heteroplasmy drifts observed in mtDNA SSRNV may complicate such estimations.

The central exons of the human MUC2 and MUC6 mucins are highly repetitive and variable in sequence between individuals

The DNA sequence of the two human mucin genes MUC2 and MUC6 have not been completely resolved due to the repetitive nature of their central exon coding for Proline, Threonine and Serine rich sequences. The exact nucleotide sequence of these exons has remained unknown for a long time due to limitations in traditional sequencing techniques. These are still very poorly covered in new whole genome sequencing projects with the corresponding protein sequences partly missing. We used a BAC clone containing both these genes and third generation sequencing technology, SMRT sequencing, to obtain the full-length contiguous MUC2 and MUC6 tandem repeat sequences. The new sequences span the entire repeat regions with good coverage revealing their length, variation in repeat sequences and their internal organization. The sequences obtained were used to compare with available sequences from whole genome sequencing projects indicating variation in number of repeats and their internal organization between individuals. The lack of these sequences has limited the association of genetic alterations with disease. The full sequences of these mucins will now allow such studies, which could be of importance for inflammatory bowel diseases for MUC2 and gastric ulcer diseases for MUC6 where deficient mucus protection is assumed to play an important role.

Discovery, Characterization, and Linkage Mapping of Simple Sequence Repeat Markers In Hazelnut

From the genome sequence of hazelnut (Corylus avellana), 192 new polymorphic simple sequence repeat (SSR) markers were developed, characterized, and used to investigate genetic diversity in 50 accessions. Next-generation sequencing allows inexpensive sequencing of plant genomes and transcriptomes, and efficient development of polymorphic SSR markers, also known as microsatellite markers, at low cost. A search of the genome sequence of ‘Jefferson’ hazelnut identified 9094 fragments with long repeat motifs of 4, 5, or 6 base pairs (bp), from which polymorphic SSR markers were developed. The repeat regions in the ‘Jefferson’ genome were used as references to which genomic sequence reads of seven additional cultivars were aligned in silico. Visual inspection for variation in repeat number among the aligned reads identified 246 as polymorphic, for which primer pairs were designed. Polymerase chain reaction (PCR) amplification followed by agarose gel separation indicated polymorphism at 195 loci, for which fluorescent forward primers were used to amplify the DNA of 50 hazelnut accessions. Amplicons were post-PCR multiplexed for capillary electrophoresis, and allele sizes were determined for 50 accessions. After eliminating three, 192 were confirmed as polymorphic, and 169 showed only one or two alleles in each of the 50 cultivars, as expected in a diploid. At these 169 SSRs, a total of 843 alleles were found, for an average of 4.99 and a range of 2 to 17 alleles per locus. The mean observed heterozygosity, expected heterozygosity, polymorphism information content, and the frequency of null alleles were 0.51, 0.53, 0.47, and 0.03, respectively. An additional 25 primer pairs produced more than two bands in some accessions with an average of 6.8 alleles. The UPGMA dendrogram revealed a wide genetic diversity and clustered the 50 accessions according to their geographic origin. Of the new SSRs, 132 loci were placed on the linkage map. These new markers will be useful for diversity and parentage studies, cultivar fingerprinting, marker-assisted selection, and aligning the linkage map with scaffolds of the genome sequence.

GtTR: Bayesian estimation of absolute tandem repeat copy number using sequence capture and high throughput sequencing

BackgroundTandem repeats comprise significant proportion of the human genome including coding and regulatory regions. They are highly prone to repeat number variation and nucleotide mutation due to their repetitive and unstable nature, making them a major source of genomic variation between individuals. Despite recent advances in high throughput sequencing, analysis of tandem repeats in the context of complex diseases is still hindered by technical limitations. We report a novel targeted sequencing approach, which allows simultaneous analysis of hundreds of repeats. We developed a Bayesian algorithm, namely – GtTR - which combines information from a reference long-read dataset with a short read counting approach to genotype tandem repeats at population scale. PCR sizing analysis was used for validation.ResultsWe used a PacBio long-read sequenced sample to generate a reference tandem repeat genotype dataset with on average 13% absolute deviation from PCR sizing results. Using this reference dataset GtTR generated estimates of VNTR copy number with accuracy within 95% high posterior density (HPD) intervals of 68 and 83% for capture sequence data and 200X WGS data respectively, improving to 87 and 94% with use of a PCR reference. We show that the genotype resolution increases as a function of depth, such that the median 95% HPD interval lies within 25, 14, 12 and 8% of the its midpoint copy number value for 30X, 200X WGS, 395X and 800X capture sequence data respectively. We validated nine targets by PCR sizing analysis and genotype estimates from sequencing results correlated well with PCR results.ConclusionsThe novel genotyping approach described here presents a new cost-effective method to explore previously unrecognized class of repeat variation in GWAS studies of complex diseases at the population level. Further improvements in accuracy can be obtained by improving accuracy of the reference dataset.

Diminishing Returns on Intragenic Repeat Number Expansion in the Production of Signaling Peptides.

Signaling peptides enable communication between cells, both within and between individuals, and are therefore key to the control of complex physiological and behavioral responses. Since their small sizes prevent direct transmission to secretory pathways, these peptides are often produced as part of a larger polyprotein comprising precursors for multiple related or identical peptides; the physiological and behavioral consequences of this unusual gene structure are not understood. Here, we show that the number of mature-pheromone-encoding repeats in the yeast α-mating-factor gene MFα1 varies considerably between closely related isolates of both Saccharomyces cerevisiae and its sister species Saccharomyces paradoxus. Variation in repeat number has important phenotypic consequences: Increasing repeat number caused higher pheromone production and greater competitive mating success. However, the magnitude of the improvement decreased with increasing repeat number such that repeat amplification beyond that observed in natural isolates failed to generate more pheromone, and could actually reduce sexual fitness. We investigate multiple explanations for this pattern of diminishing returns and find that our results are most consistent with a translational trade-off: Increasing the number of encoded repeats results in more mature pheromone per translation event, but also generates longer transcripts thereby reducing the rate of translation—a phenomenon known as length-dependent translation. Length-dependent translation may be a powerful constraint on the evolution of genes encoding repetitive or modular proteins, with important physiological and behavioral consequences across eukaryotes.