Non-coding Markers Research Articles

VARIACIÓN NO CODIFICANTE DEL CROMOSOMA X HUMANO EN POBLACIONES LATINOAMERICANAS: UNA REVISIÓN

The human X-chromosome non-coding markers, such as short tandem repeats (STRs), single nucleotide polymorphisms (SNPs), insertion-deletions (INDELs) and Alu insertions, are useful for revealing relationships among populations and for the identification of individuals. In the last decades, a number of studies have been performed to determine the genetic structure of Latin American populations by using X-chromosome markers. These studies provided useful information regarding the genetic composition of these populations and their relationship with Native American, Asian and European populations. One of the most interesting findings achieved by X-chromosome studies is the bias in the sex ratio of individuals that gave rise to the current Latin American populations, as it was previously observed through the analysis of uniparental markers, and which is undoubtedly evidenced in the differential inheritance of X-chromosome in comparison to autosomes. Besides, the genetic drift process that affected Native American populations is more pronounced in X-chromosome markers than in autosomes. The present review summarizes our current knowledge concerning X-chromosome non-coding polymorphisms studied in Latin American populations. Key words: genetic diversity, INDEL, SNP, STR, Alu insertion

Assessment for Identification of Stelechocarpus burahol and Sister Species Complex of Annonaceae Family Using trnL Intron Sequences

Stelechocarpus burahol (kepel) belongs to the Annonaceae family, and is considered to be a native species to Indonesia which is mainly distributed on the island of Java. However, the plant’s existence is currently difficult to find, so it is categorized as rare in Indonesia. A molecular approach using DNA barcoding technique is significant to assist plant identification. The gene that is widely used and proven to be accurate for Annonaceae is trnL-F. This study was aimed to evaluate the efficiency of trnL as DNA barcode for the identification of S. burahol and its relatives (Annonaceae) from Java Island. In total 10 specimens have been used in this study. Whole genome DNA was isolated by Tiangen kit and amplified by Polymerase Chain Reaction (PCR) technique using a specific primer. Results showed that trnL was easily amplified with a DNA fragment length of 500-600 bp. The trnL amplicons have successfully sequenced as indicated by the high QV20+ values. The sequence compositions were high in AT bases (63.9%). BLAST analysis of the sequences showed that S. burahol and sister species have been confirmed its identity according to the reference sequences in NCBI with query cover identities 98%-100%. This research can be concluded that trnL-F is suitable and recommended as a DNA barcode for S. burahol and its relatives. However, further research is suggested to combine analysis of both coding (rbcL, matK, etc) and non-coding (trnL) markers for better identification results.

Inhibitory role of LINC00332 in gastric cancer progression through regulating cell EMT and stemness

ObjectiveGastric cancer (GC) is one of the most common lethal malignancies worldwide. The molecular mechanisms underlying GC early detection are poorly understood. Identifying potential coding and non-coding markers and related pathways in the GC progression is essential. Some Long non-coding RNAs (lncRNAs) reportedly play vital roles during gastric GC development. However, the clinical significance and biological function of LINC00332 in GC remain largely unclear. MethodsThe gene expression patterns of GC from an RNAseq dataset (GSE122401) were retrieved from the Gene Expression Omnibus (GEO) database to recognize differentially expressed genes (DEGs) and lncRNAs (DELs) between normal and GC samples through several bioinformatic analysis. The expression of LINC00332 and MMP-13 as a target gene was quantified in fresh frozen tissues obtained from GC patients. In addition, we investigated the potential function of LINC00332 in silico and in vitro. ResultsThe expressions of LINC00332 and MMP-13 were significantly downregulated and upregulated in GC tissues, respectively. A significant inverse correlation between LINC00332 and MMP-13 mRNA expression was observed in tumor samples. The mRNA expression level of mesenchymal markers, stem cell factors, and MMP genes were significantly decreased after the LINC00332 ectopic expression, while epithelial markers expression was significantly increased. The LINC00332 overexpression markedly repressed proliferation, migration, and invasion and did not induce apoptosis in AGS cells. In addition, LINC00332 overexpression notably promoted the E-cadherin protein expression. Moreover, LINC00332 significantly decreased the cisplatin resistance. ConclusionOur findings indicated that LINC00332 may be a critical anti-EMT factor and provided a new efficient therapeutic strategy for GC treatment.

The applications and prospects of non-coding RNAs in the diagnosis and treatment of heart failure

Heart failure (HF) is a severe or advanced stage of heart disease with high mortality and readmission rates. The detection of biomarkers plays an important role in the diagnosis, treatment and clinical management of HF. In addition to the traditional biomarkers, the non-coding RNA markers such as miRNA, circRNA, and lncRNA have also shown better future applications in the diagnosis and differential diagnosis of HF, the severity and prognosis assessment, the risk assessment of cardiovascular events after discharge in patients with HF. These biomarkers will also contribute to personalized clinical management of HF. Key words: Heart failure; RNA, untranslated; microRNAs; RNA, long noncoding; Biomarkers

XIST and RPS4Y1 long non-coding RNA transcriptome as sex biomarkers in different body fluids

Background and objectivesSex determination of an individual based on biological fluid evidence is a critical issue in forensic science. RNA has proved valuable in the identification of body fluids and the estimation of stain age. However, it still could not provide sufficient information about their donor. This study aimed to evaluate the potential use of the long non-coding XIST and RPS4Y1 markers in the identification of sex from different body fluids.MethodsSaliva, semen, and peripheral and menstrual blood samples were obtained from apparently healthy volunteers. The expression of XIST and RPS4Y1 was assessed in these samples using real-time RT-PCR.ResultsXIST was detected in female saliva and peripheral and menstrual blood, while RPS4Y1 was detected in male blood and semen.ConclusionXIST and RPS4Y1 could be sex differentiation biomarkers in various body fluids.

Implications of management on immunogenetic variation in the European grayling (Thymallus thymallus)

Implications of management on immunogenetic variation in the European grayling (Thymallus thymallus)

Integral Phylogenomic Approach over Ilex L. Species from Southern South America.

The use of molecular markers with inadequate variation levels has resulted in poorly resolved phylogenetic relationships within Ilex. Focusing on southern South American and Asian species, we aimed at contributing informative plastid markers. Also, we intended to gain insights into the nature of morphological and physiological characters used to identify species. We obtained the chloroplast genomes of I. paraguariensis and I. dumosa, and combined these with all the congeneric plastomes currently available to accomplish interspecific comparisons and multilocus analyses. We selected seven introns and nine IGSs as variable non-coding markers that were used in phylogenomic analyses. Eight extra IGSs were proposed as candidate markers. Southern South American species formed one lineage, except for I. paraguariensis, I. dumosa and I. argentina, which occupied intermediate positions among sampled taxa; Euroasiatic species formed two lineages. Some concordant relationships were retrieved from nuclear sequence data. We also conducted integral analyses, involving a supernetwork of molecular data, and a simultaneous analysis of quantitative and qualitative morphological and phytochemical characters, together with molecular data. The total evidence tree was used to study the evolution of non-molecular data, evidencing fifteen non-ambiguous synapomorphic character states and consolidating the relationships among southern South American species. More South American representatives should be incorporated to elucidate their origin.

Conserved Nonexonic Elements: A Novel Class of Marker for Phylogenomics.

Noncoding markers have a particular appeal as tools for phylogenomic analysis because, at least in vertebrates, they appear less subject to strong variation in GC content among lineages. Thus far, ultraconserved elements (UCEs) and introns have been the most widely used noncoding markers. Here we analyze and study the evolutionary properties of a new type of noncoding marker, conserved nonexonic elements (CNEEs), which consists of noncoding elements that are estimated to evolve slower than the neutral rate across a set of species. Although they often include UCEs, CNEEs are distinct from UCEs because they are not ultraconserved, and, most importantly, the core region alone is analyzed, rather than both the core and its flanking regions. Using a data set of 16 birds plus an alligator outgroup, and 3600–3800 loci per marker type, we found that although CNEEs were less variable than bioinformatically derived UCEs or introns and in some cases exhibited a slower approach to branch resolution as determined by phylogenomic subsampling, the quality of CNEE alignments was superior to those of the other markers, with fewer gaps and missing species. Phylogenetic resolution using coalescent approaches was comparable among the three marker types, with most nodes being fully and congruently resolved. Comparison of phylogenetic results across the three marker types indicated that one branch, the sister group to the passerine falcon clade, was resolved differently and with moderate (70%) bootstrap support between CNEEs and UCEs or introns. Overall, CNEEs appear to be promising as phylogenomic markers, yielding phylogenetic resolution as high as for UCEs and introns but with fewer gaps, less ambiguity in alignments and with patterns of nucleotide substitution more consistent with the assumptions of commonly used methods of phylogenetic analysis.

Unstable Linkage of Molecular Markers with Sex Determination Gene in Pacific Salmon (Oncorhynchus spp.).

In the present study, we tested the congruence between the sdY sex-specific marker and other commonly used male markers, located on the Y-chromosome, with the sex phenotypes in 5 species of Pacific salmon in Asian waters, including Chinook, chum, sockeye, masu, and pink salmon. We found that the localization of the sex-specific marker of both males and females of these species is not consistent with the phenotypic sex. Also, no linkage was found between noncoding markers and the sdY gene in the same species samples. Possible genetic and physiological mechanisms underlying this discrepancy are discussed.

Identification, Validation and Classification of the Genus Phyllanthus in Nigeria Using ITS Genetic Marker and the Taxonomic Implication

An extraction, purification, PCR amplification and sequencing of DNA from five species of Phyllanthus in Nigeria namely P. amarus Schum and Thonn, P. urinaria Linn., P. odontadenius Mull-Arg., P. niruroides Mull-Arg. and P. muellerianus (O. Ktze) Excel belonging to the family of Phyllanthaceae were carried out using nuclear ribosomal Internal Transcribed Spacer (ITS 4-5) genetic marker to identify unknown Phyllanthus species. The nuclear region revealed that the Phyllanthus species were able to be amplified optimally for sequencing. The results of the nucleotide sequences were further compared on Basic Local Alignment Sequence Tool (BLAST) on GenBank and BoldSystems for validation. Results revealed that the closely related species, P. niruroides Mull_Arg. and P. odontadenius Mull-Arg. had no DNA record to separate them on both GenBank and BoldSystems while P. amarus Schum and Thonn, P. muellerianus (O. Ktze) Excel and P. urinaria Linn. were clearly compatible with other works. The sequence data were analyzed and classified with tree-based analyses of Mr.Bayes 3.2.1. in order to reveal their phylogenetic relationship. Results of the nucleotide sequences and fragment analysis were published on BoldSystems for barcoding as non-coding marker translation matrix.

105 Identification and characterization of long noncoding RNA markers for prostate cancer detection

105 Identification and characterization of long noncoding RNA markers for prostate cancer detection

A tiered barcode authentication tool to differentiate medicinal Cassia species in India.

DNA barcoding is a desirable tool for medicinal product authentication. DNA barcoding is a method for species identification using short DNA sequences that are conserved within species, but variable between species. Unlike animals, there is no single universal DNA barcode locus for plants. Coding markers, matK and rbcL, and noncoding markers, trnH-psbA (chloroplast) and ITS2 (nuclear), have been reported to be suitable for the DNA barcoding of plants with varying degree of success. Sixty-four accessions from 20 species of the medicinal plant Cassia were collected, and analyzed for these 4 DNA barcoding markers. PCR amplification was 100% successful for all 4 markers, while intra-species divergence was 0 for all 4 Cassia species in which multiple accessions were studied. Assuming 1.0% divergence as the minimum requirement for discriminating 2 species, the 4 markers could only differentiate 15 to 65% of the species studied when used separately. Adding indels to the divergence increased the percentage of species discrimination by trnH-psbA to 90%. In 2-locus barcoding, while matK+rbcL (which is recommended by Consortium for the Barcoding of Life) discriminated 90% of the species, the other combinations of matK+ITS and rbcL+trnH-psbA showed 100% species discrimination. However, matK is plagued with primer issues. The combination of rbcL+trnH-psbA provided the most accurate (100% species ID) and efficient tiered DNA barcoding tool for the authentication of Cassia medicinal products.

Allelic variation in a cellulose synthase gene (PtoCesA4) associated with growth and wood properties in Populus tomentosa.

Lignocellulosic biomass from trees provides a renewable feedstock for biofuels, lumber, pulp, paper, and other uses. Dissecting the mechanism underlying natural variation of the complex traits controlling growth and lignocellulose biosynthesis in trees can enable marker-assisted breeding to improve wood quality and yield. Here, we combined linkage disequilibrium (LD)-based association analysis with traditional linkage analysis to detect the genetic effect of a Populus tomentosa cellulose synthase gene, PtoCesA4. PtoCesA4 is strongly expressed in developing xylem and leaves. Nucleotide diversity and LD in PtoCesA4, sampled from the P. tomentosa natural distribution, revealed that PtoCesA4 harbors high single nucleotide polymorphism (SNP) diversity (πT = 0.0080 and θw = 0.0098) and low LD (r2 ≥ 0.1, within 1400 bp), demonstrating that the potential of a candidate-gene-based LD approach in understanding the molecular basis underlying quantitative variation in this species. By combining single SNP, multi-SNP, and haplotype-based associations in an association population of 460 individuals with single SNP linkage analysis in a family-based linkage populations (1200 individuals), we identified three strong associations (false discovery rate Q < 0.05) in both populations. These include two nonsynonymous markers (SNP49 associated with α-cellulose content and SNP59 associated with fiber width) and a noncoding marker (SNP18 associated with α-cellulose content). Variation in RNA transcript abundance among genotypic classes of SNP49 was confirmed in these two populations. Therefore, combining different methods allowed us to examine functional PtoCesA4 allelic variation underlying natural variation in complex quantitative traits related to growth and lignocellulosic biosynthesis.

Parasite-mediated selection of major histocompatibility complex variability in wild brandt’s voles (Lasiopodomys brandtii) from Inner Mongolia, China

BackgroundGenes of the major histocompatibility complex (MHC) exhibit high levels of variability, which is believed to have arisen through pathogen-mediated selection. We investigated the relationship between parasite load and genetic diversity at selectively neutral, non-coding markers (microsatellites) and adaptive genetic variation at a functionally important part of the MHC in six independent natural populations of Brandt’s voles (Lasiopodomys brandtii) from two regions of the Xilingol Grassland area of Inner Mongolia.ResultsTwo-hundred and fifty-two voles were screened for gastrointestinal parasites, and were assessed for genetic variation. Parasite screening was done through non-invasive fecal egg counts, while allelic diversity was determined via single-stranded conformation polymorphism and DNA sequencing. We detected eight distinct helminth egg morphotypes. A total of 10 microsatellite loci were genotyped and 19 unique MHC class II B alleles were isolated. The rate of nonsynonymous substitutions (dN) exceeded the rate of synonymous substitutions (dS) at putative antigen binding sites of DRB. Neutral and adaptive genetic diversity differed between the six vole populations. To test the main pathogen-driven selection hypotheses for the maintenance of host MHC diversity and parasite species-specific co-evolutionary effects, multivariate approaches (generalized linear mixed models) were used to test for associations between the MHC class II DRB genotype and infections with nematodes. We found no evidence for heterozygote advantage, and overall heterozygosity was lower than expected in the MHC alleles. We identified an association between the parasite load and specific MHC alleles in the voles, and this pattern varied between geographic regions.ConclusionsThe results suggest that MHC variability in Brandt’s voles is maintained by rare allele advantage and fluctuating selection, but the data failed to show any heterozygote advantage effect. Our results add to a growing body of evidence showing that the mode and relative strength of pathogen-driven selection acting on MHC diversity varies within specific wild populations. In addition, our study contributes to the understanding of what maintains MHC diversity, of host-pathogen coevolution and of how genetic diversity is maintained in voles.

Isolation and characterization of novel microsatellite loci for Asian sea bass, Lates calcarifer from genome sequence survey database

Asian sea bass, Lates calcarifer is also known as Barramundi, is an economically important finfish cultured and marketed in southern Asia. It has an extensive natural range from India through the Indo–malaysian archipelago, east to northern Australia and north to southern China (Greenwood 1976; Moore and Reynolds 1982). In Malaysia, domestication of Asian sea bass using cage nets has been established by the Fisheries Department since the 1970s, although locally produced fry only became available in the mid 1980s (Awang 1986). Microsatellite markers are valuable as genetic markers since they are codominant, highly abundant and have high levels of polymorphism (McCouch et al. 1997; Hayden and Sharp 2001). Molecular markers can be divided into type I (coding) markers which are associated with genes of known functions and type II (noncoding) markers which are associated with anonymous genomic sequences (O’Brien 1991). In general, most microsatellites represent type II markers. Nongene sequences are free to mutate, causing high levels of polymorphism, whereas sequences within protein-coding regions generally show low levels of polymorphism due to functional selection pressure (Chistiakov et al. 2006) and are more difficult to develop (Liu et al. 1999). Large-scale sequencing projects have generated the genome sequence survey (GSS) database, offering an alternative means to develop microsatellites in less time at lower cost. GSS sequence databases (www.genome.ukm.my/cbass/) provide a rich source for isolating thousands of sequences containing putative microsatellites, and offer the possibility of developing hundreds of polymorphic microsatellite markers (Chistiakov et al. 2006). Microsatellites for this particular species are extensively available (Yue et al. 2002; Sim and

Phylogenetic Relationships among the Colobine Monkeys Revisited: New Insights from Analyses of Complete mt Genomes and 44 Nuclear Non-Coding Markers

BackgroundPhylogenetic relationships among Asian and African colobine genera have been disputed and are not yet well established. In the present study, we revisit the contentious relationships within the Asian and African Colobinae by analyzing 44 nuclear non-coding genes (>23 kb) and mitochondrial (mt) genome sequences from 14 colobine and 4 non-colobine primates.Principal FindingsThe combined nuclear gene and the mt genome as well as the combined nuclear and mt gene analyses yielded different phylogenetic relationships among colobine genera with the exception of a monophyletic ‘odd-nosed’ group consisting of Rhinopithecus, Pygathrix and Nasalis, and a monophyletic African group consisting of Colobus and Piliocolobus. The combined nuclear data analyses supported a sister-grouping between Semnopithecus and Trachypithecus, and between Presbytis and the odd-nosed monkey group, as well as a sister-taxon association of Pygathrix and Rhinopithecus within the odd-nosed monkey group. In contrast, mt genome data analyses revealed that Semnopithecus diverged earliest among the Asian colobines and that the odd-nosed monkey group is sister to a Presbytis and Trachypithecus clade, as well as a close association of Pygathrix with Nasalis. The relationships among these genera inferred from the analyses of combined nuclear and mt genes, however, varied with the tree-building methods used. Another remarkable finding of the present study is that all of our analyses rejected the recently proposed African colobine paraphyly and hybridization hypothesis and supported reciprocal monophyly of the African and Asian groups.SignificanceThe phylogenetic utility of large-scale new non-coding genes was assessed using the Colobinae as a model, We found that these markers were useful for distinguishing nodes resulting from rapid radiation episodes such as the Asian colobine radiation. None of these markers here have previously been used for colobine phylogenetic reconstruction, increasing the spectrum of molecular markers available to mammalian systematics.

Microsatellites and Alu elements from the human MHC in Valencia (Spain): analysis of genetic relationships and linkage disequilibrium

Two different sets of noncoding markers (microsatellites and Alu elements) from the human chromosome six were analysed in 106 individuals from Valencia (Spain), with the aim of exploring the effect of evolutionary forces on the genetic variability of the major histocompatibility complex (MHC) and assessing the potential usefulness of these genetic loci in phylogenetic studies. Linkage disequilibrium (LD) analyses revealed statistically significant associations among markers located in the MHC class I region, and also between the microsatellite D6S2792 and several genetic loci from MHC class I, II and III regions. Results of the Ewens-Watterson test indicated that only D6S2792 showed significant departure from selective neutrality. Despite the paucity of haplotype data in the literature, results of the phylogenetic analyses at world scale (Alu elements) showed that the genetic relationships of Valencia were mainly determined by the ethnic ancestry of the populations considered, whereas at European scale (microsatellites) population affinities were strongly influenced by geography. Our findings suggest that noncoding markers from the MHC such as Alu and microsatellite loci might have a potential value as lineage (ancestry) markers in investigations into evolutionary, medical and forensic perspectives.

Abstract 3802: EWSR1: Identification and functional characterization of a novel target gene locus in Lynch syndrome

Abstract Background: Lynch syndrome (also Hereditary Non-Polyposis Colon Cancer, HNPCC) represents the most common, autosomal dominantly inherited cancer predisposition worldwide and accounts for 3-5% of the total colorectal cancer (CRC) burden. It is caused by germline mutations in DNA mismatch repair (MMR) genes (mainly MLH1 and MSH2). MMR deficiency results in microsatellite instability (MSI), i.e. genome-wide accumulation of somatic alterations at repetitive DNA sequence motifs, mostly non-coding microsatellite markers. MSI, used as a diagnostic tool to identify HNPCC-related CRCs, can also affect repeat tracts within or immediately adjacent to the coding sequence of so-called target genes (like TGFbRII, BAX, etc) which are thought to fuel carcinogenesis in HNPCC. In search for novel target gene loci we identified a large poly-T tract, (T)16, in the 3’ untranslated region (3’UTR) of the Ewing sarcoma breakpoint region 1 (EWSR1) gene. Aims: To determine i) type and frequency of instability at the EWSR1 (T)16 in 78 HNPCC and 85 sporadic colorectal cancers and ii) its possible effect on EWS expression Materials and methods: We determined the length of the EWSR1 3’UTR tract motif, (T)16, by PCR amplification and fragment analysis of 78 CRCs from HNPCC patients with identified germline mutation (MLH1: n=50; MSH2: n=28), 85 sporadic microsatellite-stable CRCs as well as 5 CRC cell lines (2 MMR proficient, 3 MMR deficient). EWS protein expression was assessed by immunohistochemistry (IHC) on a tissue microarray and immunoreactivity scored semi-quantitatively. Statistical comparisons were performed using Chi-square or Student's t test where appropriated (two-tailed p values, considering p&amp;lt;0.05 to be statistically significant). Results: Novel alleles at the EWSR1 3’UTR (T)16 tract were observed in all (n=78) HNPCC but none (0/85) of the sporadic CRCs. The type (insertion/deletion) and frequency of somatic alterations were: ins1-2 (2.5%), del1-3 (22.6%), del4 (34.4%), del5 (28.54%) and del&amp;gt;6 (11.92%). Similar observations were made for the MMR-deficient cell lines (HCT116/MLH1: del3/del4; LoVo/MSH2: del4) whereas the MMR-proficient ones were stable. RNA secondary structure prediction suggested gross structural alterations for deletions &amp;gt;4 Ts. IHC showed significant downregulation of EWS expression in sporadic CRCs (p&amp;lt;0.005), but no difference in HNPCC CRCs (p=0.7). Since the (T)16 tract may represent a binding site for AU-rich element binding proteins it could have an effect on EWSR1 mRNA stability/translation (currently being tested). Conclusion: The (T)16 tract in the 3’UTR of the EWSR1 gene represents a novel target gene locus in Lynch syndrome allowing for highly sensitive and specific identification of MMR-deficient CRCs. Moreover, IHC analysis suggests a regulatory role of this locus on EWS protein expression in HNPCC colorectal cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3802. doi:10.1158/1538-7445.AM2011-3802

Remnant native Phragmites australis maintains genetic diversity despite multiple threats

Over the past century, an increasing number of species have been negatively impacted by anthropogenic factors such as habitat disturbance and introduced species. One such plant, Phragmites australis subsp. americanus is a perennial emergent grass found in tidal and inland marshes of the Atlantic coast of the United States. While rarely dominant, it grows in mixed communities and across much of this area its distribution has been reduced dramatically, likely due to eutrophication and the invasion of conspecific P. australis introduced from Europe. In this study, two noncoding cpDNA markers and six microsatellite loci were used to characterize genetic diversity among 58 remnant native P. australis stands from North Carolina to Maine. Five chloroplast DNA haplotypes were identified along with 42 multilocus genotypes. Bayesian exploration detected no population structure (e.g., optimal K = 1), indicating that these individuals form a single population. The analysis also detected no presence of hybrids of native and introduced P. australis in the samples, despite the close proximity of individuals to each other in many cases. These results suggest that the genetic composition of native P. australis across the region remains homogeneous and pure, providing managers with justification for its conservation and a potentially large source of germplasm for use in restoration activities.

Genetic and morphological divergence among Gravel Bank Grasshoppers, Chorthippus pullus (Acrididae), from contrasting environments

Gravel Bank Grasshopper (Chorthippus pullus) populations inhabit two contrasting environments, pebbly gravel banks with scarce vegetation cover in mountainous areas along the Alps and lowland grasslands dominated by Common Heather (Calluna vulgaris). Heath populations of C. pullus have been rediscovered only recently, and show a distribution scattered across Central Europe. The wings are reduced in this species; thus, it has low potential for long-distance dispersal. We used sequence data on a newly developed non-coding nuclear marker from three gravel-bank and four heath populations to test whether grasshoppers from the two environments represent distinct lineages. Gravel-bank populations were studied in southern Germany (Bavaria), heath populations in eastern Germany (Brandenburg and Saxony) and Ukraine. We compared those genetic data with an analysis of variation in a suite of morphometric traits. Finally, we combined genetic and morphometric data to reconstruct a plausible scenario for the ecological shift observed in C. pullus. Our newly developed marker did not sort populations from contrasting environments in two monophyletic lineages. Nevertheless, we found a general lack of gene flow between the gravel-bank and heath populations. There was pronounced variation among populations in morphometric traits. That variation was partially partitioned by habitat type, and populations from the same habitat tended to be more similar than those from different habitats. Our data suggest that heath populations originated through northward expansion from multiple southern European refugia, and that the gravel-bank populations represent one of these sources. Patterns of genetic and morphometric divergence suggest that gravel-bank and heath populations may be in the process of incipient speciation.