Markers For Population Genetic Studies Research Articles

The complete mitochondrial genome of the duskytail grouper Epinephelus bleekeri (Serranidae: Epinephelinae)

The complete mitochondrial genome of Epinephelus bleekeri was first presented in this study. It is 17,227 bp in length, consisted of 13 protein-coding genes, 22 tRNA genes, 2 rRNA genes and a non-coding control region. The overall base composition of the H-strand is 26.32% A, 28.65% T, 28.72% G and 16.31% C. With the exception of ND6 and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. In particular, gene duplication is found on the tRNA-Asp gene, and five tandem repeat of this gene in E. bleekeri is different from most other vertebrates. The control region is rich in A + T (67.12%) and poor in G (14.00%), and tandem repeat sequences are observed in this non-coding region, indicating a useful marker for population genetic studies.

Sequence variation in Toxoplasma gondii rop17 gene among strains from different hosts and geographical locations.

Genetic diversity of T. gondii is a concern of many studies, due to the biological and epidemiological diversity of this parasite. The present study examined sequence variation in rhoptry protein 17 (ROP17) gene among T. gondii isolates from different hosts and geographical regions. The rop17 gene was amplified and sequenced from 10 T. gondii strains, and phylogenetic relationship among these T. gondii strains was reconstructed using maximum parsimony (MP), neighbor-joining (NJ), and maximum likelihood (ML) analyses. The partial rop17 gene sequences were 1375 bp in length and A+T contents varied from 49.45% to 50.11% among all examined T. gondii strains. Sequence analysis identified 33 variable nucleotide positions (2.1%), 16 of which were identified as transitions. Phylogeny reconstruction based on rop17 gene data revealed two major clusters which could readily distinguish Type I and Type II strains. Analyses of sequence variations in nucleotides and amino acids among these strains revealed high ratio of nonsynonymous to synonymous polymorphisms (>1), indicating that rop17 shows signs of positive selection. This study demonstrated the existence of slightly high sequence variability in the rop17 gene sequences among T. gondii strains from different hosts and geographical regions, suggesting that rop17 gene may represent a new genetic marker for population genetic studies of T. gondii isolates.

Toxoplasma gondii rhoptry protein 38 gene: sequence variation among isolates from different hosts and geographical locations

Toxoplasma gondii is an obligate intracellular parasite that is able to infect almost all mammalian species, and may lead to toxoplasmosis of the host. In the present study, we examined sequence variation in rhoptry protein 38 (ROP38) genes among T. gondii isolates collected from different hosts and geographical regions. The complete ROP38 gene from 13 T. gondii isolates was amplified and sequenced. The results of sequence alignments showed that the lengths of the entire ROP38 gene ranged from 2646 to 2650 bp, with a sequence variation of 0.2-1.1%, among the 13 T. gondii isolates. This result indicated low sequence variation in the ROP38 gene. Phylogenetic analysis of ROP38 sequences using Bayesian inference showed that the clustering of the 13 T. gondii isolates was not consistent with their respective genotypes. This result indicates that the ROP38 gene is not a suitable genetic marker for population genetic studies of different T. gondii genotypes from different hosts and geographical locations, but may represent a potential vaccine candidate against toxoplasmosis, and hence worthy of further research.

Microsatellite Marker Discovery using Single Molecule Real-Time Circular Consensus Sequencing on the Pacific Biosciences RS

Microsatellite sequences are important markers for population genetics studies. In the past, the development of adequate microsatellite primers has been cumbersome. However with the advent of next-generation sequencing technologies, marker identification in genomes of non-model species has been greatly simplified. Here we describe microsatellite discovery on a Pacific Biosciences single molecule real-time sequencer. For the Greater White-fronted Goose (Anser albifrons), we identified 316 microsatellite loci in a single genome shotgun sequencing experiment. We found that the capability of handling large insert sizes and high quality circular consensus sequences provides an advantage over short read technologies for primer design. Combined with a straightforward amplification-free library preparation, PacBio sequencing is an economically viable alternative for microsatellite discovery and subsequent PCR primer design.

Isolation and characterization of polymorphic microsatellite loci from Clathrina aurea (Porifera, Calcarea)

Although microsatellites are the most popular molecular markers for population genetic studies, only a few loci have been characterized for sponges (Porifera). In the present work, we describe the isolation and characterization of seven polymorphic microsatellite loci from an enriched library of the marine calcareous sponge Clathrina aurea. This species is considered endemic to Brazil, where it is widely distributed. However, it was recently found in Peruvian waters. Seven loci were tested in 30 individuals of one population in southeastern Brazil (Cabo Frio). The number of alleles ranged from 5 to 18 (average 12.3 ± 3.9). The species presented high average values of observed and expected heterozygosities: 0.672 ± 0.25 and 0.837 ± 0.08, respectively. No linkage disequilibrium between all pairs of loci was found, but two loci in this population showed a significant excess of homozygotes. This is the second set of loci ever characterized for a calcareous sponge. These loci will be useful for population genetics studies among Brazilian populations, to elucidate the possible introduction of this species in the Pacific Ocean and to analyze other still poorly investigated issues in sponges.

Isolation and characterization of 7 microsatellite loci in the Caribbean gorgonian Antillogorgia elisabethae

We report 7 new polymorphic microsatellite loci of the Caribbean gorgonian Antillogorgia elisabethae, and assess their utility as highly polymorphic markers for population genetic studies. We also found an indel of 4 base pairs in one of the microsatellite flanking region that can be used as an independent locus. A. elisabethae is of special interest because it is actively harvested in The Bahamas, thus characterization of gene flow and genetic diversity is critical for its conservation and management.

New microsatellite loci for Prosopis alba and P. chilensis (Fabaceae).

• Premise of the study: As only six useful microsatellite loci that exhibit broad cross-amplification are so far available for Prosopis species, it is necessary to develop a larger number of codominant markers for population genetic studies. Simple sequence repeat (SSR) markers obtained for Prosopis species from a 454 pyrosequencing run were optimized and characterized for studies in P. alba and P. chilensis.• Methods and Results: Twelve markers that were successfully amplified showed polymorphism in P. alba and P. chilensis. The number of alleles per locus ranged between two and seven and heterozygosity estimates ranged from 0.2 to 0.8. Most of these loci cross-amplify in P. ruscifolia, P. flexuosa, P. kuntzei, P. glandulosa, and P. pallida.• Conclusions: These loci will enable genetic diversity studies of P. alba and P. chilensis and contribute to fine-scale population structure, indirect estimation of relatedness among individuals, and marker-assisted selection.

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome-derived SSRs (CG-SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty-six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG-SSRs showed that these populations did not deviate from linkage equilibrium or Hardy-Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG-SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.

Complete mitochondrial genome of the brain coral Platygyra carnosus

Despite the importance of many species of Faviidae as structural-forming corals, only five species in three genera had a sequenced mitochondrial genome. We report the sixth complete mitochondrial genome in this family from the brain coral Platygyra carnosus, which is 16,463 bp in length and AT rich (67.0%). It has the typical coral mitochondrial gene arrangement, consisting of 13 protein-coding genes, 2 ribosomal RNA (rRNA) genes and 2 transfer RNA (tRNA) genes (tRNA-Met and tRNA-Trp). The ND5 gene is interrupted by a large group I intron which contains 10 protein-coding genes and 12S rRNA. Compared with related species from the same family, the three non-coding regions that are longer than 200 bp are less conserved in sequences than the coding regions, indicating potential markers for population genetic studies.

Isolation and characterization of microsatellite loci for a bioenergy grass, Miscanthus sacchariflorus (Poaceae).

• Premise of the study: Microsatellite loci were developed for the biomass C4 grass, Miscanthus sacchariflorus, and proved to be suitable markers for population genetic studies and germplasm management of this species.• Methods and Results: Twenty-three polymorphic microsatellite loci were identified from an enriched genomic library of M. sacchariflorus. The polymorphism was assessed in 50 individuals from two populations in China. The number of alleles per locus varied from two to 18, with a mean of 8.13. The observed and expected heterozygosities ranged from 0.2 to 1.0 and from 0.198 to 0.898, respectively.• Conclusions: These new markers will be useful for further investigation of genetic diversity and population genetic structure as well as molecular breeding of Miscanthus species.

Isolation and characterization of EST-based microsatellite markers for Scatophagus argus based on transcriptome analysis

In the present study, expressed sequence tag-based microsatellite markers were developed by 454 pyrosequencing for the marine fish Scatophagus argus. A total of 510,939 reads were obtained, and 4,322 di-nucleotides, 1,354 tri-nucleotides, 219 tetra-nucleotides, 30 penta-nucleotides and 9 hexa- nucleotides were detected. Among these repeated sequences, 51 microsatellites where repeat units were greater than 4 were randomly selected to test the variability in natural populations. 12 polymorphic markers were identified and characterized. The number of alleles per locus varies from 3 to 6, and observed and expected heterozygosities ranged from 0.102 to 0.658 and from 0.124 to 0.691, respectively. No genetic linkage and significant departure from Hardy–Weinberg equilibrium were detected among these loci. High-throughput pyrosequencing allows fast and efficient isolation of microsatellite markers on a large scale, and can provide enough microsatellite markers for population genetic studies and genetic mapping.

Genetic diversity among Toxoplasma gondii strains from different hosts and geographical regions revealed by sequence analysis of GRA5 gene

BackgroundToxoplasma gondii is a highly prevalent protozoan parasite infecting a wide range of animals and humans. The epidemiological and biological diversity of T. gondii has resulted in a high genetic variation and unusual population structure in this parasite. This study examined sequence diversity in dense granule 5 (GRA5) gene among T. gondii isolates from different hosts and geographical regions.MethodsThe entire genome region of the GRA5 gene was amplified and sequenced from 14 T. gondii isolates, and phylogenetic relationship among these T. gondii isolates was reconstructed using Bayesian inference (BI) and maximum parsimony (MP) based on the GRA5 sequences.ResultsThe complete sequence of the GRA5 gene was 1614 bp in length for strains TgCatBr5 and MAS, but 1617 bp for the other 12 strains. Sequence analysis identified 41 (0–1.7%) variable nucleotide positions among all isolates, with 18 variations of these being in the coding region. Variable positions in the coding region resulted in 11 amino acid substitutions, and a deletion of 3 bp in the strains TgCatBr5 and MAS leading to the deletion of one amino acid. Sequence variations resulted in the existence of polymorphic restriction sites for endonucleases AatII and MluI, allowing the differentiation of the three major clonal lineage types I, II and III by PCR-RFLP. Phylogenetic analyses using BI and MP supported the clear differentiation of the examined T. gondii strains into their respective genotypes.ConclusionsThis study demonstrated the existence of sequence variability in the GRA5 gene sequence among T. gondii isolates from different hosts and geographical regions, which allowed the differentiation of the examined T. gondii strains into their respective genotypes, suggesting that this highly polymorphic GRA5 locus may provide a new genetic marker for population genetic studies of T. gondii isolates.

Complete mitochondrial genome of the dotted gizzard shad Konosirus punctatus (Teleostei, Clupeidae)

The complete mitochondrial genome of the dotted gizzard shad Konosirus punctatus (Teleostei, Clupeidae) has been determined. The entire sequence is 16,706 bp in length, with a gene content (13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and 1 control region) and organization similar to that observed in most other vertebrates. Overall base composition of the light strand was represented as 25.8% of A, 29.0% of C, 25.1% of T and 20.1% of G. With the exception of ND6 and eight tRNA genes, all other mitochondrial genes are encoded on the heavy strand. Tandem repeat sequences were observed in the control region, indicating a useful marker for population genetic studies.

Development and Characterization of 18 Novel EST-SSRs from the Western Flower Thrips, Frankliniella occidentalis (Pergande)

The western flower thrips, Frankliniella occidentalis (Pergande), is an invasive species and the most economically important pest within the insect order Thysanoptera. For a better understanding of the genetic makeup and migration patterns of F. occidentalis throughout the world, we characterized 18 novel polymorphic EST-derived microsatellites. The mutational mechanism of these EST-SSRs was also investigated to facilitate the selection of appropriate combinations of markers for population genetic studies. Genetic diversity of these novel markers was assessed in 96 individuals from three populations in China (Harbin, Dali, and Guiyang). The results showed that all these 18 loci were highly polymorphic; the number of alleles ranged from 2 to 15, with an average of 5.50 alleles per locus. The observed (HO) and expected (HE) heterozygosities ranged from 0.072 to 0.707 and 0.089 to 0.851, respectively. Furthermore, only two locus/population combinations (WFT144 in Dali and WFT50 in Guiyang) significantly deviated from Hardy–Weinberg equilibrium (HWE). Pairwise FST analysis showed a low but significant differentiation (0.026 < FST < 0.032) among all three pairwise population comparisons. Sequence analysis of alleles per locus revealed a complex mutational pattern of these EST-SSRs. Thus, these EST-SSRs are useful markers but greater attention should be paid to the mutational characteristics of these microsatellites when they are used in population genetic studies.

An Introduction to Microsatellite Development and Analysis

The choice of molecular markers is the first step when selecting experimental plans in the field of population genetics. The popular molecular markers in population genetic studies are mainly allozyme, RAPD, RFLP, AFLP, microsatellite, SNP and ISSR. Among these, microsatellites are frequently found in nuclear, chloroplast and mitochondrial genome, showing a high level of polymorphism and nuclear microsatellites are codominant. Thus, it is a favorable molecular marker for population structure analyses and genetic diversity studies. Microsatellites are composed of tandem repeated 1~6 base pair nucleotide motifs and can be easily amplified by PCR reactions using locus specific primers. Because microsatellites have low cross-species transferability, however, they are only applicable between phylogenetically close species. In wild plants, the lack of genomic information and the high development cost of the microsatellite obstruct the wider use of microsatellites in plant population genetics research. In this review, we introduce the basis for microsatellite markers, the development process, and analytical methods as well as evolutionary models and their applications. In addition, possible genotyping errors which lead to erroneous conclusions are discussed.

Development and multiplexing of microsatellite markers using pyrosequencing in the clonal plant Comarum palustre (Rosaceae)

Microsatellites represent one of the most commonly used genetic markers for population genetic studies. Traditionally, their development is quite time consuming, requiring construction of a genomic library enriched for repeated motifs. Using pyrosequencing, a fast and cost-effective new generation sequencing technique, we produced 24,340,862 bases in 63,860 short fragment reads, including 1170 dinucleotide motifs with a minimum of six repeats and 1383 trinucleotide motifs with a minimum of four repeats for the Marsh Cinquefoil, Comarum palustre L., an endangered marsh pioneer species. We selected 58 loci with SSR (Short Sequence Repeat) segments (at least 10 repeats) for a preliminary screening. Out of them, we screened 29 loci on a capillary sequencer after ligation in a vector and PCR using T7 forward primer labelled with FAM fluorescent dye and the specific unlabeled reverse primers. This procedure allowed us to screen large number of candidate loci with the same labelled primer and unlabelled specific primers. Finally, we characterized 20 polymorphic microsatellite markers, nine dinucleotides and 11 trinucleotides. We used these markers to assess genetic diversity and clonal structure in two Belgian populations. All loci showed a maximum of two alleles per individual, suggesting that they are from a diploid genome. One genet was detected in a newly extending population while 53 different genets in a long-term ecologically managed population. The number of alleles per locus ranged from 6 to 14 in this old population with an expected heterozygosity, ranging from 0.5964 to 0.8278. These preliminary results show a genet size up to 7.2 m.

Identification of lignin genes and regulatory sequences involved in secondary cell wall formation in Acacia auriculiformis and Acacia mangium via de novo transcriptome sequencing

BackgroundAcacia auriculiformis × Acacia mangium hybrids are commercially important trees for the timber and pulp industry in Southeast Asia. Increasing pulp yield while reducing pulping costs are major objectives of tree breeding programs. The general monolignol biosynthesis and secondary cell wall formation pathways are well-characterized but genes in these pathways are poorly characterized in Acacia hybrids. RNA-seq on short-read platforms is a rapid approach for obtaining comprehensive transcriptomic data and to discover informative sequence variants.ResultsWe sequenced transcriptomes of A. auriculiformis and A. mangium from non-normalized cDNA libraries synthesized from pooled young stem and inner bark tissues using paired-end libraries and a single lane of an Illumina GAII machine. De novo assembly produced a total of 42,217 and 35,759 contigs with an average length of 496 bp and 498 bp for A. auriculiformis and A. mangium respectively. The assemblies of A. auriculiformis and A. mangium had a total length of 21,022,649 bp and 17,838,260 bp, respectively, with the largest contig 15,262 bp long. We detected all ten monolignol biosynthetic genes using Blastx and further analysis revealed 18 lignin isoforms for each species. We also identified five contigs homologous to R2R3-MYB proteins in other plant species that are involved in transcriptional regulation of secondary cell wall formation and lignin deposition. We searched the contigs against public microRNA database and predicted the stem-loop structures of six highly conserved microRNA families (miR319, miR396, miR160, miR172, miR162 and miR168) and one legume-specific family (miR2086). Three microRNA target genes were predicted to be involved in wood formation and flavonoid biosynthesis. By using the assemblies as a reference, we discovered 16,648 and 9,335 high quality putative Single Nucleotide Polymorphisms (SNPs) in the transcriptomes of A. auriculiformis and A. mangium, respectively, thus yielding useful markers for population genetics studies and marker-assisted selection.ConclusionWe have produced the first comprehensive transcriptome-wide analysis in A. auriculiformis and A. mangium using de novo assembly techniques. Our high quality and comprehensive assemblies allowed the identification of many genes in the lignin biosynthesis and secondary cell wall formation in Acacia hybrids. Our results demonstrated that Next Generation Sequencing is a cost-effective method for gene discovery, identification of regulatory sequences, and informative markers in a non-model plant.

Distinctive mitochondrial genome of Calanoid copepod Calanus sinicus with multiple large non-coding regions and reshuffled gene order: Useful molecular markers for phylogenetic and population studies

BackgroundCopepods are highly diverse and abundant, resulting in extensive ecological radiation in marine ecosystems. Calanus sinicus dominates continental shelf waters in the northwest Pacific Ocean and plays an important role in the local ecosystem by linking primary production to higher trophic levels. A lack of effective molecular markers has hindered phylogenetic and population genetic studies concerning copepods. As they are genome-level informative, mitochondrial DNA sequences can be used as markers for population genetic studies and phylogenetic studies.ResultsThe mitochondrial genome of C. sinicus is distinct from other arthropods owing to the concurrence of multiple non-coding regions and a reshuffled gene arrangement. Further particularities in the mitogenome of C. sinicus include low A + T-content, symmetrical nucleotide composition between strands, abbreviated stop codons for several PCGs and extended lengths of the genes atp6 and atp8 relative to other copepods. The monophyletic Copepoda should be placed within the Vericrustacea. The close affinity between Cyclopoida and Poecilostomatoida suggests reassigning the latter as subordinate to the former. Monophyly of Maxillopoda is rejected. Within the alignment of 11 C. sinicus mitogenomes, there are 397 variable sites harbouring three 'hotspot' variable sites and three microsatellite loci.ConclusionThe occurrence of the circular subgenomic fragment during laboratory assays suggests that special caution should be taken when sequencing mitogenomes using long PCR. Such a phenomenon may provide additional evidence of mitochondrial DNA recombination, which appears to have been a prerequisite for shaping the present mitochondrial profile of C. sinicus during its evolution. The lack of synapomorphic gene arrangements among copepods has cast doubt on the utility of gene order as a useful molecular marker for deep phylogenetic analysis. However, mitochondrial genomic sequences have been valuable markers for resolving phylogenetic issues concerning copepods. The variable site maps of C. sinicus mitogenomes provide a solid foundation for population genetic studies.

Isolation of microsatellites from an enriched genomic library of the plant-parasitic nematode Meloidogyne incognita and their detection in other root-knot nematode species

The root-knot nematode Meloidogyne incognita is a polyphagous pest distributed from temperate to tropical regions. However, the lack of suitable markers leads to a poor knowledge of its population genetic structure and colonization process. Here we describe the first characterization of 15 microsatellite loci from this nematode, that were developed from an enriched genomic library. Although the variability of these microsatellites was generally low, three of them exhibited a significant level of intrapopulation polymorphism, with three to seven alleles detected. The observed and expected heterozygosities ranged from 0.025 to 0.385 and from 0.024 to 0.779, respectively. Thus, these new microsatellite markers have potential value for the implementation of genotyping experiments in this nematode. Furthermore, successful cross-amplification of the variable microsatellite loci in seven other Meloidogyne species provides the opportunity of using these markers for population genetic studies in these damaging plant-parasitic nematodes.

EST-derived microsatellites in Pseudosciaena crocea and their applicability to related species

Simple sequence repeat (SSR) markers were obtained for the large yellow croaker Pseudosciaena crocea using 1 205 expressed sequences tags (ESTs) from the NCBI database. Primers for 48 ESTSSR loci were designed and screened with 30 P.crocea specimens captured from Guanjingyang sea area in Fujian Province of China. Sixteen of the loci were polymorphic, which were amplified with 3 to 11 alleles per locus and the mean of 6.13. The observed and expected heterozygosity per locus ranged from 0.091 to 0.844 (mean 0.544) and from 0.118 to 0.892 (mean 0.644), respectively. Polymorphic information content (PIC) ranged from 0.115 to 0.866 (mean 0.593). The results for cross-species amplification of the 16 large yellow croaker EST-SSRs on P. polyactis, C. niveatus, C. lucidus, A. argentatus and J. belengeri revealed that 14, 12, 11, 7 and 6 loci were successfully amplified with 1 to 10 alleles with an average of 4.5 per locus, respectively, which are suitable for population genetics studies of these species and useful for phylogenetic relationship analysis among these species. Overall, this study provides a set of type I markers for population genetics studies and genome mapping for large yellow croaker and its closely related species.