Cross-species Microsatellite Amplification Research Articles

A novel method for the identification of seahorses (genus Hippocampus) using cross-species amplifiable microsatellites

Seahorses (genus Hippocampus) are flagship species for marine conservation and evolutionary studies. Wild seahorse populations have declined sharply because of overfishing or other disrupting activities, which are attracting increasing global concern for their threatened status. For study of their population status, the identification of young seahorses by morphological examination has been controversial and remains a difficult task due to overlapping of meristic and morphological characters. Here we reported a simple and sensitive method to identify seahorse species from China based on cross-species microsatellites. The cross-species amplification success rate varied from 40.0% to 90.0% depending on the species, with a mean value of 59.6%. According to allele presence/absence of cross-species microsatellites, all the individuals representing 10 seahorse species could be accurately identified using one or more loci. In addition, we found both genome size and genetic distance between the source and target species negatively correlated with microsatellite cross-species amplification success. Moreover, a Neighbor-Joining dendrogram was constructed based on genetic similarity and suggested that the 10 seahorse species could be divided into four groups. These results demonstrate our method could improve the identification of seahorses, providing data to understand seahorse stock structure and recruitment dynamics, and therefore resources conservation.

Development of novel DNA markers for genetic analysis of grey hamsters by cross-species amplification of microsatellites.

The grey hamster has been used in biomedical research for decades. However, effective molecular methods for evaluating the genetic structure of this species are lacking, which hinders its wider usage. In this study, we employed cross-amplification of microsatellite loci of species within the same genus by polymerase chain reaction. Loci screened included 107 from the Mongolian gerbil (MG) and 60 from the Chinese hamster (CH); of these, 15 polymorphic loci were identified for the grey hamster. Of the 167 loci screened, 95 (56.9%) with clear bands on agarose gel were initially identified. After sequencing, 74 (77.9%) of these matched the criteria for microsatellite characteristics, including 41 from MG and 33 from CH. Lastly, 15 (20.3%) loci with more than two alleles for each locus were identified through capillary electrophoresis scanning. To justify the applicability of the 15 grey hamster loci, genetic indexes of grey hamsters were evaluated using 46 generations of outbred stock, established 20 years ago, from Xinjiang, China. Mean effective allele numbers and expected heterozygosity of stock were as low as, respectively, 1.2 and 0.14; these were 2.8 and 4.0 times inferior, respectively, to wild grey hamsters. This finding suggests that the genetic structure of the stock-bred population is too weak to resist artificial and natural selection, mutation and genetic drifting. In conclusion, we have developed de novo microsatellite markers for genetic analysis of the grey hamster, providing data and methodology for the enrichment of a genetic library for this species.

Microsatellite cross-species amplification and utility in southern African elasmobranchs: A valuable resource for fisheries management and conservation.

BackgroundSimilarly to the rest of the world, southern Africa’s diverse chondrichthyan fauna is currently experiencing high fishing pressures from direct and non-direct fisheries to satisfy market demands for shark products such as fins and meat. In this study, the development of microsatellite markers through cross-species amplification of primer sets previously developed for closely related species is reported as an alternative approach to de novo marker development. This included the design of four microsatellite multiplex assays and their cross-species utility in genetic diversity analysis of southern African elasmobranchs. As this study forms part of a larger project on the development of genetic resources for commercially important and endemic southern African species, Mustelus mustelus was used as a candidate species for testing these multiplex assays in down-stream applications.ResultsThirty five microsatellite primer sets previously developed for five elasmobranch species were selected from literature for testing cross-species amplification in 16 elasmobranch species occurring in southern Africa. Cross-species amplification success rates ranged from 28.6%-71.4%. From the successfully amplified microsatellites, 22 loci were selected and evaluated for levels of polymorphism, and four multiplex assays comprising of the 22 microsatellites were successfully constructed, optimised and characterised in a panel of 87 Mustelus mustelus individuals. A total of 125 alleles were observed across all loci, with the number of alleles ranging from 3–12 alleles. Cross-species amplification of the four optimised multiplex assays was further tested on 11 commercially important and endemic southern African elasmobranch species. Percentage of polymorphism ranged from 31.8%-95.5% in these species with polymorphic information content decreasing exponentially with evolutionary distance from the source species.ConclusionsCross-species amplification of the 35 microsatellites proved to be a time- and cost-effective approach to marker development in elasmobranchs and enabled the construction of four novel multiplex assays for characterising genetic diversity in a number of southern African elasmobranch species. This study successfully demonstrated the usefulness of these markers in down-stream applications such as genetic diversity assessment and species identification which could potentially aid in a more integrative, multidisciplinary approach to management and conservation of commercially important cosmopolitan and endemic elasmobranch species occurring in southern Africa.

Cross-species amplification of microsatellites in genera Megalobrama and Parabramis

Cross-species amplification of microsatellites in genera Megalobrama and Parabramis

Erratum to: Cross-species amplification of microsatellites reveals incongruence in the molecular variation and taxonomic limits of the Pilosocereus aurisetus group (Cactaceae)

Erratum to: Cross-species amplification of microsatellites reveals incongruence in the molecular variation and taxonomic limits of the Pilosocereus aurisetus group (Cactaceae)

First genetic linkage map for comparative mapping and QTL screening of brill (Scophthalmus rhombus)

Genetic maps constitute valuable tools to detect genomic regions associated with complex traits and to go forward to understand their genetic basis. Flatfish include several species of great commercial value for which increasing genomic resources are available including genetic maps and EST databases. Application of comparative mapping strategies to flatfish is relevant to obtain genetic information associated with productive traits. The brill (Scophthalmus rhombus) is a flatfish species closely related to turbot (S. maximus) whose meat is highly appreciated in the Spanish market. The Junta de Andalucía local Government has begun a program to adapt this species to captivity for its future production. In this study, we developed the first genetic map of brill using the current turbot genetic map as starting point. This strategy enabled us to select a number of homogeneously distributed markers in the turbot map and to apply cross-species microsatellite amplification to obtain informative markers. Nearly two hundred microsatellites from the framework turbot map were used for validation, and 100 markers were finally informative for mapping. The parents and offspring of the two families (54 and 88, respectively) used to construct the genetic map were genotyped with this panel. All markers, except eleven, were successfully grouped and ordered in 24 linkage groups. Linkage groups and order of markers were highly consistent with the previous turbot genetic map. Linkage map information was used to carry out a preliminary study on growth-related QTL for body weight, length and Fulton's condition factor in the two families, as the main phenotypic traits of interest in this species.

Successive virgin births of viable male progeny in the checkered gartersnake,Thamnophis marcianus

In recent years genotyping analysis using mini- and microsatellite markers has provided robust DNA-based support for facultative parthenogenesis (FP) in several lineages of squamate reptiles (snakes and lizards) and sharks. Rather than incidental cases of reproductive error, there is growing evidence that FP is an alternative reproductive strategy and an important mode of reproduction in these phylogenetically divergent vertebrate groups. Because documentation of FP in vertebrates is in its infancy, additional instances supported by molecular genetic methods provide insights that advance our general understanding of this phenomenon. Here, in a female checkered gartersnake (Thamnophis marcianus) reared in isolation since a juvenile, we describe five successive parthenogenetic litters produced over a 7-year period that resulted in several viable male progeny. Cross species microsatellite amplification was performed across 30 primer pairs derived from Thamnophis spp. and related natricines to the female and nine available progeny. Five loci proved heterozygous in the maternal sample with the progeny differentially homozygous at all but one locus. Combined with evidence pertaining to captive history and litter characteristics, our analysis supports a specific type of FP, terminal fusion automictic parthenogenesis, over the competing hypothesis of long-term sperm storage. Importantly, we document that a single individual was capable of producing successive litters composed of live parthenogens. In two cases, males achieved adulthood and showed the anatomical potential to demonstrate reproductive competence (normal looking hemipenes and testes). © 2012 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 107, 566–572.

Microsatellite cross-species amplification in the genus <i>Centaurea</i> (Compositae)

Microsatellite cross-species amplification in the genus Centaurea (Compositae).- Microsatellites are widely used for population genetic studies although the development of these species-specific markers is costly and time-consuming. One strategy for saving time and money is the use of markers developed for one species (source species) in a different species (target species). This is known as cross-amplification. In the present work, two sets of microsatellites are used to test their transferability to six narrow endemic Centaurea species: i) 16 nuclear loci previously published for three congeneric species and ii) 10 universal chloroplast markers designed from Nicotiana tabacum sequences. Seventeen of the 26 markers tested were transferable and 14 of them were also polymorphic and therefore useful for future works. Nuclear markers were more variable and thus more informative than chloroplast markers. Interspecific amplification performed better for the nuclear loci developed for different Centaurea species than for the universal chloroplast markers developed for Nicotiana tabacum. Likewise, transferability was more successful for the species from sect. Phalolepis than for sect. Lepteranthus. Therefore, our results support the idea that the success of the cross-amplification is influenced by the evolutionary distance between the target and the source species.

Cross transferability of SSRs to five species of Araucariaceae: a useful tool for population genetic studies in Araucaria araucana

Cross-species amplification of microsatellites is a common procedure to obtain suitable markers to be used in population genetic studies. Primers designed for one (source) species are used to amplify homologous loci in related (target) species. It is expected that phylogenetically close species will share a higher proportion of markers, and genetic distance could be a useful parameter to predict successful transferability between different taxonomic groups. We analyzed twenty-two primer pairs developed for Araucaria angustifolia (Bertol.) Kuntze in five target species of the Araucariaceae family. The results were summarized in vectors of presence and absence of bands and compared through the Jaccard similarity index. Using the sequences of eight published genes, genetic distances between pairs of species were estimated and related to transferability rate using Pearson correlations. Successful transfer rate ranged from 31.8 to 77.3%, being these among the highest reported for plants. The highest transfer rate was observed between the South American species. The transferability was confirmed sequencing seven fragments amplified in A. araucana (Molina) K. Koch, and using the best five to estimate genetic diversity parameters in a natural population of this Andean coniferous.

A panel of polymorphic microsatellite markers in Himalayan monal Lophophorus impejanus developed by cross-species amplification and their applicability in other Galliformes

Isolation and development of new microsatellite markers for any species is still labour-intensive and requires substantial inputs of time, money and expertise. Therefore, cross-species microsatellite amplification can be an effec- tive way in obtaining microsatellite loci for closely related taxa in bird species. We have reported microsatellite loci for Himalayan monal for the first time. Fifteen microsatellite markers developed for chicken were cross-amplified in Himalayan monal. All the tested 15 microsatellite markers were polymorphic, with mean (± s.e.) allelic number of 4± 1.51, ranging 2-7 per locus. The observed heterozygosity in the population ranged between 0.285 and 0.714, with mean (± s.e.) of 0.499±0.125, indicating considerable genetic variation in this population. While 12 loci conformed to Hardy-Weinberg equilibrium (P >0.05), 3l oci, i.e. MCW0295, MCW0081, MCW0330 deviated from it (P< 0.05). No evidence for linkage disequilibrium was observed among pair of loci. Our study show that these 15 micro- satellites loci could be employed in population genetic studies for Himalayan monal and their applicability in Jungle Bush Quail, Grey francolin and Kalij pheasant.

A New Problem with Cross-Species Amplification of Microsatellites: Generation of Non-Homologous Products

Microsatellites have been widely used in studies on population genetics, ecology and evolutionary biology. However, microsatellites are not always available for the species to be studied and their isolation could be time-consuming. In order to save time and effort researchers often rely on cross-species amplification. We revealed a new problem of microsatellite cross-species amplification in addition to size homoplasy by analyzing the sequences of electromorphs from seven catfish species belonging to three different families (Clariidae, Heteropneustidae and Pimelodidae). A total of 50 different electromorphs were amplified from the seven catfish species by using primers for 4 microsatellite loci isolated from the species Clarias batrachus. Two hundred and forty PCR-products representing all 50 electromorphs were sequenced and analyzed. Primers for two loci amplified specific products from orthologous loci in all species tested, whereas primers for the other two loci produced specific and polymorphic bands from some non-orthologous loci, even in closely related non-source species. Size homoplasy within the source species was not obvious, whereas extensive size homoplasy across species were detected at three loci, but not at the fourth one. These data suggest that amplification of products from non-orthologous loci and appearance of size homoplasy by cross-amplification are locus dependent, and do not reflect phylogenetic relationship. Amplification of non-orthologous loci and appearance of size homoplasy will lead to obvious complications in phylogenetic interference, population genetic and evolutionary studies. Therefore, we propose that sequence analysis of cross-amplification products should be conducted prior to application of cross-species amplification of microsatellites.

Polymorphic microsatellites in the Reeves's pheasant developed by cross-species amplification

Cross-species microsatellite amplification is an effective way of obtaining microsatellite loci for closely related taxa in bird species. The Reeves's pheasant, Syrmaticus reevesii, is a vulnerable species endemic to China. To improve population genetics and parentage analysis studies in this species, we obtained nine polymorphic microsatellite markers, in addition to the nine markers previously isolated, from the cross-species amplification of 52 markers. The number of alleles per locus varied between two and 12 with expected heterozygosity ranging from 0.298 to 0.714 (n = 107). The success rates of simulated paternity tests using CERVUS software improved at different confidence levels after adding these markers to the previous ones.

Isolation and characterization of polymorphic microsatellite DNA makers for Euphrasia nankotaizanensis (Orobanchaceae) and cross amplification in another Euphrasia L.

Euphrasia nankotaizanensis is an endangered flowering plant distributed restrictedly on rocky slope of high mountain peaks in central and northern Taiwan. In order to undertake a conservation program, especially given impacts of the global warming, it is essential to evaluate its genetic diversity and population structure. We described nine novel microsatellite primer pairs in E. nankotaizanensis and also examined its relative E. transmorrisonensis. The number of alleles per locus ranged from 7 to 29. Expected (H E) and observed (H O) heterozygosities ranged from 0.83 to 0.98 and 0.00 to 0.95, respectively. Eight of the nine microsatellite loci displayed significant departures from Hardy–Weinberg expectations, likely due to the loss of habitats and the small population size. Significant linkage disequilibrium (LD) was detected in most loci. Cross-species amplification of microsatellites took place at five loci. These primers may provide a tool for understanding the demography and population structure of Euphrasia species in Taiwan.

Cross-species amplification of microsatellites in crocodilians: assessment and applications for the future

Microsatellite DNA loci have emerged as the dominant genetic tool for addressing questions associated with genetic diversity in many wildlife species, including crocodilians. Despite their usefulness, their isolation and development can be costly, as well as labour intensive, limiting their wider use in many crocodilian species. In this study, we investigate the cross-species amplification success of 82 existing microsatellites previously isolated for the saltwater crocodile (Crocodylus porosus) in 18 non-target crocodilian species; Alligator sinensis, Caiman crocodylus, Caiman latirostris, Caiman yacare, Melanosuchus niger, Paleosuchus palpebrosus, Crocodylus acutus, Mecistops cataphractus, Crocodylus intermedius, Crocodylus johnstoni, Crocodylus mindorensis, Crocodylus moreletii, Crocodylus niloticus, Crocodylus novaeguineae, Crocodylus palustis, Crocodylus rhombifer, Crocodylus siamensis, and Osteolaemus tetraspis. Our results show a high level of microsatellites cross-amplification making available polymorphic markers for a range of crocodilian species previously lacking informative genetic markers.

Microsatellite cross-species amplification in the genus Littorina and detection of null alleles in Littorina saxatilis

Microsatellite DNA is widely used as population genetic marker, but the cost of using microsatellites is high, as they usually need to be developed and optimized for each species separately. Cross-species amplification of microsatellites is therefore commonly applied to bring down the cost, but it can also involve genotyping errors. We studied cross-species amplification of microsatellites in four species of the Atlantic group of Littorina (Neritrema): L. saxatilis (Olivi, 1792), L. obtusata (Linnaeus, 1758), L. fabalis (Turton, 1825) and L. arcana Hannaford Ellis, 1978 to investigate whether markers originally developed for a more distantly related Pacific species [L. subrotundata (Carpenter, 1864)] suffered from more amplification problems than markers developed for one of the species in the Atlantic group (L. saxatilis). We also compared variation in amplification success among the species and among different regions in the NE Atlantic. Approximately half of the 12 primers developed for L. subrotundata and the seven primers developed for L. saxatilis were successfully amplified in other species of the subgenus. The success was dependent on phylogenetic distance among species within the subgenus. On the other hand, the variation in performance of the loci between geographically remote populations of the same species was as high as variation among the species. In earlier studies statistical analyses indicated that several loci showed a heterozygote deficiency due to null alleles. The presence of null alleles was confirmed by a segregation analysis of the microsatellite loci in eight half-sib families of L. saxatilis.

Characterization and cross-species amplification of microsatellites from the endangered Hawksbill turtle (Eretmochelys imbricate)

Twelve novel polymorphic microsatellites were isolated from the endangered Hawksbill turtle (Eretmochelys imbricate). Eight of 12 markers were used to study genetic diversity of two sea turtle species: E. imbricate and green sea turtle (C. mydas). In E. imbricate, the average allele number of the eight microsatellites was 6.25/locus with a range of 3–13. The average expected and observed heterozygosity was 0.66 and 0.63 respectively. In C. mydas, the average allele number of the eight markers was 11.63/locus. The observed heterozyosity (0.68) was lower than the expected heterozyosity (0.79). Most of 12 microsatellites amplified specific and polymorphic PCR products in other six turtle species. Hence, the developed microsatellites would facilitate studies on genetic diversity and population structure of E. imbricate and other marine turtle species.

Utility of pairwise mtDNA genetic distances for predicting cross-species microsatellite amplification and polymorphism success in fishes

In many studies involving microsatellites cross-species amplification, primers designed for one (source) species are used to amplify homologous loci in related (target) species. However, it is not clear how closely related the species must be to attain significant success. Genetic divergence is a clear and easy way to assess similarity between species and provides an accurate measure of their evolutionary distance. Eight Mediterranean target species of the family Serranidae were analysed using twelve primers developed for Serranus cabrilla. Additionally, two mitochondrial genes (12S rRNA and 16S rRNA) were chosen on the basis of their extensive use in phylogenetic and evolutionary analyses to compute genetic divergence between the species. Significant negative correlations were found between genetic divergence and both cross-species amplification and maintained polymorphism of microsatellite markers, which could be generalized by gathering information from different fish studies. The success of obtaining amplifiable and polymorphic microsatellite loci can be a priori approximated knowing the mtDNA genetic divergence between a given source and target species using our inferred regression equations.

Cross-species microsatellite amplification inVasconcelleaand related genera and their use in germplasm classification

To generate inexpensive and efficient DNA markers for addressing a number of population genetics problems and identification of wild hybrids in Vasconcellea, we have evaluated the use of simple sequence repeat (SSR) primers previously developed for other species. A set of 103 Vasconcellea accessions and some individuals of the related genera Carica and Jacaratia were analyzed with 10 primer pairs directing amplification of chloroplast microsatellites in Nicotiana tabacum and 9 nuclear SSR primer pairs recently identified in Vasconcellea x heilbornii. Heterologous amplification of chloroplast SSRs was successful for 8 of the 10 loci, of which 6 showed polymorphism. Seven of the 9 nuclear SSR primer pairs were useful in Vasconcellea and often also in Jacaratia and Carica, all revealing polymorphism. Exclusive haplotypes for each described taxon were identified based on chloroplast microsatellite data. Clustering based on separate nuclear and chloroplast data resulted in a clear grouping per taxon, but only low resolution was obtained above species level. The codominancy of nuclear SSRs and the general high polymorphism rate of SSR markers will make them more useful in future population genetics studies and diversity assessment in conservation programs.

A low rate of cross-species microsatellite amplification success in Ranid frogs

A low rate of cross-species microsatellite amplification success in Ranid frogs

A wide-range survey of cross-species microsatellite amplification in birds.

The possibility to perform cross-species microsatellite amplification in birds was surveyed by analysing sets of primers developed from the swallow and the pied flycatcher genomes on a panel of 48 different bird species. In total, 162 cases (species/marker combinations) of heterologous amplification were recorded. Ten amplification products were sequenced and all were found to be true homologues of the original loci. There was a significant and negative relationship between microsatellite performance and evolutionary distance between the original species and the tested species. As a rough indicator of expected cross-species microsatellite performance we estimate that 50% of markers will reveal polymorphism in a species with a DNA-DNA hybridization delta T(m)H value of 5 separating it from the original species. This corresponds to a divergence time of = 11 million years before present for passerine birds. The established relationship between performance and evolutionary distance agrees very well with data obtained from some mammalian species. The proportion of polymorphic loci among those markers that amplified decreased with increasing genetic distance, suggesting that few long repeats are preserved during evolution. One of the swallow markers, HrU2, amplified a specific product in all species analyzed and will thus allow access of nuclear sequence data over a broad range of species. The only predictor of cross-species performance was the amount of non-specific amplification seen in the original species. An analysis of 10 species from within the family Hirundinidae with the swallow primers consistently revealed extensive polymorphism with average probabilities of identical genotypes ranging from 6 x 10(-4) to 6 x 10(-7). There were distinct allele frequency differences between the Hirundinidae species and we envisage that microsatellite cross-species amplification will be a useful tool in phylogeny construction and in species identification.