Application Of Microsatellite Markers Research Articles

Development and application of microsatellite markers in Hippophae rhamnoides subsp. sinensis Rousi (Hippophae rhamnoides L.) based on transcriptome sequencing.

Hippophae rhamnoides subsp. sinensis Rousi is a cold- and drought-tolerant pioneer species with significant economic and ecological value. Evaluating its genetic diversity and population structure is of great importance for guiding the development and utilization of resources. In this study, a total of 41,804 SSRs were generated by transcriptome sequencing of Hippophae rhamnoides subsp. sinensis Rousi. Among the different SSR motif types, mononucleotide repeats (26,972) were the most abundant, followed by trinucleotides, tetranucleotides, and pentanucleotides. 200 pairs of SSR primers were selected to detect polymorphisms, of which 15 pairs primers were selected as validated polymorphic SSRs used for genetic diversity and population structure analysis. A total of 63 alleles were identified with 15 pairs primers, with Nei's genetic diversity index ranged from 0.27 to 0.83 (average: 0.54), and the expected heterozygosity ranged from 0.16 to 0.73 (average: 0.46). The polymorphism information content ranged from 0.23 to 0.81 (average: 0.48). Genetic structure analyses showed that the 10 populations could be broadly categorized into two groups. AMOVA denoted that genetic variations primarily originated from within the populations, with minimal differences observed between the groups, accounting for only 7% of the total genetic variation. This implies that mutation in H. rhamnoides subsp. sinensis Rousi mainly occurred within the populations. The results showed that the 10 populations of H. rhamnoides subsp. sinensis Rousi are rich in genetic diversity, with low levels of population differentiation and a high degree of gene exchange, which should be taken into consideration for the future work of germplasm resource preservation and seedling breeding.

Genetic Profile of Local Buffalo (Bubalus bubalis) Populations in Pacitan and Tuban, East Java, Indonesia Measured by the Molecular Marker of INRA032 Locus

This study focused on the application of microsatellite markers at the INRA032 locus for genetic diversity assessment in buffalo (Bubalus bubalis) populations in Pacitan and Tuban Regencies, East Java, Indonesia. The total number of samples used was 16, each population represented by 8 samples. Genetic diversity assessment parameters include allele frequency, the Polymorphism Information Content (PIC) and heterozygosity. The results showed that based on the INRA032 locus, the Tuban buffalo population had a higher allele frequency range (0.08 to 0.33) than the Pacitan population (0.18 to 0.31). The average PIC value in both populations was 0.39, so it can be concluded that the INRA032 locus is informative enough to detect polymorphisms in both populations. The percentage heterozygosity of the Pacitan buffalo population is 88%, which is higher than the Tuban population at 50%, suggesting that the genetic diversity of the two populations is still quite high despite the decreasing trend in population numbers.

Identification of a novel DFNA5 mutation, IVS7-2 a > G, in a Chinese family with non-syndromic sensorineural hearing loss

Background To date, seven DFNA5 mutations have been reported in families with autosomal dominant non-syndromic hearing loss worldwide. All the mutations cause exon 8 skipping at the mRNA level, that led to the protein truncated and the protein could exert a gain of ototoxic function. Objective In this study, we found an autosomal-dominant non-syndromic hearing loss Chinese pedigree which spanned four generations and comprised 43 members. We want to identify the causative gene and mutation. Methods Application of microsatellite markers on DFNA 23 loci preliminary screening of 25 genes, data were analyzed by linkage analysis. Results We mapped the locus to the region between D7S629 and D7S516 (two-point lod-score of 5.39) with the application of 8 microsatellite markers. By direct sequencing of candidate genes in mapping region, we identified a novel missense mutation ivs7-2 A > G in DFNA5 gene, which was faithfully cosegregated with hearing loss in the family. Conclusion and significance The missense mutation in intron 7 of DFNA5 causes skipping of exon 8, resulting in premature termination of the open reading frame. This type of mutation has repeatedly confirmed that it provides more evidence for the previous view and provides a more solid foundation for future research.

Genetic variations among different generations and cultured populations of Nile Tilapia (Oreochromis niloticus) in Ghana: Application of microsatellite markers

Genetic diversity between and within breeds and populations of fish provides valuable information on the probable response to selection, and towards sustainable aquaculture production. However, due to the widespread introductions of O. niloticus to many regions in Africa, the genetic diversity of wild and cultured populations is now threatened. In the present study, microsatellite (SSRs) markers were used to assess genetic diversity among different Generations and cultured populations of O. niloticus in Ghana. Five Generations of a particular improved strain of O. niloticus, together with three different aquaculture populations were screened with 5sets of microsatellite primers, which produced stable and reproducible amplification and polymorphic patterns. Shannon's information index (I) values provided evidence of high genetic diversity among the Generations and the 3 cultured populations. Whereas no significant genetic differentiation (FST = 0.005), thus, structuring existed among the different Generations of the Akosombo strain, moderate genetic structuring (FST = 0.082) was found among the three different cultured population. Nei's D, phylogenetic tree analysis, also placed the AK and the two other cultured populations of O. niloticus into two different clusters, suggestive of high mixing of stocks and sharing of different genetic backgrounds. Analysis of Molecular variance (AMOVA), showed more genetic divergence existing within populations (95%), than among populations (5%). The microsatellite marker technique employed in this study was found to be a simple and efficient method for genetic diversity studies. The present study thus provides valuable base information regarding the population structure of cultured populations of O. niloticus in Ghana, which would enhance further fish population genetic studies.

Development and application of microsatellite markers within transcription factors in flare tree peony (Paeonia rockii) based on next-generation and single-molecule long-read RNA-seq

Tree peonies native to China are a precious crop with ornamental, medicinal and edible oil properties, of which flare tree peony (Paeonia rockii) is one of the most significant germplasms in Paeonia. The development and application of expressed sequence tag-simple sequence repeat (EST-SSR) markers are very valuable for genetic and breeding applications, but EST-SSR resources for the genus Paeonia are still limited. In this study, we first reported the development of SSRs within transcription factors (TFs) in P. rockii based on next-generation sequencing (NGS) and single-molecule long-read sequencing (SMLRS). A total of 166 EST-SSRs containing six nucleotide repeat types were identified from 959 candidate TFs associated with yield, with an average of one SSR per 5.83 unigenes. In total, 102 (61.45%) pairs of primers produced amplification products in the two RNA-seq cultivars. Among them, 58 (56.86%) pairs of primers from 18 gene families (AP2, bHLH, HSF, etc.) were identified to be polymorphic both in the parents of a linkage mapping population and in eight randomly selected accessions of P. rockii. Further, the 58 EST-SSRs indicated a high level of informativeness with PIC values ranging from 0.32 to 0.91 (mean 0.70) after assessment in 37 tree peony accessions. Transferability studies indicated that the amplification ratio of the 58 pairs of primers ranged from 89.66 to 100% across seven species of Paeonia. In addition, a genetic relationship study was performed in 62 accessions. Cluster analysis using the neighbour-joining (NJ) tree demonstrated that major clusters corresponded to the known pedigree trees. Taken together, these newly developed EST-SSRs have a potential use in the conservation of tree peony germplasm and marker-assisted selection (MAS) breeding.

Application of microsatellite markers for hybrid verification and genetic analysis of oil palm (Elaeis guineensis Jacq.)

The legitimacy of parents and progenies used in crop improvement programmes is vital for any meaningful progress in selection. While acknowledging the shortcomings of controlled pollination in oil palm breeding and commercial seed production, the legitimacy of 20 oil palm progenies from the Nigerian Institute for Oil Palm Research (NIFOR) breeding programme was determined using 16 fluorescently-labeled microsatellite markers. The genotyping of parents and progenies was conducted by capillary electrophoresis using the ABI 3730 DNA Genetic Analyzer (Applied Biosystems, USA). Results revealed a complementary expression of the parents’ alleles in 18 out of the 20 individual progenies screened, confirming their hybridity and genetic identity. The two illegitimate progenies detected could be attributed to pollination and planting errors, respectively. A subset of three sufficiently informative loci (sMg00016, sMg00179 and sMo00102) was identified for routine quality control genotyping. The detection of illegitimate progenies provided ample evidence to substantiate the importance of assessing hybrid fidelity in breeding programmes. Furthermore, the usefulness of microsatellite markers as a reliable technique for routine assessment and unambiguous identification of oil palm crosses was established. The implications of microsatellite- based hybrid identification in oil palm varietal improvement programmes have been adequately discussed.
 Key words: Contamination, Genetic identity, Illegitimacy, NIFOR, Parent-Progeny relationships, seed production

The genetic diversity of wild and cultivated Manila clam ( Ruditapes philippinarum ) revealed by 29 novel microsatellite markers

Background: Microsatellite loci often used as a genetic tool for estimating genetic diversity population variation in a wide variety of different species. The application of microsatellite markers in genetics and breeding includes investigating the genetic differentiation of wild and cultured populations, assessing and determining the genetic relationship of different populations. The aim of this work is to develop several microsatellite markers via highthroughput sequencing and characterize these markers in commercially important bivalve Ruditapes philippinarum. Results: Among the two populations of R. philippinarum studied, 110 alleles were detected. The number of alleles at the cultured population ranged from 3 to 17 (mean NA = 6.897) and wild population ranged from 2 to 15 (mean NA = 6.793). The observed and expected heterozygosities of cultured population ranged from 0.182 to 0.964, and from 0.286 to 0.900, with an average of 0.647 and 0.692, respectively. The observed and expected heterozygosities of wild population ranged from 0.138 to 1.000, and from 0.439 to 0.906, with an average of 0.674 and 0.693, respectively. The polymorphism information content ranged from 0.341 to 0.910 with an average of 0.687. Sixteen and thirteen microsatellite loci deviated significantly from Hardy-Weinberg equilibrium after correction for multiple tests in cultured and wild population, respectively. Conclusions: Twenty-nine novel microsatellite loci were developed using Illumina paired-end shotgun sequencing and characterized in two population of R. philippinarum.

Development and cross-validation of microsatellite markers for Rauvolfia weddeliana Müll.Arg. (Apocynaceae) species complex

Rauvolfia weddeliana is an endemic species restricted to plateau landscapes in South American savannahs. Rapid loss of habitat and expansion of agriculture in Central-West Brazil have critically reduced the original extent of savannahs, representing a major threat to its biotic diversity. Due to the discontinuous distribution of R. weddeliana and the vulnerability of its habitats, it is crucial to estimate the genetic diversity of remaining populations. The application of microsatellite markers is a useful approach with relative low cost and high informative potential for studies related to conservation genetics and population genetics. The development of specific libraries for endangered species may aid future studies about the connectivity of populations, reproductive biology, and genetic diversity. We developed microsatellite markers for R. weddeliana and tested the transferability of the markers to a closely related species, R. gracilis. Ten microsatellite markers were identified, and a set of primers for their amplification is presented. Most identified motifs were dimers, with lengths from 18 to 74 base pairs. Nine markers presented high informative potential (PIC > 0.5). The set of markers developed in this study will support further investigations in population genetics of R. weddeliana and possibility of closely related species.

Genome-wide development and utilization of novel intron-length polymorphic (ILP) markers in Medicago sativa

Alfalfa (Medicago sativa L.) is the most widely cultivated forage legume around the world. Though development and application of microsatellite markers in large-scale was reported in this species, a systematic investigation and large-scale exploitation of intron-length polymorphic (ILP) markers has not been conducted. In the present study, the RNA-Seq sequences of alfalfa were aligned with the genomic sequences of Arabidopsis to predict the position of introns and develop ILP markers in alfalfa. A total of 693 putative ILPs were identified, and 502 ILP markers were successfully developed. Furthermore, 100 ILP markers exhibited relatively high levels of transferability to leguminous (40.0%–83.0%) and non-leguminous (21.0%–22.0%) species. Polymorphisms in 40 randomly selected MsILP markers were evaluated in 21 alfalfa accessions and collectively yielded 169 alleles with an average of 4.7 alleles per locus. The polymorphism information content (PIC) ranged from 0.15 to 0.87 with an average of 0.60, which indicated a high level of polymorphism in the MsILP markers. For the first time, we developed large-scale ILP markers in alfalfa and demonstrated their utility in transferability, which will be valuable for genetic relationship assessments, comparative genomic studies and marker-assisted breeding of leguminous and non-leguminous species.

Genetic Diversity and Redundancy Among Potato Accessions in the Montenegrin Collection as Revealed by Microsatellite Markers

Potato was introduced in Montenegro in the middle of the eighteenth century. Since then it has become the most important crop in plant production. During the period between 2008 and 2010 a total of 52 potato accessions was collected across Montenegro and stored in a national gene bank. In the study reported here 23 accessions from the collection were examined using microsatellite (also known as simple sequence repeats or SSRs) molecular markers with the aim to explore genetic diversity and redundancy within the germplasm. The accessions were selected on the basis of preliminary characterization of all 52 accessions for 11 lightsprout traits. Molecular characterization of 23 accessions by 12 SSR markers was carried out at SASA (Science and Advice for Scottish Agriculture) that manages a database of more than 3000 genetic profiles of potato from Europe and abroad. Comparison of SSR genetic profiles of Montenegrin collection against the existing SASA database allowed us to test the authenticity of the Montenegrin accessions. Out of the 23 accessions examined, 13 showed distinct genetic profiles of which seven showed perfect matching with known cultivars, two profiles showed strong similarity to another two cultivars, and four profiles were found unique with regards to the SASA database. Application of microsatellite markers in this study provided valuable information on the extent of genetic diversity residing within Montenegrin potato germplasm; it gave clear indications of the scale of redundancy within the collection; and helped clarify the identity of the accessions. Four accessions within the collection might incorporate unique variation and will be subjected to further agronomical examinations to assess their potential for breeding purposes.

Development and characterization of microsatellite markers in the African forest elephant (Loxodonta cyclotis)

BackgroundAfrican elephants comprise two species, the savanna elephant (Loxodonta africana) and the forest elephant (L. cyclotis), which are distinct morphologically and genetically. Forest elephants are seriously threatened by poaching for meat and ivory, and by habitat destruction. However, microsatellite markers have thus far been developed only in African savanna elephants and Asian elephants, Elephas maximus. The application of microsatellite markers across deeply divergent lineages may produce irregular patterns such as large indels or null alleles. Thus we developed novel microsatellite markers using DNA from two African forest elephants.FindingsOne hundred microsatellite loci were identified in next generation shotgun sequences from two African forest elephants, of which 53 were considered suitable for testing. Twenty-three microsatellite markers successfully amplified elephant DNA without amplifying human DNA; these were further characterized in 15 individuals from Lope National Park, Gabon. Three of the markers were monomorphic and four of them carried only two alleles. The remaining sixteen polymorphic loci carried from 3 to 8 alleles, with observed heterozygosity ranging from 0.27 to 0.87, expected heterozygosity from 0.40 to 0.86, and the Shannon diversity index from 0.73 to 1.86. Linkage disequilibrium was not detected between loci, and no locus deviated from Hardy–Weinberg equilibrium.ConclusionsThe markers developed in this study will be useful for genetic analyses of the African forest elephant and contribute to their conservation and management.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-016-2167-3) contains supplementary material, which is available to authorized users.

Application of microsatellite markers in identifying genetic relationship among different swine populations in Sri Lanka

Five different swine populations in Sri Lanka including Village pigs, wild boar and exotic breeds such as Large White, Landrace and Duroc were selected for this genetic study. Fifteen microsatellite markers recommended by the FAO (Food and Agriculture Organization) and ISAG (International Society of Animal Genetics) were employed to analyze genetic distance among different swine populations. A total of 120 samples of pigs were collected from various locations in Sri Lanka where village pigs and wild boar could be found. The MICROSAT and PHYLIP computer packages were used to estimate the allelic frequency and construct the phylogenetic tree. The phylogenetic tree was constructed using Neighbor-joining approach with 1000 bootstrap values, The results of the study revealed that 138 alleles were found at 15 loci screened across five swine populations Among them 11 were polymorphic and rest were monomorphic. The polymorphic alleles ranged between 2-4 alleles per locus. The genetic distance between village pig and wild boar (0.091) was closer than between Large White (0.261), Landrace (0.241) and Duroc (0.268). According to the dendogram the village pig and wild boar populations clearly diverged front exotic breeds. In the phylogenetic tree, high distances observed were separating exotic pig populations from the other two populations of village pigs and wild boar. Further, the village pigs and wild boar clustered separately. Based on these findings it is proposed that the village pigs and wild boars may be recognized as distinct population for breeding or conservation purpose. AGRIEAST 2010 (9) p.6-10

Application of microsatellite markers as potential tools for traceability of Girgentana goat breed dairy products

In livestock, breed assignment may play a key role in the certification of products linked to specific breeds. Traceability of farm animals and authentication of their products can contribute to improve breed profitability and sustainability of animal productions with significant impact on the rural economy of particular geographic areas and on breed and biodiversity conservation. With the goal of developing a breed genetic traceability system for Girgentana dairy products, the aim of this study was to identify specific microsatellite markers able to discriminate among the most important Sicilian dairy goat breeds, in order to detect possible adulteration in Girgentana dairy products. A total of 20 microsatellite markers were analyzed on 338 individual samples from Girgentana, Maltese, and Derivata di Siria goat breeds. Specific microsatellite markers useful for traceability of dairy products were identified. Eight microsatellite markers showed alleles present at the same time in Maltese and Derivata di Siria and absent in Girgentana and, therefore, they were tested on DNA pools of the three breeds. Considering the electropherograms' results, only FCB20, SRCRSP5, and TGLA122 markers were tested on DNA samples extracted from cheeses of Girgentana goat breed. These three microsatellite markers could be applied in a breed genetic traceability system of Girgentana dairy products in order to detect adulteration due to Maltese and Derivata di Siria goat breeds.

Development of 5123 Intron-Length Polymorphic Markers for Large-Scale Genotyping Applications in Foxtail Millet

Generating genomic resources in terms of molecular markers is imperative in molecular breeding for crop improvement. Though development and application of microsatellite markers in large-scale was reported in the model crop foxtail millet, no such large-scale study was conducted for intron-length polymorphic (ILP) markers. Considering this, we developed 5123 ILP markers, of which 4049 were physically mapped onto 9 chromosomes of foxtail millet. BLAST analysis of 5123 expressed sequence tags (ESTs) suggested the function for ∼71.5% ESTs and grouped them into 5 different functional categories. About 440 selected primer pairs representing the foxtail millet genome and the different functional groups showed high-level of cross-genera amplification at an average of ∼85% in eight millets and five non-millet species. The efficacy of the ILP markers for distinguishing the foxtail millet is demonstrated by observed heterozygosity (0.20) and Nei's average gene diversity (0.22). In silico comparative mapping of physically mapped ILP markers demonstrated substantial percentage of sequence-based orthology and syntenic relationship between foxtail millet chromosomes and sorghum (∼50%), maize (∼46%), rice (∼21%) and Brachypodium (∼21%) chromosomes. Hence, for the first time, we developed large-scale ILP markers in foxtail millet and demonstrated their utility in germplasm characterization, transferability, phylogenetics and comparative mapping studies in millets and bioenergy grass species.

Thrips tabaci population genetic structure and polyploidy in relation to competency as a vector of tomato spotted wilt virus.

Knowledge of population-level genetic differences can help explain variation among populations of insect vectors in their role in the epidemiology of specific viruses. Variation in competency to transmit Tomato spotted wilt virus (TSWV) that exists among populations of Thrips tabaci has been associated with the presence of cryptic species that exhibit different modes of reproduction and host ranges. However, recent findings suggest that vector competency of T. tabaci at any given location depends on the thrips and virus populations that are present. This study characterizes the population genetic structure of T. tabaci collected from four locations in North Carolina and examines the relationship between population genetic structure and variation in TSWV transmission by T. tabaci. Mitochondrial COI sequence analysis revealed the presence of two genetically distinct groups with one characterized by thelytokous, parthenogenetic reproduction and the other by arrhenotokous, sexual reproduction. Using a set of 11 microsatellite markers that we developed to investigate T. tabaci population genetic structure, we identified 17 clonal groups and found significant genetic structuring among the four NC populations that corresponded to the geographic locations where the populations were collected. Application of microsatellite markers also led to the discovery of polyploidy in this species. All four populations contained tetraploid individuals, and three contained both diploid and tetraploid individuals. Analysis of variation in transmission ofTSWV among isofemale lines initiated with individuals used in this study revealed that ‘clone assignment,’ ‘virus isolate’ and their interaction significantly influenced vector competency. These results highlight the importance of interactions between specific T. tabaci clonal types and specific TSWV isolates underlying transmission of TSWV by T. tabaci.

Cross-species amplification of Megalobrama amblycephala EST-SSR in M.pellegrini and identification of their reciprocal hybrids

To investigate the applicability of microsatellite markers developed from Megalobrama amblycephala in M.pellegrini and find the molecular markers to identify the M.amblycephala,M.pellegrini and their reciprocal hybrids,90 pairs of M.amblycephala EST-SSR primers were synthesized for cross-species amplification on the genome DNA of M.pellegrini,and 26 pairs of microsatellite primers of M.amblycephala and M.pellegrini were screened for PCR amplification on the genome DNA of M.amblycephala,M.pellegrini and their reciprocal hybrids.The results revealed that 76 pairs(84.4%)of the primers were amplified successfully.Fifty pairs were screened from these 76 primers to test the polymorphism in M.pellegrini population and 13 markers were polymorphic with an average of 3.2 alleles per locus,the mean observed heterozygosity being 0.72 and the mean PIC being 0.42.The results of PCR amplification on the genome DNA of M.amblycephala,M.pellegrini and their reciprocal hybrids indicated that 4 primer pairs had no amplified products on M.amblycephala,and 2 primer pairs had no amplified products on M.pellegrini.Among the 20 loci which had effective amplified products,9 specific loci could identify M.amblycephala,M.pellegrini and the reciprocal hybrids,respectively.Combination of the other 17 specific loci also distinguished M.amblycephala,M.pellegrini and the reciprocal hybrids effectively.However,the reciprocal hybrids[M.amblycephala(♀)×M.pellegrini(♂),M.pellegrini(♀)×M.amblycephala(♂)]could not be identified through these markers used in this study.

Application of microsatellite markers developed for arvicoline species in a population genetic study of the root vole Microtus oeconomus

Using a root vole Microtus oeconomus (Pallas, 1776) population in NE Poland we applied 31 microsatellite markers previously developed for root voles and closely related species, with the aim to improve the population genetic tools in this species. Here we present 16 polymorphic microsatellite markers grouped into four sets suitable for simultaneous amplification and genetically sex identification in M. oeconomus. The number of alleles per locus in 227 individuals varied from 7 to 26 with a low frequency of null alleles, expected heterozygosity ranged from 0.758 to 0.927, and observed heterozygosity from 0.722 to 0.947. Two loci showed significant deviation from Hardy-Weinberg equilibrium (p<0.05) and all loci showed independent inheritance. We expect these markers to be useful for studies of genetic population structure and kinship of M. oeconomus populations.

Strong genetic divergence indicates that congeneric damselflies Coenagrion puella and C. pulchellum (Odonata: Zygoptera: Coenagrionidae) do not hybridise

Coenagrionid damselflies are in general decline in the British Isles. Numerous factors have been implicated in the loss of these species including recent speculation that hybridisation between congeners may result in species decline. Here we use a panel of 12 microsatellite loci to examine levels of genetic divergence and the likely occurrence of hybridisation in five populations of Coenagrion puella and C. pulchellum using samples from four sites in south-east England. Coenagrion puella and C. pulchellum were highly genetically divergent, and there was no evidence of hybridisation between any of the populations examined, even where C. puella and C. pulchellum were sympatric. There was some suggestion that C. pulchellum was less genetically diverse than C. puella, though this may have been a result of ascertainment bias associated with cross-species application of microsatellite markers. We conclude that there is no evidence that hybridisation between C. puella/pulchellum could be responsible for the on-going demographic decline in C. pulchellum. Nevertheless, further genetic studies such as this one are likely to provide estimates of diversity, population structure and dispersal capacity that will be invaluable in future conservation management strategies for coenagrionid damselflies.

Microsatellite Markers for Characterization of Grape Genetic Resources and Identification of QTLs for Important Agronomical Traits

ABSTRACTThe great economic importance of grapevine relies on the production of fresh fruits and refined products (wines, juices, raisins). The elevating trade requirements for the quality of grape products enforces the application of up to date methods and approaches for characterization of grape genetic resources and improvement of grape breeding process. Here we describe the recent development and directions of research, related to application of microsatellite markers for cultivar identification to genome mapping in Bulgaria. Information on the utilization of the microsatellite markers for precise identification of grape cultivars, verification of synonyms/homonyms, parentage analysis as well as for practical bulk testing of planting materials from nurseries is provided. A recently initiated research directed to mapping of QTLs for fungal disease resistance is outlined.

Isolation of novel microsatellite loci in the Rocky Mountain apollo butterfly, Parnassius smintheus.

Microsatellite DNA loci have emerged as arguably the most popular molecular markers for ecological and evolutionary studies. They are prized for their high variability, co-dominant expression, and broad distribution throughout the genome. They have been widely used to study population genetic structure, kinship, and paternity. Butterflies have served as model systems in ecological and evolutionary studies, in part because they are often intimately associated with specific habitats, they are easily monitored and handled, and knowledge of their natural history is extensive (BOGGS et al. in press). Given the utility of butterflies as model systems in ecology and evolution, and the value of microsatellite markers as analytical tools in these fields, the application of microsatellite markers to studies of butterfly biology is very promising. Unfortunately, progress in this area has been hampered by the difficulty of isolating useful microsatellite markers from butterfly genomes (MEGLECZ and SOLIGNAC 1998; KEYGHOBADI et al. 1999; NEVE and MEGLECZ 2000). Very few microsatellite loci have been reported for butterflies or in fact for any Lepidoptera (PALO et al. 1995; BOGDANOWICZ et al. 1997; MEGLECZ and SOLIGNAC 1998; KEYGHOBADI et al. 1999; REDDY et al. 1999; HARPER et al. 2000; ANTHONY et al. 2001) and isolation and characterization of microsatellite markers is clearly more difficult in these insects than in most other organisms (MEGLECZ and SOLIGNAC 1998; NEVE and MEGLECZ 2000). Here, we describe four novel microsatellite loci useful for population studies in the butterfly, Parnassius smintheus (Doubleday). This brings the total number of usable loci for this species to eight, the largest number currently reported for any species of butterfly.