Efficiency of semi-automated fluorescent multiplex PCRs with 11 microsatellite markers for genetic studies of deer populations

Thirty bovine and eight ovine microsatellite primer pairs were tested on four tropical deer species: Eld's and Swamp deer (highly threatened) and Rusa and Vietnamese Sika deer (economically important). Thirty markers gave an amplified product in all four species (78.9%). The number of polymorphic microsatellite markers varied among the species from 14 in Eld's deer (47%) to 20 in Swamp deer (67%). Among them, 11 microsatellite loci were multiplexed in three polymerase chain reactions (PCRs) and labelled with three different fluorochromes that can be loaded in one gel-lane. To test the efficiency of the multiplex, primary genetic studies (mean number of alleles, expected heterozygosities and Fis values) were carried out on four deer populations. Parentage exclusion probability and probability of identity were computed and discussed on a Swamp deer population. These multiplexes PCRs were also tested on several other deer species and subspecies. The aim of this study is to establish a tool useful for genetic studies of population structure and diversity in four tropical deer species which with few modifications can be applied to other species of the genus Cervus.     

Multiplex genotyping of novel tetranucleotide microsatellites from a marine foodfish species crimson red snapper (Lutjanus erythropterus)

Crimson red snapper (Lutjanus erythropterus) is an important marine foodfish species in Asia with great potentials for aquaculture. We isolated nine polymorphic microsatellites, among which eight were tetranucleotide repeats, and one was CA‐microsatellite. The average allele number present in 48 individuals was 11.1/locus, ranging from three to 33. The expected heterozygosity ranged from 0.49 to 0.97 with an average of 0.74. Six of the nine markers conformed to the Hardy–Weinberg expectations. No pairwise markers showed the possibility of linkage. Protocols for two multiplex polymerase chain reactions (PCRs), each amplifying two and seven markers, respectively, were presented. The developed microsatellites and optimized multiplex PCRs will be useful for studying population structure of wild stocks and parentage assignment for cultured populations.     

Multiplex panels of polymorphic microsatellite loci for two cryptic bat species of the genus Pipistrellus,developed by cross‐species amplification within the family Vespertilionidae

The paper presents multiplex panels of polymorphic microsatellites for two closely related cryptic species Pipistrellus pipistrellus and Pipistrellus pygmaeus. We tested the cross‐species amplification of 34 microsatellite loci, originally developed for five vespertilionid bat species. Ten and nine polymorphic loci in P. pipistrellus (mean number of alleles per locus = 10.5) and P. pygmaeus (8.1), respectively, in three multiplex polymerase chain reactions per species were amplified. All loci can be analysed in a single fragment analysis and can be used as markers to the study of evolution and the ecology of structured populations of socially living bats.     

Application of bovine microsatellite markers for genetic diversity analysis of Swiss yak (Poephagus grunniens)

In order to assess the applicability of bovine microsatellite markers for population genetic studies in Swiss yak, 131 bovine microsatellite markers were tested on a panel of 10 animals. Efficient amplification was observed for 124 markers (94.6%) with a total of 476 alleles, of which 117 markers (94.3%) were polymorphic. The number of alleles per locus among the polymorphic markers ranged from two to nine. Seven loci (ILSTS005, BMS424B, BMS1825, BMS672, BM1314, ETH123 and BM6017) failed to amplify yak genomic DNA. Two cattle Y-chromosome specific microsatellite markers (INRA126 and BM861) amplified genomic DNA from both male and female yaks. However, two additional markers on cattle Y-chromosome (INRA124 and INRA189) amplified DNA from only males. Of the polymorphic markers, 24 microsatellites proposed by CaDBase for within- and cross-species comparisons and two additional highly polymorphic markers (MHCII and TGLA73) were used to investigate the genetic variability and the population structure of a Swiss yak herd that included 51 additional animals. The polymorphic information content ranged from 0.355 to 0.752, while observed heterozygosity (HO) ranged from 0.348 to 0.823. Furthermore, a set of 13 markers, organized into three multiplex polymerase chain reactions, was evaluated for routine parentage testing. This set provided an exclusion probability in a family of four yaks (both parents and two offspring) of 0.995. These microsatellites serve as useful tools for genetic characterization of the yak, which continues to be an important domestic livestock species.     

Bovine microsatellite loci are highly conserved in red deer (Cervus elaphus), sika deer (Cervus nippon) and Soay sheep (Ovis aries)

We tested 174 bovine microsatellite primer pairs for use in a primitive breed of sheep and two species of deer. Of 173 markers, 127 (73·4%) gave a product in Soay sheep ( Ovis aries ) of which 54 (42·5%) were polymorphic. One hundred and twenty-nine of 174 (74·1%) markers gave a product in red deer ( Cervus elaphus ) of which 72 (55·8%) were polymorphic. In sika deer ( Cervus nippon ) 126 of 171 (73·7%) microsatellite primers gave a product with 47 (37·3%) polymorphic. The proportion of bovine microsatellite loci conserved across artiodactyl species was significantly greater in this study than previously reported. Reasons for this high degree of microsatellite conservation are discussed. We suggest that a high resolution comparative map of the artiodactyls can be constructed using microsatellites.     

De novo discovery and multiplexed amplification of microsatellite markers for black alder (Alnus glutinosa) and related species using SSR-enriched shotgun pyrosequencing

Recent developments in sequencing technologies and bioinformatics analyses provide an unprecedented opportunity for cost and time effective high quality microsatellite marker discovery in nonmodel organisms for which no genomic information is available. Here, we use shotgun pyrosequencing of a microsatellite-enriched library to develop, for the first time, microsatellite markers for Alnus glutinosa, a keystone tree species of European riparian woodland communities. From a total of 17?855 short sequences, we identified 590 perfect microsatellites from which 392 had designed primers. A subset of 48 loci were tested for amplification, 12 of which were polymorphic in A. glutinosa. These 12 loci were successfully coamplified in a single multiplex polymerase chain reaction experiment and validated for population genetics applications. In addition, 10 and 8 of these microsatellites were found to be transferable to the related A. incana and A. cordata species. The developed multiplex of 12 microsatellite markers therefore provides new opportunities for experimental evolutionary and forest genetics research in Alnus.     

Cross-amplification tests of ungulate primers in the endangered Neotropical pampas deer (Ozotoceros bezoarticus)

In cross-species amplification tests of 15 ungulate primers in pampas deer, five were retained to form a small panel of highly polymorphic loci that could be used to efficiently screen populations of this endangered species. The polymerase chain reactions were performed incorporating the universal fluorescent labeled M13 (-21) primer. In 69 pampas deer, average allelic diversity was 15, expected heterozygosity was 0.869 and the mean polymorphic information content value was 0.847. Paternity exclusion probabilities over loci were NE-1P = 0.01336 and NE-2P = 0.00135, and combined non-exclusion probability of identity was P(ID) = 3 x 10(-8).     

Microsatellite DNA markers for the ground beetle Pterostichus oblongopunctatus F.

Six microsatellite DNA loci were isolated from the ground beetle Pterostichus oblongopunctatus F. (Coleoptera, Carabidae) to study population structure. Each locus was polymorphic, with the number of alleles ranging from three to six. Observed heterozygosity varied between 0.20 and 0.67. All six loci were tested for amplification in the closely related P. quadrifoveolatus and they were shown to be polymorphic. The primers developed were used in multiplex polymerase chain reactions.     

Thirty‐four polymorphic microsatellites for European roe deer

The European roe deer (Capreolus capreolus) is an interesting model for molecular ecology studies because of its abundance and adaptability across a range of environments (including human‐modified habitats), and because of its increasing impacts on agricultural crops and on regenerating forests. We identify polymorphic microsatellites in two managed populations of roe deer in France by using cross‐species amplification of primers from other Cervids and from Bovids. Of the 62 primer pairs tested, 45 amplified microsatellites in roe deer, and 34 were polymorphic. Eleven primer pairs were selected for multiplex gel‐loading for routine genotyping of the studied populations.     

Polymorphic sequence‐characterized codominant loci in the chestnut blight fungus,Cryphonectria parasitica

Studies on the population biology of the chestnut blight fungus, Cryphonectria parasitica, have previously been carried out with dominant restriction fragment length polymorphism (RFLP) fingerprinting markers. In this study, we describe the development of 11 codominant markers from randomly amplified polymorphic DNAs (RAPDs). RAPD fragments were cloned and sequenced, and polymerase chain reaction (PCR) primers were designed flanking insertions/deletions. Primers labelled with fluorescent dyes were combined in multiplex reactions to assay five or six loci simultaneously in a capillary sequencing system. These codominant markers have the potential to complement RFLP methods for studying C. parasitica.     

Development and multiplex PCR amplification of novel microsatellite markers in the White‐tailed Sea Eagle,Haliaeetus albicilla (Aves: Falconiformes,Accipitridae)

We report the development of 14 novel polymorphic microsatellite markers cloned from the White‐tailed Sea Eagle, Haliaeetus albicilla, a formerly threatened raptor that has received much conservation attention throughout Eurasia. We also present a protocol for multiplex polymerase chain reaction (PCR) amplification of the loci. Among 40 unrelated H. albicilla individuals from southern Sweden, the markers produced two to eight alleles per locus, and average observed and expected heterozygosities were 0.463 and 0.468, respectively. We further present five microsatellite markers that appeared monomorphic in H. albicilla, but which may be of interest for use in other raptor species.     

Characterization of novel microsatellites and development of multiplex PCR for large‐scale population studies in wild cherry,Prunus avium

Primers were developed for 14 microsatellite or simple sequence repeat (SSR) loci identified from a Prunus avium‘Charger’ genomic DNA library. In a survey of 16 wild cherry accessions 10 of the loci revealed polymorphisms of between two and six alleles. The remaining loci were found to be monomorphic. Seven polymorphic loci identified in this study and four polymorphic loci previously reported in sweet cherry were mapped and found to be unlinked. Two multiplex polymerase chain reactions (PCR) were optimized to enable the characterization of all 11 unlinked, polymorphic SSR loci.     

Isolation and characterization of polymorphic microsatellite loci for the dace complex: Leuciscus leuciscus (Teleostei: Cyprinidae)

Ten novel polymorphic microsatellites were isolated from the dace complex (Leuciscus leuciscus), which is a European cyprinid species. Four multiplex polymerase chain reaction sets were optimized in order to genotype 26 polymorphic loci in all, including 16 previously published cyprinid-specific loci. The level of genetic diversity was assessed in 142 dace individuals. We also successfully applied 26 of the microsatellites to 10 related species. These primers thus will be useful to assess population structure of the dace and other cyprinid species, with application for conservation issues and phylogeographical approaches.     

<Emphasis Type="Italic">Anaplasma phagocytophilum</Emphasis> infection in a multi-species deer community in the New Forest,England

The tick-transmitted Anaplasma phagocytophilum has been recorded in a range of mammal species and causes granulocytic ehrlichiosis in humans, horses, and companion animals as well as tick-borne fever in ruminants. Although deer and other ruminant species are known to be natural hosts, the distribution among sympatric deer populations is unexplored. Blood from 80 deer of four species were screened using an A. phagocytophilum-specific real-time polymerase chain reaction. Overall, 29% (19–40) of deer tested positive. Fallow deer (Dama dama), the most numerous species, had significantly lower prevalence (21%) than roe (Capreolus capreolus), red (Cervus elaphus), or sika (Cervus nippon) deer (average 50%). It is suggested that patterns of habitat use influence infection levels in different deer species. The role of deer as reservoirs of anaplasmosis remains unknown; however, prevalence in deer could be a useful index of local infection pressure and the risk of disease in domestic animals and humans.     

Development of microsatellite markers in Abies nordmanniana (Stev.) Spach and cross‐species amplification in the Abies genus

Abies nordmanniana (Stev.) Spach is a widely grown Christmas tree in Denmark, from where it is exported to most of Europe. An ongoing breeding programme is taking place, and as a tool for that, we report the development of five polymorphic nuclear microsatellite markers. To investigate the potential for transferring the markers to other species in the Abies genus, polymerase chain reaction amplification was tested in 19 other species. In general, amplification occurred in a very high proportion of the tested species.     

Microsatellite markers for the palaeo-temperature indicator Pentapharsodinium dalei (Dinophyceae)

Pentapharsodinium dalei is a widely distributed cold-water dinoflagellate, which is used in palaeoecology as an indicator of relatively warmer conditions in polar and sub-polar regions. This species has been proposed to be one of the first indicators of global warming at high latitudes. We developed the first microsatellite markers for P. dalei to facilitate the study of spatial and temporal population genetic changes. Single cysts were isolated from surface sediments in Koljö Fjord, Sweden. After cyst germination, single vegetative cells were isolated for establishing monoclonal cultures. Six dinucleotide polymorphic microsatellite markers were developed as multiplex polymerase chain reactions and were genotyped in 32 strains. The number of alleles per locus varied between 4 and 12, and the estimated gene diversity varied from 0.588 to 0.891. The haploid state of the vegetative cells was confirmed. The six selected microsatellites will be useful to explore population dynamics in P. dalei from contemporary planktonic and revived benthic samples to enable, for example, detailed studies into the evolutionary consequences of anthropogenic and climate-driven habitat changes.     

Development of new microsatellite loci and multiplex reactions for muskellunge (Esox masquinongy)

The muskellunge (Esox masquinongy) is a valued fisheries species throughout its native range. Numerous studies have documented performance and phenotypic differences among muskellunge populations, but genetic markers for assessment have been lacking. We characterized 14 microsatellite loci and developed five multiplex polymerase chain reactions. Successful amplification of northern pike (Esox lucius) was observed for seven loci. These microsatellites will be useful for analysing population structure, performance characteristics of propagated strains, and helping to develop and monitor hatchery management guidelines for muskellunge.     

A deer (subfamily Cervinae) genetic linkage map and the evolution of ruminant genomes

Comparative maps between ruminant species and humans are increasingly important tools for the discovery of genes underlying economically important traits. In this article we present a primary linkage map of the deer genome derived from an interspecies hybrid between red deer (Cervus elaphus) and Père David's deer (Elaphurus davidianus). The map is approximately 2500 cM long and contains >600 markers including both evolutionary conserved type I markers and highly polymorphic type II markers (microsatellites). Comparative mapping by annotation and sequence similarity (COMPASS) was demonstrated to be a useful tool for mapping bovine and ovine ESTs in deer. Using marker order as a phylogenetic character and comparative map information from human, mouse, deer, cattle, and sheep, we reconstructed the karyotype of the ancestral Pecoran mammal and identified the chromosome rearrangements that have occurred in the sheep, cattle, and deer lineages. The deer map and interspecies hybrid pedigrees described here are a valuable resource for (1) predicting the location of orthologs to human genes in ruminants, (2) mapping QTL in farmed and wild deer populations, and (3) ruminant phylogenetic studies.     

Genetic Variations Revealed by Microsatellite Markers ina Small Population of the Sika Deer (Cervus nippon)on Kinkazan Island, Northern Japan

Genetic variations within a population of the Japanese sika deer, Cervus nippon, on Kinkazan Island were studied by microsatellite analysis. Seventeen pairs of polymerase chain reaction primers designed for several species of ungulates successfully amplified polymorphic microsatellite DNA in sika deer. About 20% of the Kinkazan population was sampled and genotyped for nine diagnostic microsatellite loci. Alleles at those loci in the Kinkazan population were found to be under the Hardy-Weinberg equilibrium. To determine whether the Kinkazan deer have a reduced level of genetic variability, an average heterozygosity in the population was calculated and compared with the values determined for other populations from Hyogo, Yamaguchi, Shimane, Tsushima, and Nagasaki. Neither the observed nor the expected heterozygosity in the Kinkazan deer significantly differed from that in the other populations. Our result indicated that, despite its small population size, the Kinkazan deer preserve extensive microsatellite variations.