Identification and characterization of nuclear microsatellites in Mediterranean cedars (Cedrus sp.)

We developed primers for the amplification of six polymorphic nuclear microsatellites in Mediterranean Cedrus taxa. Microsatellites originated from two Cedrus atlantica genomic libraries enriched for TC (four markers) and TG (two markers) motifs. No distortion from Mendelian segregation was observed. There were up to eight alleles per locus and mean expected heterozygosity was 0.75. Four microsatellites also amplified in the Himalayan cedar. These markers should be helpful for species identification, diversity studies, parentage analysis and genome mapping and to monitor biodiversity in endangered Mediterranean Cedrus forests.     

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.     

Development and characterization of microsatellites from Senegal sole (Solea senegalensis)

To assist in the domestication of the sole fish Solea senegalensis, 15 microsatellites were developed from its genome (13 di‐ and two trinucleotide). These loci, along with three from S. solea that cross‐amplified, were characterized on a sample of 20 individuals of S. senegalensis from a hatchery stock. All loci were polymorphic displaying from two to 15 alleles/locus (mean 6.5). The results obtained from the hatchery population screening indicate that they might be useful for studies of genetic structuring and/or kinship analysis in S. senegalensis.     

Population genetic analysis and origin discrimination of snow crab (Chionoecetes opilio) using microsatellite markers

Major habitats for the snow crab Chionoecetes opilio are mostly found within the northwest Atlantic and North Pacific Oceans. However, the East Sea populations of C. opilio, along with its relative the red snow crab (C. japonicas), are two of the most important commercial crustacean species for fisheries on the east coast of the Korean Peninsula. The East Sea populations of C. opilio are facing declining resources due to overfishing and global climate change. Thus, an analysis of population structure is necessary for future management. Five Korean and one Russian group of C. opilio were analyzed using nine microsatellite markers that were recently developed using next-generation sequencing. No linkage disequilibrium was found between any pair of loci, indicating that the markers were independent. The number of alleles per locus varied from 4 to 18 with a mean of 12, and allelic richness per locus ranged from 4.0 to 17.1 across all populations with a mean of 9.7. The Hardy–Weinberg equilibrium test revealed significant deviation in three out of nine loci in some populations after sequential Bonferroni correction and all of them had higher expected heterozygosity than observed heterozygosity. Null alleles were presumed in four loci, which explained the homozygosity in three loci. The pairwise fixation index (F _ST ) values among the five Korean snow crab populations did not differ significantly, but all of the pairwise F _ST values between each of the Korean snow crab populations and the Russian snow crab population differed significantly. An UPGMA dendrogram revealed clear separation of the Russian snow crab population from the Korean snow crab populations. Assignment tests based on the allele distribution discriminated between Korean and Russian origins with 93 % accuracy. Therefore, the snow crab populations around the Korean Peninsula need to be managed separately from the populations in Bering Sea in global scale resource management. Also, this information can be used for identification of snow crab origin which is problematic in worldwide crab trade.     

Isolation and characterization of microsatellites in lane snapper (Lutjanus synagris), mutton snapper (Lutjanus analis), and yellowtail snapper (Ocyurus chrysurus)

Polymerase chain reaction primer pairs for a total of 25 nuclear‐encoded microsatellites (loci) were developed from genomic DNA libraries of lane snapper (Lutjanus synagris), mutton snapper (Lutjanus analis), and yellowtail snapper (Ocyurus chrysurus). The microsatellites include 24 perfect (21 dinucleotide and three trinucleotide) and one imperfect (combination tetranucleotide/tetranucleotide) repeat motifs. A total of 32 individuals of each species were assayed for allelic variation at all 25 microsatellites; reliable amplification products were generated for lane snapper (25 loci), mutton snapper (21 loci), and yellowtail snapper (24 loci). Significant deviations from Hardy–Weinberg expectations, following Bonferroni corrections, were found for one microsatellite in lane and yellowtail snappers, and for three microsatellites in mutton snapper. All pairwise comparisons of microsatellites (all three species) did not deviate significantly from genotypic equilibrium.     

Isolation and characterization of microsatellites in trembling aspen (Populus tremuloides)

 We have identified, isolated, and characterized microsatellite/simple sequence repeat (SSR) loci in trembling aspen (Populus tremuloides) by screening partial genomic libraries. We have also examined the compatibility and use of the P. tremuloides SSR primers to resolve microsatellites in other Populus species. Fourteen microsatellites were identified from 1600 clones screened. The TC/AG microsatellites were the most abundant. A total of 29 alleles were detected in 36 P. tremuloides individuals at the four SSR loci (two each of di- and tri-nucleotide repeats) characterized. The number of alleles at the SSR loci ranged from 5 to 11, with an average of 7.25 alleles per locus, and the observed heterozygosity ranged from 0.19 to 0.82, with a mean of 0.46 per locus. Although the highest polymorphism was observed for a dinucleotide SSR locus, the trinucleotide SSR loci showed substantial polymorphism. There were 34 unique multilocus genotypes among the 36 P. tremuloides individuals examined, and 89% of the individuals had unique multilocus genotypes. Two pairs of SSR primers were successful in PCR, amplifying genomic DNA and resolving microsatellites of comparable size from Populus deltoides, P. nigra, P.×canadensis, and P. maximowiczii. The microsatellite DNA markers developed could be used for clonal fingerprinting, certification of controlled crosses, genome mapping, marker-assisted early selection, genetic diversity assessments, and conservation and sustainable management of poplar genetic resources. Received: 14 November 1997 / Accepted: 17 November 1997     

Chloroplast microsatellites in Prunus,Rosaceae

Ten chloroplast microsatellite markers were developed from Japanese plum (Prunus salicina) based on nucleotide sequences of c. 4300 bp from six chloroplast regions. Out of 10 microsatellites, seven markers contained mononucleotide repeats. Almost all microsatellites displayed discrete amplified fragments for 17 species in Prunus. The microsatellites generated 46 different fragment types and differentiated all used species. Polymorphism was also observed within species for all microsatellites.     

In silico mining of microsatellites and analysis of genetic diversity among inter‐ and intra‐generic aphids of the subfamily Aphidinae

Nearly 5 000 aphid species damage crops, either by sucking plant sap or as disease‐transmitting vectors. Microsatellites are used for understanding molecular diversity and eco‐geographical relationships among aphid species. Expressed sequence tag (EST)‐microsatellite motifs were identified through an in silico approach using inbuilt simple sequence repeat mining tools in aphid EST dataset. Microsatellite mining revealed one in every five aphid genes as containing a repeat motif, and out of 9 290 EST microsatellites mined from Aphis gossypii Glover and Acyrthosiphon pisum (Harris) (both Hemiptera: Aphididae), 80% were of A and/or T (AT, ATA, AAT, AATA, and ATTT) motifs, and the rest contained G and/or C motifs. All microsatellite sequences were annotated using BLAST. Primers for EST microsatellites were designed using the Primer 3.0 tool. 106 primer pairs of both dinucleotide repeats (DNRs) and trinucleotide repeats (TNRs), representing open reading frames (ORFs) and untranslated regions (UTRs), were synthesized to amplify 15 aphid species belonging to the subfamily Aphidinae, collected from diverse hosts. Four hundred forty‐five polymorphic alleles were amplified. Fifty TNR and 23 DNR microsatellites amplified across the species studied. Polymorphism information content values of microsatellites ranged from 0.23 to 0.91, amplifying 2–16 alleles. Genetic similarity indices were estimated using the ‘NTSYS‐pc’ software package. Unweighted pair group with arithmetic mean and principal component analysis resolved taxonomic relationships of the aphid species studied. The new aphid microsatellites developed will provide valuable information to researchers to study Indian aphid species diversity and genetic relationships.     

PERMANENT GENETIC RESOURCES: A set of 12 microsatellite loci for genetic studies of Leishmania braziliensis

Twelve microsatellite loci of Leishmania braziliensis were examined, nine of which were developed in this work. Fifty‐six Leishmania braziliensis were genotyped with these microsatellite loci. The 12 loci studied were polymorphic with the number of alleles ranging from five to 19, with a mean of 9.7 ± 4.1 and the observed heterozygosity averaging 0.425 ± 0.202. The important heterozygote deficits we observed (F_IS = 0.41, P value = 0.004) appear incompatible with the heterozygote excess expected in clonal diploids. This last result could revive the clonality/sexuality debate regarding Leishmania. This work validates the potential use of these microsatellites for population genetics analysis.     

Polymorphic microsatellites identified by cross-species amplifications in the European Coot Fulica atra

We tested the cross-amplification of 26 microsatellites developed for passerines and an additional three developed for Gallinula species in eight European Coots from two populations. Sixteen microsatellite markers successfully amplified, of which nine were polymorphic with 2–6 alleles (mean 3.7 alleles) and an expected heterozygosity (H _e) ranging from 0.375 to 0.805 (mean H _e = 0.589). On average, we found 2.22 alleles/locus and a mean H _e of 0.440 in one nest, and 2.56 alleles/locus and a mean H _e of 0.494 in the other one. These nine polymorphic markers could be of potential use in studies of genetic variability, population structure and reproductive strategy of European Coots.     

Development of microsatellite markers and analysis of intraspecific genetic variability in chickpea (Cicer arietinum L.)

Paucity of polymorphic molecular markers in chickpea (Cicer arietinum L.) has been a major limitation in the improvement of this important legume. Hence, in an attempt to develop sequence-tagged microsatellite sites (STMS) markers from chickpea, a microsatellite enriched library from the C. arietinum cv. Pusa362 nuclear genome was constructed for the identification of (CA/GT) _n and (CT/GA) _n microsatellite motifs. A total of 92 new microsatellites were identified, of which 74 functional STMS primer pairs were developed. These markers were validated using 9 chickpea and one C. reticulatum accession. Of the STMS markers developed, 25 polymorphic markers were used to analyze the intraspecific genetic diversity within 36 geographically diverse chickpea accessions. The 25 primer pairs amplified single loci producing a minimum of 2 and maximum of 11 alleles. A total of 159 alleles were detected with an average of 6.4 alleles per locus. The observed and expected heterozygosity values averaged 0.32 (0.08–0.91) and 0.74 (0.23–0.89) respectively. The UPGMA based dendrogram was able to distinguish all the accessions except two accessions from Afghanistan establishing that microsatellites could successfully detect intraspecific genetic diversity in chickpea. Further, cloning and sequencing of size variant alleles at two microsatellite loci revealed that the variable numbers of AG repeats in different alleles were the major source of polymorphism. Point mutations were found to occur both within and immediately upstream of the long tracts of perfect repeats, thereby bringing about a conversion of perfect motifs into imperfect or compound motifs. Such events possibly occurred in order to limit the expansion of microsatellites and also lead to the birth of new microsatellites. The microsatellite markers developed in this study will be useful for genetic diversity analysis, linkage map construction as well as for depicting intraspecific microsatellite evolution.     

Novel microsatellite markers for Bactris gasipaes (Palmae)

Ten new polymorphic microsatellites were isolated and characterized in Bactris gasipaes using a microsatellite enrichment protocol and selective hybridization with oligonucleotide probes. The loci are highly polymorphic, with a mean of 14.6 alleles per locus and a mean expected heterozygosity of 0.83 among 62 individuals of the Pampa Hermosa landrace. These microsatellites will be useful for population genetic analysis and germplasm characterization for heart‐of‐palm breeding.     

Eleven new microsatellites of the sea bass (Dicentrarchus labrax L.)

Eleven polymorphic microsatellites were isolated from the sea bass, Dicentrarchus labrax, using a microsatellite enrichment protocol and selective hybridization with an (AC)₁₂ probe. The loci showed different variation patterns in 21 unrelated sea bass individuals, with a mean number of alleles of 8.6 and a mean observed heterozygosity of 0.68. These microsatellite markers should be useful for population genetic analysis and biodiversity studies of sea bass.     

Isolation and characterization of polymorphic microsatellites in a sex‐reversal fish,rice field eel (Monopterus albus)

The rice field eel (Monopterus albus) is a fish of economic importance in China and some Asian countries. From a (GT)_n‐enriched genomic library, 30 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Thirteen loci exhibited polymorphism with two to 13 alleles (mean 7.9 alleles/locus) in a test population and observed heterozygosity ranged from 0.3125 to 0.9688 (mean 0.7140). These loci should provide sufficient level of genetic variation to study the fine‐scale population structure and reproductive ecology of the species.     

Isolation and characterization of polymorphic microsatellites in a Yangtze River fish,brass gudgeon (Coreius heterodon Bleeker)

The brass gudgeon (Coreius heterodon) is a fish of economic importance in the Yangtze River. From a (GATA)_n‐enriched genomic library, 25 microsatellites were developed by employing the fast isolation by AFLP of sequences containing repeats (FIASCO) protocol. Nine loci exhibited polymorphism with two to 12 alleles (mean 3.9 alleles/locus) in a test population, and observed heterozygosity ranged from 0.1111 to 0.9630 (mean 0.4426). Three of the nine loci showed polymorphism in a congeneric species, the largemouth bronze gudgeon Coreius guichenoti. These loci should provide sufficient level of genetic diversity to evaluate the fine‐scale population structure of C. heterodon.     

Microsatellite DNA markers for population‐genetic studies of Atlantic bluefin tuna (Thunnus thynnus thynnus) and other species of genus Thunnus

Twenty‐five microsatellites from Atlantic bluefin tuna (Thunnus thynnus thynnus) were characterized. All 25 microsatellites were polymorphic; the number of alleles among up to 56 individuals surveyed ranged from two to 23. Atlantic bluefin tuna are highly exploited and major questions remain as to stock structure and abundance in the eastern and western North Atlantic. The microsatellites will be useful in testing stock‐structure hypotheses and in generating estimates of effective population size. The polymerase chain reaction primer sets developed also amplified identifiable alleles in three other species of genus Thunnus: T. albacares (yellowfin tuna), T. alalunga (albacore tuna) and T. obesus (bigeye tuna).     

The period of occurrence,density, and distribution of larvae of three commercial crab species in Peter the Great Bay,Sea of Japan

Based on materials from plankton surveys carried out in 2004–2009, the period of occurrence, density, and distribution of larvae of three commercial species of crabs in the Peter the Great bay and adjacent areas of Sea of Japan were studied. The larvae of the horsehair crab Erimacrus isenbeckii (Brandt, 1848) occurred in the plankton from mid-March to early June, within the range of water temperature from −1 to 10.8°C. The larvae of the helmet crab Telmessus cheiragonus (Tilesius, 1812) appeared in the plankton in mid-April and occurred to the end of June within the temperature range from 2.8 to 13.0°C. The larvae of the snow crab Chionoecetes opilio (O. Fabricius, 1788) appeared in the plankton in mid-April as well, but some individuals sporadically occurred until early August. All the species of crabs produced one generation of larvae for their reproduction season. The terms of larval stay in plankton depended on water temperature and the duration of the pelagic period increased in colder years. In that area, the larvae of C. opilio were the most abundant (up to 41 ind./m³) and the zoea density of horsehair and helmet crabs was significantly lower (no more than 2 ind./m³). The larvae of C. opilio occurred over the entire area of the Peter the Great bay; the greatest aggregations of their early stages were observed in its southwestern open part. The maximum density of E. isenbeckii zoea was recorded in the south of the Amursky bay and in the Posyet bay. Individual larvae of T. cheiragonus occurred in the Posyet bay and in the southern part of the Amursky and Ussuriisky bays. The late-stage larvae of all crab species were concentrated in areas of the coastal circulation.     

Primers for 10 polymorphic microsatellites from Caulanthus amplexicaulis var. barbarae,and cross‐amplification in other species within the Streptanthoid Complex (Brassicaceae)

Ten microsatellite primer pairs, developed from Caulanthus amplexicaulis var. barbarae (J. Howell) Munz, specifically amplified target loci across a diverse range of taxa from the Streptanthoid Complex, Brassicaceae. All primer pairs produced amplification products from at least 22 of the 23 accessions tested. Product size variation and observable heterozygosity were consistent with what would be expected from the amplification of polymorphic microsatellites across the complex. Our findings demonstrate that microsatellites developed for C. amplexicaulis var. barbarae will be useful for population‐genetic studies in taxa throughout the Streptanthoid Complex.     

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.