Genomewide gene‐associated microsatellite markers for the model invasive ascidian,Ciona intestinalis species complex

The vase tunicate, Ciona intestinalis species complex, has become a good model for ecological and evolutionary studies, especially those focusing on microevolution associated with rapidly changing environments. However, genomewide genetic markers are still lacking. Here, we characterized a large set of genomewide gene‐associated microsatellite markers for C. intestinalis spA (=C. robusta). Bioinformatic analysis identified 4654 microsatellites from expressed sequence tags (ESTs), 2126 of which successfully assigned to chromosomes were selected for further analysis. Based on the distribution evenness on chromosomes, function annotation and suitability for primer design, we chose 545 candidate microsatellites for further characterization. After amplification validation and variation assessment, 218 loci were polymorphic in at least one of the two populations collected from the coast of Arenys de Mar, Spain (N = 24–48), and Cape Town, South Africa (N = 24–33). The number of alleles, observed heterozygosity and expected heterozygosity ranged from 2 to 11, 0 to 0.833 and 0.021 to 0.818, and from 2 to 10, 0 to 0.879 and 0.031 to 0.845 for the Spanish and African populations, respectively. When all microsatellites were tested for cross‐species utility, only 60 loci (25.8%) could be successfully amplified and all loci were polymorphic in C. intestinalis spB. A high level of genomewide polymorphism is likely responsible for the low transferability. The large set of microsatellite markers characterized here is expected to provide a useful genomewide resource for ecological and evolutionary studies using C. intestinalis as a model.     

The highly polymorphic microsatellite markers for the greater long‐tailed hamster (Tscherskia triton)

We isolated 25 dinucleotide microsatellite loci from the greater long‐tailed hamster (Tscherskia triton) populations in North China. We developed the amplification conditions of polymerase chain reaction for producing high‐resolution genetic markers for each locus. We found 10 microsatellite loci were highly polymorphic in 90 individual hamsters from three areas of North China, and the number of alleles in each locus varied from three to 11. These markers are potential tools for studying the genetic variation of the natural populations of this species.     

Development and characterization of microsatellite markers in Eucommia ulmoides Oliver (Eucommiaceae)

Twenty‐three microsatellite markers were developed from an AC‐enriched genomic library of Eucommia ulmoides, an economically important tree species for both herbal medicine and organic chemical industry in China. Nineteen microsatellite loci were found polymorphic by testing 36 individuals from 10 populations, with two to 14 alleles per locus. The expected heterozygosity ranged from 0.054 to 0.874. This set of microsatellite markers has provided a useful tool for the ongoing efforts in studying population genetic structure of E. ulmoides.     

Isolation and Characterization of Microsatellite Loci for Striped Bass (Morone saxatilis)

Genetic variation has been difficult to detect in striped bass (Morone saxatilis). Therefore, we identified and characterized 13 microsatellite loci to provide additional genetic markers for striped bass. Microsatellites were identified by screening a striped bass genomic library or by using primers developed for European sea bass (Dicentrarchus labrax) microsatellite loci. We found that 6 of the 13 microsatellite loci were polymorphic in DNA samples obtained from wild populations of striped bass. The number of alleles per locus varied from 3 to 12, and the observed heterozygosities ranged from 0.55 to 0.78. These results indicate that microsatellite loci provide more alleles and higher heterozygosities than other genetic markers developed for striped bass. Received November 9, 1999; accepted February 11, 2000.     

Isolation and characterization of microsatellite loci of Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta)

Lycopodium fordii Bak. (Lycopodiaceae, Pteridophyta) is a gardening plant with a native, fragmentary distribution in Taiwan. In this study, we described the development of eleven microsatellite loci in L. fordii for genetic studies. These new markers were tested in 16 individuals of three populations. The number of alleles ranged from 1 to 5 and the expected heterozygosity from 0.41746 to 0.72222. Four of the nine polymorphic loci were significantly deviated from Hardy-Weinberg expectations due to the heterozygote deficiency. The microsatellite markers have also been proved as informative genetic markers for other 15 Lycopodium species.     

Development of microsatellite markers for the tropical moss,Acanthorrhynchium papillatum

We isolated and characterized microsatellite loci for the palaeotropic moss, Acanthorrhynchium papillatum. Eight loci tested on 98 gametophytic samples generated four to 26 alleles per locus with genetic diversities ranging from 0.578 to 0.936. These microsatellite loci are now being used as genetic markers for studies on the effects of deforestation on moss populations in South‐East Asia.     

Genetic diversity of Ligula intestinalis (Cestoda: Diphyllobothriidea) based on analysis of inter‐simple sequence repeat markers

In order to investigate the genetic diversity of Ligula intestinalis populations, nine inter‐simple sequence repeat (ISSR) markers were applied to populations from nine geographical areas around the world and 10 host species. The 110 loci selected from the ISSR patterns produced revealed high variability among the analysed samples, with a polymorphism of 100% and a global coefficient of gene differentiation estimated by Nei’s index (G_ST) of 0.776. Major genetic differentiation was found to be correlated to five broad geographical regions (Europe, China, Canada, Australia and Algeria). Nevertheless, no significant genetic variation was found among European isolates, although they originated from disparate geographical localities and/or unrelated hosts. Classical classification methods: maximum parsimony and factorial correspondence analysis were compared with an advanced statistical method: the self‐organizing map (SOM). The results demonstrated that the ISSR approach is rapid and inexpensive and provides reliable markers to assess genetic diversity of L. intestinalis. Furthermore, SOM artificial neuronal networks are considered to provide an efficient alternative tool for mapping the genetic structures of parasite populations.     

Morphological and microsatellite diversity associated with ecological factors in natural populations of Medicago laciniata Mill. (Fabaceae)

Genetic variability in 10 natural Tunisian populations of Medicago laciniata were analysed using 19 quantitative traits and 12 polymorphic microsatellite loci. A large degree of genetic variability within-populations and among-populations was detected for both quantitative characters and molecular markers. High genetic differentiation among populations for quantitative traits was seen, with Q _ST = 0.47, and F _ST = 0.47 for microsatellite markers. Several quantitative traits displayed no statistical difference in the levels of Q _ST and F _ST . Further, significant correlations between quantitative traits and eco-geographical factors suggest that divergence in the traits among populations may track environmental differences. There was no significant correlation between genetic variability at quantitative traits and microsatellite markers within populations. The site-of-origin of eco-geographical factors explain between 18.13% and 23.40% of genetic variance among populations at quantitative traits and microsatellite markers, respectively. The environmental factors that most influence variation in measured traits among populations are assimilated phosphorus (P₂0₅) and mean annual rainfall, followed by climate and soil texture, altitude and organic matter. Significant associations between eco-geographical factors and gene diversity, H _e , were established in five-microsatellite loci suggesting that these simple sequence repeats (SSRs) are not necessarily biologically neutral.     

Polymorphic microsatellite loci in the sponge Crambe crambe (Porifera: Poecilosclerida) and their variation in two distant populations

Seven polymorphic microsatellite loci were characterized in the marine encrusting sponge Crambe crambe from a partial genomic DNA‐enriched library. Preliminary data on allelic variation of these loci in two distant populations of C. crambe are presented to assess their potential utility as high‐resolution genetic markers for this species. The number of alleles per locus ranged between three and 16 and the distributions of allele frequencies differed considerably between the two populations, indicating a marked genetic differentiation between them. These are the first microsatellite loci reported from any species in the phylum.     

Isolation and characterization of 12 microsatellite loci for cross-species amplification in the cyprinid gudgeons Squalidus chankaensis and S. japonicus

Twelve dinucleotide markers were successfully isolated and characterized from a microsatellite‐enriched genomic library obtained for the gudgeon Squalidus chankaensis biwae. These markers were also available for the congeners S. c. tsuchigae and S. japonicus from Japan, which had five to 46 alleles and an expected heterozygosity ranging from zero to 0.946. Linkage equilibrium was observed at all loci, and most loci did not show significant deviation from Hardy–Weinberg equilibrium. The isolated microsatellite markers will be useful for genetic diversity studies of Squalidus populations.     

Genetic diversity of the Chestnut blight fungus Cryphonectria parasitica in four French populations assessed by microsatellite markers

Microsatellites are powerful markers to infer population genetic parameters. Here, 13 microsatellite loci isolated from a genomic and a cDNA library of Cryphonectria parasitica were used to characterize the genetic diversity and structure of four French populations. Twelve of these loci were polymorphic within populations, and average gene diversity (H_e) was estimated to be 0.35. There was a lower genetic diversity in a south-eastern population relative to three south-western populations. In these three populations, microsatellite genotypic diversity was higher than vegetative compatibility type diversity. A high genetic differentiation (G_ST = 0.27) suggested a low gene flow and/or founder effects of French populations which are in agreement with low dispersal of spores and different introductions of this species in southern France. This study demonstrates the significance of these microsatellite loci to assess gene flow and reproductive system in this important pathogen.     

Development of microsatellite markers in the toxic dinoflagellate Alexandrium tamarense (Dinophyceae)

Outbreaks of paralytic shellfish poisoning caused by the toxic dinoflagellate Alexandrium tamarense (Dinophyceae) are currently a serious problem from an economic and food hygiene point of view throughout the world. We isolated 13 polymorphic microsatellite loci from this species. These loci provided microsatellite markers with high polymorphism ranging from four to 15 alleles per locus and gene diversity between 0.632 and 0.974. The markers are available for more detailed investigations of genetic structure and gene flow of A. tamarense populations.     

Characterization of microsatellite loci in silver carp (Hypophthalmichthys molitrix), and cross‐amplification in other cyprinid species

Captive populations of silver carp (Hypophthalmichthys molitrix), a major aquaculture species in Asia, would undoubtedly benefit from genetic monitoring and improvement programs. We report the isolation and preliminary characterization of 16 microsatellite loci derived from both conventional and microsatellite‐enriched libraries. Inheritance studies confirmed the allelic nature of observed polymorphisms at all loci, while identifying null alleles at two loci. These loci, having varying degrees of polymorphism, should provide useful markers for applied genetic studies. A high degree of cross‐amplification among 10 other cyprinid species suggests that these loci may have more widespread utility.     

Comparison of genetic variation and differentiation using microsatellite markers among three rare threatened and one widespread toad lily species of Tricyrtis section Flavae (Convallariaceae) in Japan

Genetic diversities were examined using six microsatellite markers amplifiable in three rare and one widespread species of Tricyrtis section Flavae, which are endemic to Japan. Contrary to a general expectation, the three rare species, Tricyrtis flava, Tricyrtis ohsumiensis and Tricyrtis perfoliata, have comparable genetic variation at the species level to that of the widespread Tricyrtis nana. This is probably because T. nana has not sufficiently recovered genetic diversity from the bottleneck at speciation or because recent range contractions have occurred in the three rare species. Genetic diversity at the population level was smaller in the putative selfing species T. nana than in the other three outcrossing species. Compared with a preceding study using allozyme markers, the genetic diversity in microsatellite loci was considerably larger, probably resulting from higher mutation rates at the microsatellite loci. Owing to the high genetic diversity of the microsatellite markers, genetic differentiation among populations could be estimated even in T. nana with little allozyme polymorphism.     

Development of compound microsatellite markers in the marine phytoplankton Alexandrium tamiyavanichii (Dinophyceae)

We isolated 24 polymorphic microsatellite markers from the toxic dinoflagellate A. tamiyavanichii. These loci provide a class of highly variable genetic markers, as the number of alleles ranged from 2 to 6, and the estimate of gene diversity was from 0.269 to 0.739 across the 24 microsatellites. We consider that these loci have a potentially to reveal the genetic structure and gene flow among A. tamiyavanichii populations.     

Isolation and characterization of microsatellite loci in the highland papaya Vasconcellea × heilbornii V. Badillo (Caricaceae)

Nine polymorphic microsatellite loci were identified in the tropical plant Vasconcellea ×heilbornii and used to estimate allelic diversity in two populations of southern Ecuador. Allelic richness ranged from two to five alleles, observed heterozygosity ranged from 0.150 to 0.947 and expected heterozygosity ranged from 0.186 to 0.701. Most of these markers also amplified microsatellite loci in two other Vasconcellea species (Vasconcellea stipulata and Vasconcellea cundinamarcensis). Hence, these markers will be useful for population genetic analysis and the evaluation of genetic diversity and gene flow in these species.     

New microsatellite primers for Daphnia galeata mendotae

In order to study genetic changes in populations of Daphnia galeata mendotae, I characterized seven polymorphic microsatellite loci. Primers to amplify these loci were tested on individual eggs from the resting egg bank of Onondaga Lake, NY. Levels of polymorphism and cross‐amplification in D. g. galeata indicate that they will be useful markers for ecological genetic studies on both adults and diapausing eggs of these species.     

Development of microsatellite markers for southern catfish (Silurus meriaionalis) and cross‐species amplification in northern sheatfish (Silurus soldatovi)

Forty microsatellite markers were developed from a (CA)_n enrichment library created from the DNA of southern catfish (Silurus meriaionalis). Also, the population structure of northern sheatfish (Silurus soldatovi) was examined by 24 microsatellite loci. They are polymorphic in at least one of the two geographically distant populations sampled from the Heilongjiang River and the Songhuajiang River in North China, respectively. Unbiased expected heterozygosity levels varied from 0.435 to 0.946 and number of alleles per locus varied from three to 20. Results indicated that these microsatellite loci were highly polymorphic and could be used as genetic markers.     

Identification and characterization of nuclear microsatellite loci in the aquatic moss Platyhypnidium riparioides (Brachytheciaceae)

Eight microsatellite loci from the aquatic moss Platyhypnidium riparioides were identified using the method of microsatellite‐enriched libraries. Polymorphism was assessed in a sample of four populations of 20 individuals each from four streams of the Meuse hydrographic basin in southern Belgium. The markers amplified three to seven alleles per locus. Comparison of observed and expected heterozygosities as well as F‐statistics (F_ST = 0.62) reveals a significant genetic differentiation among populations. These markers will be useful for further investigation of population genetic structure and diversity at different nested spatial scales.     

Microsatellite polymorphism and population subdivision in natural populations of European sea bass Dicentrarchus labrax (Linnaeus, 1758)

Polymorphism of microsatellite markers was used to study the genetic variability and structure in natural populations of European sea bass Dicentrarchus labrax. The data consisted of six microsatellite loci analysed for 172 individuals from three samples collected in the Golfe-du-Lion (France) and one sample collected in the Golfo-de-Valencia (Spain). Our goals were (i) to assess the level of genetic variability as revealed by these markers, (ii) to estimate the genetic differentiation among natural populations within a restricted area, and (iii) to evaluate how microsatellite loci fit the predictions of the two most widely used mutation models (the infinite allele model and the stepwise mutation model). As expected, our results indicate that the genetic polymorphism is very high when compared with previously used genetic markers, the mean expected heterozygosity per locus ranging between 0.69 and 0.93. We also found that all loci but one fitted the infinite allele model better. Using this model as a lower limit, we could extrapolate from the observed diversity effective population sizes on the order of 35 000 individuals. Our results also suggest that there may be a slight genetic differentiation between the two gulfs (F_ST= 0.007, P < 0.05), indicating that the corresponding populations are likely to be dynamically independent. This finding for a species with high dispersal abilities, if confirmed, has important beatings on fish-stock assessment.