Microsatellite DNA markers for the study of Atlantic salmon (Salmo salar) kinship,population structure,and mixed‐fishery analyses

Eleven microsatellite DNA loci were identified and characterized for Atlantic salmon (Salmo salar) collected from the Penobscot River, Maine, USA and the River Nith, Scotland, UK. The markers revealed high levels of genetic diversity (seven to 48 alleles per locus), heterozygosity (to 100%), and allelic heterogeneity (all comparisons). Considerable differentiation was observed as the genetic distance (chord) between the two collections was 0.680 and the pairwise F_ST, 0.12, was highly significant. These findings are consistent with patterns of continental‐level differentiation observed previously using an alternate suite of microsatellite loci. Locus‐by‐locus analyses of molecular variance suggested that most markers were suitable for delineating kinships and population genetic structure.     

Isolation and characterization of novel tetranucleotide microsatellite DNA markers for the spotted salamander,Ambystoma maculatum

Fifteen tetranucleotide microsatellite loci were identified and characterized for spotted salamanders (Ambystoma maculatum) collected from three vernal pools in the south‐eastern USA. These markers revealed a high degree of genetic diversity (7–32 alleles per locus), heterozygosity (31.6–86.3%) and allelic heterogeneity (91% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.50 and 0.65, and all F_ST values (0.08–0.14) were statistically significant. Moreover, genotypic assignment tests correctly classified all individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and mating system.     

Novel tetranucleotide microsatellite DNA markers for the wood frog,Rana sylvatica

Fifteen tetranucleotide microsatellite loci were identified and characterized for wood frogs (Rana sylvatica) collected from three vernal pools in the southeastern US. These markers revealed a high degree of genetic diversity (nine to 34 alleles per locus), heterozygosity (30.6–92.3%) and allelic heterogeneity (69% of comparisons were statistically significant). Considerable differentiation among populations was observed as genetic distances (chord) ranged between 0.40 and 0.55 and all F_ST values (0.02–0.05) were statistically significant. Genotypic assignment tests correctly classified 103 of 113 individuals to their respective collection. These markers should prove useful for investigating fine‐scale population structure and metapopulation dynamics.     

Microsatellite DNA markers for the study of horseshoe crab (Limulus polyphemus) population structure

Twenty‐two microsatellite DNA loci were identified and characterized for horseshoe crabs (Limulus polyphemus) collected from two Atlantic coast and one Gulf of Mexico site. These markers revealed a high degree of genetic diversity (8–35 alleles per locus), heterozygosity (25.0% to 100.0%), and allelic heterogeneity (69.8% of comparisons). Considerable regional differentiation was observed as genetic distances (chord) ranged between 0.25 and 0.45, and all F_ST values (0.014–0.092) were significant. These preliminary findings are consistent with patterns of regional differentiation observed using allozyme variation and contradictory to findings of limited gene flow reported for sequence variation at the mitochondrial DNA COI region.     

Assessment of genetic variation between reproductive ecotypes of Klamath River steelhead reveals differentiation associated with different run-timings

Seven microsatellite DNA loci were optimized to assess genetic differentiation in coastal steelhead (Oncorhynchus mykiss irideus) sampling groups from the lower Klamath River (California, USA). Genetic relationships among three winter‐run and two summer‐run groups were investigated. The different groups displayed high levels of allelic variation. Pairwise F_ST comparisons and Nei's genetic distance supported low, yet significant, genetic differentiation between summer and winter run‐timings similar to other studies of temporal variation in salmonids. Analysis of molecular variance showed that most of the genetic variation was at the individual level (97.9%), although significant genetic variation existed between timing of runs (2.59%). Additionally, at least one locus in each group was out of Hardy–Weinberg equilibrium due to a deficiency in heterozygotes, and significant F_IS values were observed in three temporal collections. Together, these results suggest stock admixture, caused by multiple populations of origin in each sampling group, better known as the Wahlund Effect. These observations are preliminary evidence for isolation by time between Klamath River steelhead runs during distinct periods.     

Development of novel microsatellite markers for the grassland species Lolium multiflorum,Lolium perenne and Festuca pratensis

Twelve microsatellite markers were isolated from Lolium multiflorum. Allelic variability and cross‐species amplification were assessed on 16 individuals of each of the three grassland species L. multiflorum, Lolium perenne and Festuca pratensis. Cross‐species amplification success was 100% for L. perenne and 83% for F. pratensis. The number of alleles detected ranged from one to 14 with an average of 3.4. While three microsatellite loci were polymorphic in all three species, one marker produced species‐specific alleles in all three species. These microsatellite markers provide a valuable tool for population genetic studies within and among species of the Festuca–Lolium complex.     

Assessing population genetic structure of sorghum landraces from North-western Morocco using allozyme and microsatellite markers

 The level of genetic diversity and the population genetic structure of sorghum landraces from North-western Morocco have been investigated based on direct field-sampling using both allozyme and microsatellite markers. As expected, microsatellite markers showed a much higher degree of polymorphism than allozymes, but relative measures of genetic structure such as Wright’s inbreeding coefficient F _IS and Nei’s coefficient of genetic differentiation G _ST were similar for the two sets of markers. Substantial inbreeding was found to occur within fields, which confirms that sorghum is predominantly selfing under cultivation. Most of the genetic diversity in Moroccan landraces occurs within fields (more than 85%), as opposed to among fields or among regions, a result which contrasts to those of studies based on accessions from germplasm collections. It is suggested that individual fields of sorghum constitute valuable units of conservation in the context of in situ conservation practices. Received: 8 December 1998 / Accepted: 28 December 1998     

Global population structure of the spiny dogfish Squalus acanthias,a temperate shark with an antitropical distribution

The spiny dogfish (Squalus acanthias) is a temperate, coastal squaloid shark with an antitropical distribution in the Atlantic and Pacific oceans. The global population structure of this species is poorly understood, although individuals are known to undergo extensive migrations within coastal waters and across ocean basins. In this study, an analysis of the global population structure of the spiny dogfish was conducted using eight polymorphic nuclear microsatellite markers and a 566‐bp fragment of the mitochondrial ND2 gene region. A low level of genetic divergence was found among collections from the Atlantic and South Pacific basins, whereas a high level of genetic divergence was found among Pacific Ocean collections. Two genetically distinct groups were recovered by both marker classes: one exclusive to North Pacific collections, and one including collections from the South Pacific and Atlantic locations. The strong genetic break across the equatorial Pacific coincides with major regional differences in the life‐history characters of spiny dogfish, suggesting that spiny dogfish in areas on either side of the Pacific equator have been evolving independently for a considerable time. Phylogeographic analyses indicate that spiny dogfish populations had a Pacific origin, and that the North Atlantic was colonized as a result of a recent range expansion from the South American coast. Finally, the available data strongly argue for the taxonomic separation of the North Pacific spiny dogfish from S. acanthias and a re‐evaluation of the specific status of S. acanthias is warranted.     

Isolation and characterization of 149 novel microsatellite DNA markers for striped bass,Morone saxatilis,and cross‐species amplification in white bass,Morone chrysops,and their hybrid

To support detailed genetic analysis of striped bass (Morone saxatilis) and white bass (Morone chrysops), we isolated 153 microsatellite loci from repeat‐enriched striped bass DNA libraries. Of these, 147 markers amplified in striped bass (average 4.7 alleles per locus) and 133 in white bass (average 2.2 alleles per locus). One hundred twenty‐two markers amplified in their hybrid. Development of new microsatellite markers will facilitate evaluations of genetic structure in wild populations and will support pedigree analysis and linkage mapping for selective breeding.     

Characterization of microsatellite DNA markers for a mahseer species,Tor tambroides (Cyprinidae) and cross‐amplification in four congeners

This study reports the isolation and characterization of microsatellite DNA markers in a mahseer species, Tor tambroides (Pisces, Cyprinidae). Of a total of 14 loci evaluated, 10 were polymorphic in T. tambroides samples, with an average of 2.86 alleles per locus. Deviations from Hardy–Weinberg equilibrium were observed at one locus and there was no indication of linkage disequilibrium among loci. A high level of cross‐amplification among four congeners was achieved, with 12 loci successfully amplifying and 11 loci showing polymorphism in at least one other species. These markers will be a useful resource for population genetic studies and broodstock management of closely related mahseer species.     

Molecular Polymorphism of the Wheat Leaf Rust Fungus in Morocco Using Amplified Fragment Length Polymorphism

The genetic variability and collection structure of the wheat leaf rust fungus Puccinia recondita collected from four agro‐ecological areas of Morocco, Abda‐doukala, Chaouia‐Tadla, Gharb and Tangérois were investigated by amplified fragment length polymorphism (AFLP) markers. A set of five AFLP primers combinations which generated 253 polymorphic loci were used. Hierarchical partitioning revealed that bread wheat collections of Puccinia recondita form a single collection. No significant variation was observed between durum wheat collections of Puccinia recondita; they maintained most of the genetic variability within rather among collections. The distribution pattern of genetic variation of Puccinia recondita collections seems to be the result of high gene flow and the mixed reproduction system.     

Evaluating the potential of barley and wheat microsatellite markers for genetic analysis of<Emphasis Type="Italic"> Elymus trachycaulus</Emphasis> complex species

The potential of barley and wheat microsatellite markers for genetic analysis of Elymus trachycaulus complex species was evaluated. A set of 25 barley and 3 wheat microsatellite markers were tested for their ability to cross-amplify DNA from four accessions of E. trachycaulus and two accessions Pseudoroegneria spicata. Thirteen barley (52%) and two (68%) wheat primer pairs successfully amplified consistent products from both E. trachycaulus and P. spicata species. Four of the 15 successful primer pairs produced visible polymorphisms among the accessions tested. A higher successful rate of cross-species amplification of barley and wheat microsatellite markers in E. trachycaulus and P. spicata was found in this study. These primer pairs are now available for use as markers in genetic analysis of E. trachycaulus complex species. Our results suggest that publicly available wheat and barley microsatellite markers are a valuable resource for the genetic characterization of wild Triticeae species.     

Fine‐scale spatial and temporal population genetics of Aedes japonicus,a new US mosquito,reveal multiple introductions

The newly introduced mosquito Aedes japonicus has expanded from its original range in Northeastern Asia to 29 US states (including Hawaii) plus Canada and northern Europe. Our objectives were to test an earlier hypothesis of multiple introductions of this species to the Northeastern US and evaluate putative temporal changes in genetic makeup. Using a panel of seven microsatellite loci, we confirmed the existence of two abundant genetic forms in specimens originally collected in 1999–2000 (F_ST value based on microsatellite data = 0.26) that matches the disjunctive distribution of mitochondrial haplotypes. To examine the distribution of the two genetic ‘types’ across Pennsylvania we created a fine‐scale genetic map of Ae. japonicus using 439 specimens collected from 54 Pennsylvania counties in 2002–2003. We also made direct comparisons between collections in 1999–2000 and new collections made in 2004–2005 obtained from the same areas in the northeastern US. We observed that the strong association between mtDNA haplotype and microsatellite signature seen in 1999–2000 had weakened significantly by 2002 across Pennsylvania, a trend continued to some extent in 2004–2005 in PA, NJ, and NY, indicating that once easily distinguishable separate introductions are merging. The two expanding genetic forms create a complex correlation between spatial and genetic distances. The existence of multiple introductions would be obscured without sampling early and across time with highly polymorphic molecular markers. Our results provide a high‐resolution analysis of the spatial and temporal dynamics of a newly introduced disease vector and argue that successive introductions may be a common pattern for invasive mosquitoes.     

Microsatellite markers for the Indian golden silkmoth, Antheraea assama (Saturniidae: Lepidoptera)

Antheraea assama, an economically important and scientifically unexplored Indian wild silkmoth, is unique among saturniid moths. For this species, a total of 87 microsatellite markers was derived from 35 000 expressed sequence tags and a microsatellite‐enriched sub‐genomic library. Forty individuals collected from Tura and West Garo Hills region of Northeast India were screened for each of these loci. Ten loci from expressed sequence tags and one from genomic library were found to be polymorphic. These microsatellite markers will be useful resources for population genetic studies of A. assama and other closely related species of saturniids. This is the first report on development of microsatellite markers for any saturniid species.     

Development and characterization of nine polymorphic microsatellite markers in the sand goby Pomatoschistus minutus (Gobiidae)

A microsatellite‐enriched genomic library was constructed for the sand goby, Pomatoschistus minutus (Pallas 1770), and nine polymorphic DNA microsatellite markers of high quality were successfully optimized. Characterization of 96 individuals from the Vaccarès lagoon (France) showed moderate to high levels of polymorphism (two to 54 alleles). All the markers conformed to Hardy–Weinberg equilibrium and showed no evidence of null alleles, large allele dropout, stuttering and linkage disequilibrium between pairs of loci. These markers successfully amplify in three closely related species and can be employed to investigate population genetic structure and to clarify paternity in Pomatoschistus species.     

Development of sequence‐tagged microsatellite site markers for chickpea (Cicer arietinum L.)

Microsatellite loci were identified from chickpea (Cicer arietinum L.), the third most important grain legume crop in the world. A total of 13 sequence‐tagged microsatellite markers were developed using two different approaches: (i) amplification using degenerate primers and (ii) cloning of intersimple sequence repeat (ISSR)‐amplified fragments. Thirty‐five chickpea accessions were analysed, which resulted in a total of 30 alleles at the 13 loci. The observed heterozygosity ranged from 0.1143 to 0.4571 with an average of 0.2284. The cross‐species transferability of the sequence‐tagged microsatellite site (STMS) markers was checked in Cicer reticulatum, the wild annual progenitor of chickpea. These microsatellite markers will be useful for assessing the genetic diversity patterns within chickpea as well as aid in construction of intra‐ and interspecific genetic linkage maps.     

Isolation and characterization of microsatellite loci for Florida largemouth bass, Micropterus salmoides floridanus, and other micropterids

We isolated and characterized 52 novel microsatellite markers from Florida largemouth bass, Micropterus salmoides floridanus, for use in conservation, management and population genetic studies. Markers were assessed in M. s. floridanus from peninsular Florida (n = 30) and averaged eight alleles per locus with observed heterozygosity of 0.57 (range 0–0.97). Cross‐taxa amplification was successful among 88% of tested congeners. These polymorphic and potentially taxon‐diagnostic markers contribute to the limited number of microsatellites currently available for micropterids and specifically M. s. floridanus.     

Isolation and characterization of microsatellite loci for the Potentilla core group (Rosaceae) using 454 sequencing

Microsatellites are valuable markers for the analysis of genetic diversity, linkage mapping or genotyping. The limited availability of microsatellites for the genus Potentilla (Rosaceae) stipulated the isolation of markers from a representative (Potentilla pusilla Host) of the Potentilla core group that constitutes the most species‐rich evolutionary lineage within the genus. Thousand four hundred and seventy‐six simple sequence repeat (SSR) containing candidate sequences were isolated from a single‐type line using 454 sequencing. Seventy‐four functional microsatellite markers were developed from 200 sequences selected for suitable priming sites flanking microsatellite repeats referring to a 37% primer‐to‐marker conversion ratio. Seventy‐two markers were polymorphic. These numbers confirm the increased efficiency of pyrosequencing over traditional isolation techniques in the development of microsatellites. Amplification primer sequences and the sequences of corresponding target fragments are provided for all functional markers, and molecular polymorphisms estimated for four accessions of P. pusilla and among seven core group species represented by 14 individuals are reported. Cross‐species transferability ranged between 86.4% and 97.3% among the studied taxa, and 57, 11 and six of the selected primer pairs amplified fragments of expected size and number in seven, six and five of the species, respectively. Reproducibility of the molecular phenotypes was 97.0%, which was inferred using a replicate sample of P. pusilla.     

Migratory behaviour shapes spatial genetic structure of cyprinid fishes within the Lake Malawi catchment

• Genetic differences among freshwater fish populations are dependent on life‐history characteristics of the species, including the range of adult dispersal and the extent of homing to natal breeding grounds. However, the effects of variation in such characteristics on population genetic connectivity are rarely studied comparatively among closely related species.
• We studied population genetic structure within three congeneric cyprinid species from the Lake Malawi catchment that differ substantially in life‐history traits and conservation status, using a combination of microsatellite and mitochondrial DNA markers. Mpasa (Opsaridium microlepis) is a large (70 cm total length) migratory species that spawns in rivers, but as an adult is exclusively known from the main lake body. Sanjika (Opsaridium microcephalum), is a medium size (30 cm total length) species that exists in lake breeding, river‐lake migratory and apparently landlocked populations. Dwarf sanjika (Opsaridium tweddleorum) is a small non‐migratory species (15 cm total length) that persists in small tributaries surrounding the main lake and adjoining rivers.
• The results revealed striking differences among the three species in spatial genetic structuring. The river‐lake migratory mpasa showed only weak yet significant population genetic structure within the main Lake Malawi catchment, suggesting that there is no strong natal homing. The habitat‐generalist sanjika showed only weak spatial genetic differentiation at microsatellite loci within the Lake Malawi catchment, but moderate structure in mitochondrial DNA, potentially reflecting male‐biased dispersal. The river‐restricted dwarf sanjika showed strong genetic structure in both microsatellite and mitochondrial DNA, suggesting strictly limited dispersal at both adult and juvenile stages.
• We conclude that contrasting migration life histories have resulted in dramatically different patterns of population genetic structure among these congeneric species. The observed patterns demonstrate how divergent life‐history evolution may strongly influence broader patterns of population genetic connectivity in freshwater fish, with consequences for management and conservation. Specifically the results suggesting gene flow among Lake Malawi populations of mpasa, an IUCN red‐listed ‘Endangered’ species endemic to the lake catchment, imply that conservation initiatives operating at both local and catchment scales are needed to reverse local population decline.

     

BIOGEOGRAPHIC ANALYSIS OF THE GLOBALLY DISTRIBUTED HARMFUL ALGAL BLOOM SPECIES ALEXANDRIUM MINUTUM (DINOPHYCEAE) BASED ON rRNA GENE SEQUENCES AND MICROSATELLITE MARKERS1

The toxic dinoflagellate Alexandrium minutum Halim is one of three species that comprise the “minutum” species complex. This complex is notable due to its role in the etiology of paralytic shellfish poisoning (PSP). Recent increases in PSP incidence and the geographic expansion of toxin‐producing Alexandrium dinoflagellates have prompted the intensive examination of genetic relationships among globally distributed strains to address questions regarding their present distribution and reasons for their apparent increase. The biogeography of A. minutum was studied using large subunit ribosomal DNA gene (LSU rRNA) and internal transcribed spacer (ITS) sequences and genotypic data from 12 microsatellite loci. rRNA gene and ITS sequencing data distinguished between two clades, herein termed the “Global” and the “Pacific”; however, little to no resolution was seen within each clade. Genotypic data from 12 microsatellite loci provided additional information regarding genetic relationships within the Global clade, but it was not possible to amplify DNA from the Pacific clade using these markers. With the exception of isolates from Italy and Spain, strains generally clustered according to origin, revealing geographic structuring within the Global clade. Additionally, no evidence supported the separation of A. lusitanicum and A. minutum as different species. With the use of microsatellites, it is now possible to initiate studies on the origin, history, and genetic heterogeneity of A. minutum that were not previously possible using only rRNA gene sequence data. This study demonstrates the power of combining a marker with intermediate resolution (rRNA sequences) with finer‐scale markers (microsatellites) to examine intraspecies variability among globally distributed isolates and represents the first effort to employ this technique in A. minutum.