High variability and disomic segregation of microsatellites in the octoploid Fragaria virginiana Mill. (Rosaceae)

The objectives of the present study were to develop microsatellite markers for the wild strawberry, Fragaria virginiana, to evaluate segregation patterns of microsatellite alleles in this octoploid species, and assess genetic variability at microsatellite loci in a wild population. A genomic library was screened for microsatellite repeats and several PCR primers were designed and tested. We also tested the use of heterologous primers and found that F. virginiana primers amplified products in cultivated strawberry, Fragaria × ananassa Duch. and Fragaria chiloensis. Similarly, microsatellite loci developed from cultivated strawberry also successfully amplified F. virginiana loci. We investigated four microsatellite loci in detail, three developed from F. virginiana and one from cultivated strawberry. A survey of 100 individuals from a population of F. virginiana in Pennsylvania demonstrated high heterozygosities (H_e or gene diversity ranged from 0.80 to 0.88 per locus) and allelic diversity (12–17 alleles per locus), but individual plants had no more than two alleles per locus. Segregation patterns in parents and progeny of two controlled crosses at these four loci were consistent with disomic Mendelian inheritance. Together these findings suggest that the genome of F. virginiana is "highly diploidized" and at least a subset of microsatellite loci can be treated as codominant, diploid markers. Significant heterozygote deficiencies were found at three of the four loci for hermaphroditic individuals but for only one locus among females in this gynodioecious species.Communicated by J. Dvorak     

Isolation and characterization of microsatellite loci in Fagus longipetiolata Seem. (Fagaceae)

Eleven microsatellite loci have been developed from Fagus longipetiolata and the loci were characterized for 21 individuals. All eleven loci were polymorphic, with 2–8 alleles and an average of 4.8 per locus. The observed (H _O) and expected (H _E) heterozygosities were 0.053–0.714 and 0.355–0.856, respectively. There was significant deviation from Hardy–Weinberg equilibrium at two loci. No locus pair had significant linkage disequilibrium. Cross-species amplifications of the markers were also tested in three other congeneric species.     

Characterization of microsatellite markers in the mosquito Ochlerotatus caspius (Diptera: Culicidae)

Seven polymorphic microsatellite loci were developed for the mosquito species Ochlerotatus caspius, using an enriched genomic library. The number of alleles per locus varied between two and 11; the expected heterozygosity (H_E) ranged from 0.18 to 0.77. These microsatellite primers should prove useful for population genetic studies of this mosquito species.     

Development and characterization of microsatellite markers from the yellow passion fruit (Passiflora edulis f. flavicarpa)

Here we described the development of the first set of Passiflora microsatellite loci isolated from an enriched genomic library. A sample of 43 individuals from 12 accessions of the yellow passion fruit was used to characterize those loci, which revealed up to 20 alleles per locus. Two loci were monomorphic. The observed (H_O) and expected (H_E) heterozygosities were very similar, as expected for a self‐incompatible species. Allelic diversity (H_T) was 0.444. This set of markers will permit genetic structure analyses of cultivated and wild populations of Passiflora, and contribute for integrating genetic maps based on dominant markers, as they can provide bridge alleles.     

Microsatellite analysis of genetic diversity in the Chinese alligator (<Emphasis Type="Italic">Alligator sinensis</Emphasis>) Changxing captive population

Chinese alligator (Alligator sinensis) is a critically endangered species endemic to China. In this study, the extent of genetic variation in the captive alligators of the Changxing Reserve Center was investigated using microsatellite markers derived from American alligators. Out of 22 loci employed, 21 were successfully amplified in the Chinese alligator. Sequence analysis showed loci in American alligators had a bigger average size than that of the Chinese alligators and the longest allele of an individual locus almost always existed in the species with longer stretch of repeat units. Eight of the 22 loci were found to be polymorphic with a total of 26 alleles present among 32 animals scored, yielding an average of 3.25 alleles per polymorphic locus. The expected heterozygosity (H _E) ranged at a moderate level from 0.4385 to 0.7163 in this population. Compared to that in the American alligators, a lower level of microsatellite diversity existed in the Changxing population as revealed by about 46% fewer alleles per locus and smaller H _E at the homologous loci. The average exclusion power and the ability to detect shared genotypes and multiple paternity were evaluated for those markers. Results suggested that when the polymorphic loci were combined, they could be sensitive markers in genetic diversity study and relatedness inference within the Chinese alligator populations. The level of genetic diversity present in the current Changxing population indicated an important resource to complement reintroductions based on the individuals from the other population. In addition, the microsatellite markers and their associated diversity characterized in this population could be utilized to further investigate the genetic status of this species.     

Development of microsatellite marker loci for European hazelnut (<Emphasis Type="Italic">Corylus avellana</Emphasis> L.) from ISSR fragments

Polymorphic microsatellite markers were developed for European hazelnut (Corylus avellana L.) from the sequences of inter-simple sequence repeat (ISSR) fragments and flanking regions. Twenty-five ISSR primers were used to generate fragments for cloning. Of the 520 unique sequences obtained, 41 contained long internal repeats (≥20 bp) with flanking sequences sufficient for primer design. From these, we developed 23 new polymorphic microsatellite loci. The flanking sequences were obtained for fragment ends by chromosome walking, and an additional 47 polymorphic markers were developed. Two additional polymorphic markers were developed from a GA-enriched library. The 72 new marker loci were characterized using 50 diverse hazelnut accessions. For the internal repeat loci, the number of alleles per locus ranged from 2 to 16, with a mean of 7.52. Mean values for expected heterozygosity (H_e), observed heterozygosity (H_o), and polymorphism information content (PIC) were 0.62, 0.59, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.05 at six of the 23 loci. For the 47 marker loci developed from fragment ends, the number of alleles per locus ranged from 2 to 16, with a mean of 7.30. Mean values for H_e, H_o, and PIC were 0.62, 0.47, and 0.58, respectively. The estimated frequency of null alleles (r) was ≥0.10 at 18 of the 47 loci. Of the 70 loci developed from ISSR and flanking sequences, 50 segregated in our mapping population and were assigned to linkage groups.     

Isolation and characterization of 18 polymorphic microsatellite loci from freshwater pearl mussel (<Emphasis Type="Italic">Cristaria plicata</Emphasis>)

Cristaria plicata was an important freshwater mussel for pearl culture in China. 18 polymorphic microsatellite markers were isolated and characterized using (CA)₁₅-enriched genomic library of C. plicata. These loci showed high levels of genetic polymorphism testing on 60 individuals sampled from Poyang Lake of Jiangxi Province, China. The number of alleles per locus ranged from 4 to 18. The expected (H _E) and observed heterozygosities (H _O) were 0.7232–0.8961 and 0.0000–1.0000, respectively. Four microsatellite loci were significantly deviated from Hardy–Weinberg equilibrium and no linkage disequilibrium was found. These microsatellite loci will be useful for assessment of genetic diversity and population structure in C. plicata.     

Isolation and characterization of variable microsatellite loci in Lilium philadelphicum (Liliaceae)

Here we describe the characterization of six polymorphic microsatellite loci for Lilium philadelphicum (Liliaceae). Polymorphism levels ranged from 7 to 30 alleles per locus with a mean number of 15 alleles per locus. Observed heterozygosity (H_O) ranged from 0.17 to 0.82 and did not deviate from Hardy–Weinberg expectations in the three western Montana populations included in the analysis. These loci are proving useful in studying gene flow between populations of this species distributed across its range in North America.     

Microsatellite loci developed for black-crowned night-heron (Nycticorax nycticorax) and their cross-amplifications in Ardeidae

The present study reports isolation and characterization of 11 polymorphic microsatellite markers for Nycticorax nycticorax. A total of 82 alleles were detected with an average of 7.5 alleles per locus. The observed heterozygosity (H _O) ranged from 0.25 to 1.00, and the expected heterozygosity (H _E) ranged from 0.51 to 0.88. Analyses revealed no evidence for Linkage disequilibrium between any two loci, and only one locus was significantly deviated from HWE with the estimation of exact P values by the Markov chain method (P < 0.001). The 11 loci were successfully amplified in 11 other Ardeidae species. These results demonstrate these markers can be used for the study of intra- and inter-specific genetic diversity.     

Isolation and characterization of microsatellite markers from Brown Eared-pheasant (Crossoptilon mantchuricum)

The Brown Eared-pheasant (Crossoptilon mantchuricum) is an endangered species endemic to China. Here we developed eight microsatellite loci using a modified biotin-capture method. In the analyses of 28 individuals sampled, the number of alleles per locus ranged from four to nine. The expected (H _E) and observed (H _O) heterozygosities were 0.466–0.825 and 0.619–0.847, respectively. Results that eight microsatellite loci were highly polymorphic indicated that these markers are sufficiently powerful to address such questions as genetic diversity and population genetic structure of C. mantchuricum.     

Development of microsatellite markers for the endangered freshwater pearl mussel Margaritifera margaritifera L. (Bivalvia: Unionoidea)

Freshwater pearl mussels (Margaritifera margaritifera L.) are among the most critically endangered freshwater invertebrates. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4900 recombinant clones from two genomic libraries. Thirteen loci revealed polymorphisms as demonstrated on 42 tested individuals from four river drainages. Allelic richness ranged from two to 12 alleles and averaged 6.8 alleles per locus with heterozygosity levels varying from 0 to 0.850 for observed heterozygosity (H_O) and from 0.174 to 0.850 for expected heterozygosity (H_E). Deficiency of heterozygous genotypes was observed in eight of 13 loci.     

Polymorphic microsatellite DNA markers for the ark shell Scapharca subcrenata (bivalve: Arcidae)

We have isolated and characterized twelve novel polymorphic microsatellite loci from Scapharca subcrenata to analyse the population structure. The number of observed alleles per locus ranged from 3 to 17. Observed heterozygosity (H _O) ranged from 0.321 to 0.929. Cross-species amplification was tested successfully in three other bivalve species. These microsatellite markers will be useful for genetic diversity studies of S. subcrenata and other Lamellibranchia species.     

Development and characterization of 14 novel microsatellite markers for an invasive goby (Tridentiger bifasciatus) in water transfer system

Genetic patterns observed among populations by neutral molecular markers is a major method used to reconstruct the invasion history of invasive species. The Shimofuri goby (Tridentiger bifasciatus) has invaded several lakes along the East Route of the South-to-North Water Transfer Project, China (ENST). However, neutral molecular markers for this goby are not currently available. In this study, fourteen novel polymorphic microsatellite loci were developed through next generation sequencing technology. These markers were also tested in an invasive population (Nansi Lake, a storage lake of ENST) consisting of 54 individuals. The average number of alleles per locus was 7.6 and ranged from 4 to 13. The average H_o was 0.587 and ranged from 0.404 to 0.852, and the average H_e was 0.733 and ranged from 0.564 to 0.851. The average PIC per locus was 0.689 and ranged from 0.521 to 0.824. Four markers (TB4, TB65, TB66 and TB26) were found to deviate from HWE after correction following the False Discovery Rate method. Null alleles were found in four loci (TB11, TB65, TB66 and TB26). These highly polymorphic microsatellite markers are useful for further study to reconstruct invasion history of Shimofuri goby.     

Isolation and characterization of microsatellite loci in the two‐spotted spider mite,Tetranychus urticae (Acari: Tetranychidae)

Sixteen microsatellite loci are described for the two‐spotted spider mite Tetranychus urticae, which is an agricultural pest. The microsatellite loci were obtained through the construction of an enriched library; these loci exhibited polymorphisms (2–5 alleles per locus) and high levels of observed (0.033–0.667, average 0.415) and expected (0.033–0.602, average 0.336) heterozygosities. The isolated microsatellite markers are expected to be useful for the construction of a linkage map of this species.     

Polymorphic microsatellite markers for the critically endangered Balearic shearwater, Puffinus mauretanicus

Ten novel polymorphic microsatellite loci were isolated and characterized from the Balearic shearwater (Puffinus mauretanicus), a critically endangered seabird. The developed loci revealed a relatively low number of alleles per locus, as well as low levels of polymorphism (H_O = 0.377 ± 0.241). One of the loci appeared to be W‐linked. All polymorphic loci were successfully amplified in its closely related species, the Yelkouan shearwater (Puffinus yelkouan). These microsatellite markers would be useful for assessing population structure in the Balearic shearwater and the possible hybridization process between both shearwaters species.     

Isolation and characterization of polymorphic microsatellite markers from Australia short-finned eel (Anguilla australis Richardson)

Seventeen microsatellite DNA loci from the Australian short‐finned eel (Anguilla australis Richardson) were isolated and their amplification characteristics were described. The polymerase chain reaction primers were tested on 40 eel individuals. The primers amplified loci with relatively high numbers of alleles, ranging from five to 14 with an average of nine per locus. Mean observed heterozygosity (H_O) and expected heterozygosity (H_E) were 0.6779 and 0.7374, respectively, indicating that these markers would be useful for population studies. No loci deviated significantly from Hardy–Weinberg equilibrium (P = 0.05) and no evidence was found for genotypic disequilibrium among loci at a 5% significance level.     

Microsatellite loci transferability from Theobroma cacao to Theobroma grandiflorum

Theobroma grandiflorum (cupuassu) is an important fruit tree native to the Brazilian Amazon. Forty‐eight microsatellite loci developed for the congener Theobroma cacao were tested in cupuassu, and 29 (60.4%) produced robust alleles. The analyses of 216 cupuassu accessions using the 21 polymorphic microsatellite loci revealed a total of 113 alleles. The number of alleles per polymorphic locus ranged from two to 11, with an average of 5.38 alleles per locus. The average observed heterozygosity was 0.343, while the mean expected heterozygosity was 0.614. The successful transferability of T. cacao microsatellite primers to cupuassu was consistent with currently accepted phylogeny.     

Development and characterization of 11 novel microsatellite loci for the roundscale spearfish Tetrapturus georgii and their cross‐species amplification among other istiophorid species

Eleven novel polymorphic microsatellite loci were developed and characterized for the recently validated roundscale spearfish Tetrapturus georgii. Characterization of these markers, based on 35 roundscale spearfish from the western North Atlantic, revealed two to 21 alleles per locus with an average expected heterozygosity (H_E) of 0·09–0·94, and all loci conformed to Hardy–Weinberg expectations. Cross‐amplification of these 11 loci against all other eight known istiophorid species indicates promising prospects for the utility of these markers for istiophorids in general.     

Isolation and characterization of eight microsatellite loci from the endangered plant species Hypochaeris salzmanniana (Asteraceae)

We report the isolation and characterization of eight microsatellite loci for Hypochaeris salzmanniana, an endangered species endemic to the southwestern coast of Spain and the Atlantic coast of Morocco. A total of 32 alleles were detected across a sample of 45 individuals, with an average number of 4.0 alleles per locus. The average polymorphic information content (PIC) was 0.533 and the observed (H _O) and expected (H _E) heterozygosity values varied from 0.022 to 0.978 and from 0.434 to 0.759, respectively. Five loci exhibited significant deviation from Hardy–Weinberg equilibrium (P ≤ 0.001) and three pairs of loci showed significant linkage disequilibrium (P ≤ 0.01). The eight loci were tested for transferability in three others species (H. arachnoidea, H. glabra, and H. radicata) belonging to the same section of H. salzmanniana. With the exception of locus Hsalz7, all loci successfully amplified in the three species. These preliminary data confirm the usefulness of microsatellite markers for assessing the ecology and genetic structure of H. salzmanniana and to understand the evolution of species within the section Hypochaeris.     

Characterization of microsatellite loci developed for <Emphasis Type="Italic">Amaranthus hypochondriacus</Emphasis> and their cross-amplifications in wild species

The present study reports isolation and characterization of 12 polymorphic microsatellite markers for Amaranthus hypochondriacus. A total of 92 alleles were detected across the 20 accessions, with an average of 7.7 alleles per locus. The observed (H _O ) and expected (H _E ) heterozygosity values ranged from 0 to 0.95 and from 0.49 to 0.92, respectively. At significance threshold (P < 0.05), nine loci deviated from Hardy-Weinberg equilibrium (HWE), whereas significant linkage disequilibria (LD) were observed between five pairs of loci. The 12 loci were successfully amplified in 18 other amaranth species representing cultivated grain and vegetable species, their putative progenitors and wild species. These results demonstrate wide potential applicability of these markers for the study of intra- and inter-specific genetic diversity as well as evolutionary relationships among cultivated and wild amaranths.