Development and characterization of polymorphic microsatellite DNA markers for Sasa senanensis (Poaceae: Bambuseae)

Sasa senanensis is a dwarf bamboo species distributed on the floors of cool temperate forests in Japan and adjacent regions. We isolated eight polymorphic microsatellite loci from this species. The number of alleles ranged from two to eight and the observed heterozygosity per locus from 0.13 to 0.74. Seven of the eight loci were also polymorphic in Sasa nipponica. Most of these markers were successfully amplified in other dwarf bamboo species. These markers will be useful for investigating clonal structure and population genetics in some dwarf bamboo species.     

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 20 polymorphic microsatellite loci for Scaptodrosophila hibisci

Scaptodrosophila hibisci is an endemic Australian Drosophilidae that breeds in the flowers of native Hibiscus . Here we report the isolation and amplification of 20 polymorphic microsatellite loci . We cloned these microsatellites because loci developed for Drosophila melanogaster failed to amplify in S. hibisci . Null alleles were detected at six loci, and five were X‐linked. Two of the primer pairs amplified an unlinked ‘bonus’ locus. One locus containing juxtaposed microsatellite loci was suitable for designing an additional set of primers. Mean number of alleles per locus was 10, mean H _O and H _E per locus were 0.532 and 0.636, respectively.     

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 and characterization of novel di‐ and tetranucleotide microsatellite markers in Pacific cod (Gadus macrocephalus)

The Pacific cod (Gadus macrocephalus) supports large commercial fisheries in the northern Pacific Ocean and the Bering Sea. Here we characterize 10 polymorphic microsatellite loci isolated from enriched genomic libraries. Loci were screened on a sample of 96 spawning adults. The average number of alleles per locus was 25.3 (range 12–44), with expected heterozygosities (H_E) ranging from 0.54 to 0.97. No significant deviations of genotypic proportions from Hardy–Weinberg equilibrium or linkage equilibrium were detected. These markers will be used in future studies of population structure and mixed stock analysis of this important gadid species.     

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.     

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.     

Highly polymorphic microsatellite loci from the giant mottled eel (Anguilla marmorata)

Eighteen polymorphic microsatellite loci from the giant mottled eel (Anguilla marmorata) were identified and their characteristics were described. These markers were tested in two wild populations from China (n = 20) and Australia (n = 20) respectively. The number of alleles ranged from 9 to 14 with an average of 11.28 per locus. The mean expected heterozygosity (H_E) was 0.8696 in the population from China, 0.8823 in the population from Australia. No locus deviates significantly from Hardy–Weinberg proportions (P < 0.05).     

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.     

Characterization of new microsatellite loci in Pieris amamioshimensis (Ericaceae), a species nearly extinct in the wild

A set of 11 polymorphic microsatellite markers has been developed and characterized for the critically endangered species Pieris amamioshimensis. Fifty‐nine individuals of an ex‐situ population were used to identify these markers. The total number of alleles for each locus ranged from 3 to 9, with an average of 5.4. The expected heterozygosities (H_S) and observed heterozygosities (H_O) ranged from 0.47 to 0.77 and 0.22 to 0.88, respectively. In total, four loci exhibited significant deviations from Hardy–Weinberg equilibrium: two loci showed significant heterozygosity excess and the other two loci showed significant heterozygosity deficit. The polymorphism information content (0.43 ≤ PIC ≤ 0.73), the probability of exclusions (PE₁ = 0.9565, PE₂ = 0.9969 and PE₃ = 0.9999) and probabilities for identity (PI = 3.78 × 10^?9 and PI‐Sib = 2.35 × 10^?4) suggest that these markers are useful for estimating not only genetic diversity but also parentage, for the ex‐situ conservation management of populations.     

Clonal identification by microsatellite loci in sporadic flowering of a dwarf bamboo species, <Emphasis Type="Italic">Sasa cernua</Emphasis>

Dwarf bamboo species are monocarpic. They flower simultaneously and die after several decades. The type of flowering in the genus Sasa ranges from sporadic to gregarious. In order to determine whether or not the sporadic flowering of dwarf bamboo is fixed genetically, we investigated the distribution of clones using eight microsatellite (SSR) loci in a sporadic flowering patch of Sasa cernua Makino, a major dwarf bamboo species found in central Hokkaido. In May 2006, flowering occurred on 60.5% of living culms in a 1600 m² patch. We established a 50 × 10 m study plot in this patch and noted 1267 clumps consisting of 2529 living culms. We investigated all 1267 clumps and identified six multilocus genotypes as clones using five variable SSR loci. All flowering clumps belonged to the same clone. On the other hand, non-flowering vegetative clumps were also discovered to be of the same clone. These data suggest that all flowering culms originated from a single clone of a sporadic flowering patch of S. cernua. Clonal analysis for investigation of sporadic flowering of S. cernua revealed that only a portion of a clone flowers and dies instead of the whole clone.     

Analysis of molecular genetic diversity and population structure in Amaranthus germplasm using SSR markers

We assessed the molecular genetic diversity and population structure of Amaranthus species accessions using 11 simple sequence repeat markers. A total of 122 alleles were detected, and the number of alleles per marker (N_A) ranged from 6 to 21 with an average of 11.1 alleles. The frequency of major alleles per locus ranged from 0.148 to 0.695, with an average value of 0.496 per marker. The overall polymorphic information content values were 0.436–0.898, with an average value of 0.657. The observed heterozygosity (H_O) and expected heterozygosity (H_E) ranged from 0.056 to 0.876 and from 0.480 to 0.907, with average values of 0.287 and 0.698, respectively. The average H_O (0.240) was lower than the H_E and gene flow (Nm), and showed substantial genetic variability among all populations of amaranth accessions. The sample groupings did not strictly follow the geographic affiliations of the accessions. A similar pattern was obtained using model-based structure analysis without grouping by species type. Knowledge of the genetic diversity and population structure of amaranth can be used to select representative genotypes and manage Amaranthus germplasm breeding programs.     

Polymorphic microsatellite loci in the Coffee Berry Borer,Hypothenemus hampei (Coleoptera,Scolytidae)

Microsatellite loci were isolated from the coffee berry borer, Hypothenemus hampei, which is regarded as the major pest in coffee cultures. Seven polymorphic loci were obtained from an enriched genomic library. A low to moderate genetic diversity was observed per locus, with an observed number of alleles ranging from two to five in the 39 unrelated individuals sampled from Ethiopia. A clear deficit of heterozygotes within the population (mean heterozygosities, H_O = 0.10/H_E = 0.50) and an extreme inbreeding (F_IS, 0.70–1.00) were demonstrated. Cross‐species amplifications showed that some of the markers could be useful in two closely related Hypothenemus species.     

Development of EST‐SSRs from the Alpine Lady‐fern,Athyrium distentifolium

The utility of EST‐simple sequence repeats (EST‐SSRs) was evaluated in the fern Athyrium distentifolium. From 1152 frond cDNA clones, 165 microsatellites, including di‐, tri‐, tetra and penta‐nucleotide repeat motifs, were identified. Primer design was possible for 74 of the SSRs; subsequent screening of 10 loci on 186 individuals from six natural populations revealed between two and seven alleles per locus and expected heterozygosity (H_E) estimates ranging from 0.027 to 0.809. Eight of these loci were further examined for cross‐species and cross‐generic amplification in other Woodsiaceae species, and polymorphic products were detected. EST‐derived SSRs provide robust, informative and potentially transferable polymorphic markers suitable for biodiversity research.     

Development and characterization of new microsatellite markers for ginseng (<Emphasis Type="Italic">Panax ginseng</Emphasis> C. A. Meyer)

Panax ginseng C.A. Meyer, commonly known as Korean or Asian ginseng, is a perennial herb native to Korea and China. Its roots are highly prized for several medicinal properties. The present study describes development and characterization of twenty-two polymorphic microsatellite markers for this species. A total of 99 alleles were detected with an average of 4.5 alleles per locus across 20 accessions. Values for observed (H _O ) and expected (H _E ) heterozygosities ranged from 0.05 to 1.00 and from 0.18 to 0.73, respectively. Eleven loci deviated from Hardy–Weinberg equilibrium (P < 0.001). Significant (P < 0.05) heterozygote deficiency was observed at 13 loci. Exact test for linkage disequilibrium showed significant values (P < 0.05) between 12 pairs of loci. These microsatellite markers provide powerful tools for understanding population and conservation genetics of this species and also for genetic differentiation and authentication of different Panax species being used in commercial ginseng products.     

Characterization of 27 novel microsatellite loci in Sinibotia superciliaris (Günther 1892) and cross‐species transferability in Sinibotia reevesae (Chang 1944)

The Chinese golden loach, Sinibotia superciliaris and its congener Sinibotia reevesae, are endemic to China and extraordinary similar in morphological traits. However, few genetic studies have been conducted on them, especially those based on nuclear markers. Here, we developed 27 novel polymorphic microsatellite markers from S. superciliaris and further tested the cross‐species transferability of those loci in S. reevesae. The cross‐species amplification test showed that 19 loci demonstrate polymorphism in S. reevesae. The mean number of alleles (N_A) were both 3 in S. superciliaris and S. reevesae, the mean expected heterozygosities (H_E) were 0.57 and 0.54, the Shannon‐Wiener Diversity Indices (SW) were 0.84 and 0.82, and the evenness (E) were 0.09 and 0.08, respectively. The polymorphic information content (PIC) showed a high level of polymorphism for the two species (mean 0.54 and 0.50, respectively). In addition, the cross‐species transferability (100%) of those markers was clearly high, which confirmed that the microsatellite markers developed here could be used effectively for other related species.     

Isolation and characterization of polymorphic microsatellite loci from Wilsonia backhousei (Convolvulaceae)

Wilsonia backhousei is a clonal saltmarsh plant restricted to the southern latitudes of Australasia and threatened in New South Wales. We have identified eight informative microsatellite loci in the species from (AG)_n‐ and (AC)_n‐enriched libraries. In 48 samples from two populations we detected an average of five alleles per locus (range 2–8, average H_E = 0.45), of which 72% were unique to one population or the other. Six of the eight loci were also amplifiable in Wilsonia rotundifolia under the same reaction conditions. The markers will be excellent tools for use in the management and conservation of both species.     

Identification and characterization of nuclear microsatellite loci in Abies alba Mill.

Eleven polymorphic nuclear microsatellite markers for Abies alba Mill. were developed from an enriched genomic library. An average of 5.2 alleles per locus and a mean expected heterozygosity of 0.532 were found in a sample of 24 Abies alba individuals from different populations within Europe. These loci can be used in studies of genetic diversity for parentage analysis and for estimation of gene flow in silver fir populations. Moreover, successful amplifications were obtained for eight other Mediterranean Abies species, suggesting that these loci may be useful for similar applications in other fir species.     

Isolation and characterization of 15 polymorphic microsatellite markers for the fig-pollinating wasp,Blastophaga nipponica (Hymenoptera: Agaonidae)

We developed microsatellite markers for the fig-pollinating wasp Blastophaga nipponica Grandi using a dual-suppression-PCR technique. Twenty-one candidates of microsatellite loci were obtained, of which 15 yielded scorable patterns. The degree of polymorphism for the 15 loci was further characterized using summary statistics describing the genetic variation in 60 individuals from three natural populations in Japan. All 15 loci were polymorphic and yielded 2–27 alleles per locus. Overall observed heterozygosity (H _O) and expected heterozygosity (H _E) were 0.465 and 0.631, respectively. As expected, based on the inbreeding tendency of this species, the mean inbreeding coefficient (F _IS) was high (= 0.255). These markers will contribute to studies on the population structure of this species.