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.     

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.     

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.     

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 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 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 dwarf bamboo species Sasa cernua and Sasa kurilensis (Poaceae) in northern Japan

Ten polymorphic microsatellite markers were isolated from the dwarf bamboo species Sasa cernua and Sasa kurilensis. The applicability of these markers was confirmed by genotyping of open‐pollinated seeds and leaf samples from natural populations. Genotypes of seeds from each culm shared at least one allele from the maternal parent without contradiction. All 10 loci were polymorphic in S. cernua with 2–15 alleles (average H_E = 0.532). Eight loci were polymorphic in S. kurilensis with 2–10 alleles (average H_E = 0.532). These markers will be useful in detailing the extent of clonal and sexual reproduction in these species.     

Genetic variation and differentiation in Nordic populations of Elymus alaskanus (Scrib. ex Merr.) Löve (Poaceae)

 To gain information on the extent and nature of genetic variation in Elymus alaskanus, levels and distribution of genetic variation were assessed within and among 13 populations originating from Iceland, Norway, Sweden and Russia using allozymes. The results showed that four (30.7%) of the 13 loci were polymorphic within the species, while the mean percentage of polymorphic loci within the populations was 1.9%. The mean number of alleles per locus for the species was 1.8 and 1.02 across the populations. Genetic diversity at the species level was low (H _es=0.135), and mean population diversity was notably lower (H _ep=0.005). A high degree of genetic differentiation was observed among populations. The salient points emerging from this study are: (1) statistically significant differences were found in allele frequencies among populations for every polymorphic locus (P<0.001), (2) the high mean coefficient of gene differentiation (G _ST) showed that 95% of the total allozyme variation was attributable to differences among populations, and (3) relatively high genetic distances between the populations were obtained (mean D=0.16). The Norwegian populations had the highest genetic diversity as compared with the other populations. Geographical comparisons revealed three different groups of populations clearly differentiated, i.e. Scandinavia (Norway and Sweden), Iceland and Russia. Cluster and principal coordinates analyses revealed the same genetic patterns of relationships among populations. Generally, this study indicates that E. alaskanus contains low allozymic variation in its populations. The implications of these results for the conservation of the species are discussed. Received: 23 October 1998 / Accepted: 19 December 1998     

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.     

Development of microsatellite loci for Pelophylax plancyi and cross-amplification in other ranid species

Thirteen polymorphic microsatellite loci were obtained from the gold-striped pond frog, Pelophylax plancyi. The number of alleles per locus ranged from 5 to 13 with an average of 8.38. The observed and expected heterozygosities (H _O and H _E) ranged from 0.533 to 0.890 and from 0.620 to 0.897, with averages of 0.686 and 0.751, respectively. In order to assess interspecific amplification, all primer pairs were tested with the same PCR conditions on three other Pelophylax species: Pelophylax hubeiensis, Pelophylax fukienensis, Pelophylax nigromaculata and two species from the genus Hylarana and Hoplobatrachus, respectively. All of these loci can be amplified successfully in the Pelophylax species, with eight loci amplified in all species tested.     

Genetic and clonal diversity in Korean populations ofVitex rotundifolia (Verbenaceae)

Vitex rotundifolia L.f. is a woody perennial and has sexual and asexual modes of reproduction. Allozyme study was conducted on 550 plants in 13 Korean populations. The levels of genetic variability and divergence within and among populations, respectively, are considerably lower and higher than the mean values for woody plants with similar life history tralts. Mean percentage of polymorphic loci (P _P), mean number of alleles per locus (A _P), and mean genetic diversity (He _P) within populations ofV. rotundifolia were: 16.7%, 1.21, and 0.047. On average, about 79% of the total variation inV. rotundifolia was common to all populations (meanG _ST=0.208). In addition, significant differences in allele frequencies among populations were found in all polymorphic loci examined (P<0.001). On the other hand, levels of genotypic diversity within and among populations were moderate. About 44% (18/41) of multilocus genotypes were “local genotypes” (genotypes occurring in only one population), whereas only one “widespread genotype” (genotypes occurring in more than 75% of the populations) were detected. The mean number of multilocus genotypes per population (G) and mean genotypic diversity index (D _G) were 8.4 and 0.74, respectively. Most common multilocus genotypes found in populations were homozygous for five polymorphic loci. The abundance of ramets of these genets is responsible for the low levels of expected heterozygosity within populations. The results indicate that clonal reproduction may act as an enhancer of genetic drift by reducing effective size of local populations ofV. rotundifolia.     

Generation and characterization of 24 novel EST derived microsatellites from tea plant (Camellia sinensis) and cross-species amplification in its closely related species and varieties

A total of 31 expressed sequence tag (EST)-derived polymorphic microsatellites from tea plant, Camellia sinensis (L.) O. Kuntze, were generated and characterized using the ESTs of the author’s EST sequencing project and other sources. A set of 40 accessions tea germplasms had been used to examine the diversity. Among the 31 microsatellite loci, 24 had two to eight polymorphic alleles. Observed heterozygosity (H _o) were relatively higher (on an average of 0.533), varying from 0.175 (primer 21) to 0.950 (primer 228). Cross-species polymorphic amplification in other four species and two varieties of section Thea (L.) Dyer genus Camellia L. was successful for the 24 loci. Contribution of the 24 novel EST-SSR primers presented here will provide necessary and powerful molecular tools for management and conservation studies on the tea germplasms in the future. Li-Ping Zhao and Zhen Liu contributed equally to this work.     

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.     

Low genetic differentiation among populations ofArabis serrata (Brassicaceae) along an altitudinal gradient

We investigated the extent of genetic differentiation among populations of fujihatazao,Arabis serrata along an altitudinal gradient at Mt. Fuji in Shizuoka Prefecture. This species is a perennial plant, widely distributed in Japan forming small isolated populations. However, at Mt. Fuji, this species constitutes a large population distributed from 1440 to 2400 m altitude. A total of 411 individuals were sampled from ten subpopulations. Eighteen loci were detected on eleven enzyme systems. Eleven loci were monomorphic and seven loci were polymorphic with a mean of 2.11 alleles per loci. Nei's genetic distance (mean 0.01) and genetic identity (mean 0.968) were very similar among populations indicating a low genetic differentiation. The total genetic diversity (H _T ) estimated for the polymorphic loci was, in average, 0.396. The mean gene differentiation (G_ST=0.091) was very low. Gene frequency of seven polymorphic loci was analyzed by spatial autocorrelation methods based on Moran's indexes. Only Pgi-3 exhibited a significant negative autocorrelation (−0.160;P<0.05); other loci values ranged from −0.134 to 0.027. Gene flow estimated by indirect methods varied between genes but most of the values were high (meanNm=20.8) suggesting that subpopulations at different altitudes are probably connected. Despite plants at different altitudes present different ecological traits (e.g., differences in phenology, growth and reproductive traits), subpopulations ofA. serrata are still low differentiated, at least for the loci studied. This may be explained by the recent origin of some habitats (e.g., second crater and surrounded areas) in this locality.     

Isolation and characterization of 12 microsatellite loci for Michelia coriacea (Magnoliaceae), a critically endangered endemic to Southeast Yunnan, China

By using a modified biotin–streptavidin capture method, a total of 12 microsatellite loci were developed and characterized for Michelia coriacea (Magnoliaceae), a critically endangered endemic to Southeast Yunnan, China. The number of alleles (A) ranged from two to six in 30 samples of this species. The ranges of observed (H _O ) and expected (H _E ) heterozygosities were 0.033–0.8000 and 0.033–0.7910, respectively. Cross-species amplification in M. yunnanensis showed that a subset of these markers holds promise for congeneric species. These polymorphic SSR markers would be useful tools for population genetics studies on M. coriacea and other congeneric species.     

Isolation and characterization of new microsatellite markers from the Japanese scallop (<Emphasis Type="Italic">Patinopecten yessoensis</Emphasis>)

Twenty polymorphic microsatellite loci were isolated from the Japanese scallop (Patinopecten yessoensis) using a method of hybridizing twice. The genetic diversity was analyzed by these microsatellite markers in field specimens from Zhangzidao Island in Liaoning Province, China. Observed heterozygosity (H _o), expected heterozygosity (H _e), number of effective alleles (N _e), and polymorphism information contents (PIC) were calculated. The heterozygote deficiency or excess was detected by the fixation index (Fis). The Hardy–Weinberg equilibrium was tested by possibilities (P _HWE) using chi-square test. Results indicate that the average of allele numbers was 6.0. The mean values of the parameters H _o, H _e, N _e, and PIC were 0.7921, 0.7076, 4.4347, and 0.6684, respectively. Only 4 of 20 microsatellite markers showed heterozygote deficiency including loci FJ262370, FJ262381, FJ262384, and FJ262400. The P _HWE value indicates the wild population deviated somewhat from the Hardy–Weinberg equilibrium. These newly isolated markers increase the available molecular resources of the Japanese scallop.     

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.     

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.     

Allozyme diversity in the federally threatened golden paintbrush, <Emphasis Type="Italic">Castilleja levisecta</Emphasis> (Scrophulariaceae)

Castilleja levisecta (Scrophulariaceae), the golden paintbrush, is an insect-pollinated herbaceaous perennial found in the Pacific Northwest. Currently restricted to two island populations off British Columbia and nine populations (eight on islands) in Washington, C. levisecta is a rare species threatened with extinction. Allozymes were used to describe genetic diversity and structure in these eleven populations. Despite its threatened status and small geographic range, exceptionally high levels of genetic diversity are maintained within C. levisecta. All sixteen of the loci resolved were polymorphic within the species (P_s=100%), while the mean percentage of loci polymorphic within populations (P_p) was 65.7%. The mean number of alleles per polymorphic locus (AP_s) was 2.94 within the species and averaged 2.38 within populations (AP_p). Genetic diversity (H_es) was 0.285 for the species, whereas mean population genetic diversity (H_ep) was 0.213. Smaller populations had, on average, fewer observed alleles and less genetic diversity. A significant negative correlation (r = –0.72) was found between genetic identity and geographic distance, indicating reduced gene flow between distant populations. The most geographically isolated population was one of the larger populations, one of the most genetically diverse and the most genetically divergent. A wide range of pairwise population genetic identities (I = 0.771 – 0.992) was found, indicating considerable genetic divergence between some populations. Overall, 19% of the total genetic diversity was distributed among populations. Results of this survey indicate that genetic augmentation of existing populations is unnecessary. The high allelic diversity found for the species and within its populations holds promise for conservation and restoration efforts to save this rare and threatened plant species.     

Isolation and characterization of microsatellite loci in the endangered freshwater fish <Emphasis Type="Italic">Varicorhinus alticorpus</Emphasis> (Cyprinidae)

Varicorhinus alticorpus (Cyprinidae) is an endangered freshwater fish with a scattered distribution in the southern and eastern regions of Taiwan. We described the development of nine polymorphic microsatellite loci in V. alticorpus for genetic studies. These new markers were tested in 20 individuals of the rare species. The number of alleles ranged from 4 to 13. Expected (H _E) and observed (H _O) heterozygosities ranged from 0.692 to 0.892 (averaged at 0.821) and from 0.000 to 0.350 (averaged at 0.088), respectively. All loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. This suite of highly polymorphic microsatellites provides the first chance to undertake a conservation program for this species in Taiwan. It is essential to evaluate its genetic diversity and the fine-scale population structure. Tzen-Yuh Chiang, Teh-Wang Lee, and Feng-Jiau Lin contributed equally to this work.