Microsatellite variability and heterozygote excess in Elymus trachycaulus populations from British Columbia in Canada

Microsatellite markers were used to assess the genetic diversity and population structure in four populations of Elymus trachycaulus from British Columbia and one population of Elymus alaskanus from Northwest Territories. Fourteen microsatellite loci were used in this study. Our results indicated that E. trachycaulus is highly polymorphic, with an average percentage of polymorphic loci of 96.5% over the four populations. Average expected heterozygosity values (H_E or gene diversity) varied from 0.418 to 0.585 with a mean of 0.497. Most of the genetic variation was found within populations (85%) and the differentiation among populations was found to be 15% (F_st = 0.15). Interpopulation genetic distances corresponded well with the geographic distance between the population sites of origin, as well as morphological characteristics. Tests for Hardy–Weinberg equilibrium (HWE) for all loci and all populations revealed that all loci significantly differ from HWE. Subsequent analysis indicated that departure from HWE at some loci was due to an excess of heterozygotes. Possible explanations for heterozygote excess are discussed. The most likely reason for observed heterozygote excess could be due to the polyploidy nature of the species.     

Genetic variability of the tokay gecko based on microsatellite analysis

The black-spotted tokay gecko and red-spotted tokay gecko have different distribution areas and are significantly different in appearance but are classified into the same species Gekko gecko. Twelve microsatellite loci were isolated, characterized and evaluated from wild black-spotted tokay geckos for the first time. Of them, nine loci were successfully amplified in red-spotted tokay geckos using multiplex polymerase chain reactions (PCRs). A total of 208 different alleles were observed in the 70 wild black-spotted and red-spotted tokays, and the average number of alleles per locus was 17.3. The average values for observed heterozygosity, expected heterozygosity and polymorphism information content were 0.762, 0.891 and 0.871, respectively, which showed that the wild G. gecko population had a high level of genetic variability. Both black-spotted tokays and red-spotted tokays showed a significant (P < 0.001) deficit of heterozygotes. The red-spotted tokay (H_E = 0.881, A = 16.4) had a higher level of genetic variability than black-spotted tokay (H_E = 0.804, A = 10.7). The pairwise F_ST (P < 0.001) estimates of the two types of tokay were 0.143, which indicated that there was a significant level of genetic differentiation between the two.     

High allozyme diversity and unidirectional linear migration patterns within a population of tetraploid Isoetes sinensis, a rare and endangered pteridophyte

Isoetes sinensis (Isoetaceae), an aquatic quillwort which occurs only in two fragmented sites of China as an allotetraploid, is critically endangered. Genetic variation among eight subpopulations of I. sinensis was examined in the Xin’an River (119°14′–15′E, 29°28′N) by using allozyme polymorphism. Eighteen loci of 10 enzyme systems were examined and used for the analysis of population genetic parameters. As expected for allotetraploids, fixed heterozygosity was found at four loci. A high level of genetic diversity was observed in the population, with mean number of alleles per loci of 1.8, and mean percentage of polymorphic loci of 55.6%, which were much higher than the average values in fern species. The genetic variation within each subpopulation was not positively correlated with its size, which may be explained by high gene flow (Nm = 2.57), clonal reproduction and fixed heterozygosity of allopolyploid. The I. sinensis population contained high clonal diversity (PD = 0.39, D = 0.95), indicating the successful seedling recruitment of the population. Significant positive relationship was detected between clonal diversity and the size of subpopulation. Partitioning the genetic diversity indicated that 91.1% of the genetic variation was within subpopulations and only 8.9% existed among subpopulations. The migration pattern of I. sinensis along the Xin’an River is best explained by a source–sink model, but with unidirectional gene flow among subpopulations underlined by hydrochoric force. The results were then discussed in relation to both in situ and ex situ conservation efforts of the population.     

Population genetics studies of half-smooth tongue sole Cynoglossus semilaevis using ISSR markers

Inter-simple sequence repeat (ISSR) analysis was performed in order to evaluate the genetic diversity of wild and hatchery samples of half-smooth tongue sole Cynoglossus semilaevis. A group of 200 genotypes belonging to four wild samples, Laizhou (LZ), Weihai (WH), Qingdao (QD), Rizhao (RZ) and one hatchery sample, Mingbo (MB) were screened using 15 different ISSR primers. A total of 137 loci were produced in the five studied samples. 41.80%, 45.26%, 44.27%, 42.86% and 41.59% of these loci were polymorphic over all the genotypes tested in LZ, WH, QD, RZ and MB samples, respectively. The number of polymorphic loci detected by single primer combination ranged from 2 to 7. The average heterozygosity of LZ, WH, QD, RZ and MB samples were 0.0710, 0.0814, 0.0793, 0.0727 and 0.0696, respectively. The WH sample showed a higher genetic diversity including total number of ISSR bands (P < 0.05), total number of polymorphic bands (P < 0.05), average heterozygosity (P < 0.05) and total number of genotypes (P < 0.05) than all the other samples. Among the five studied samples, the hatchery sample (MB) showed the lowest genetic viability.     

Genetic diversity and population structure of the endangered and medically important Rheum tanguticum (Polygonaceae) revealed by SSR Markers

Rheum tanguticum (Polygonaceae), an endangered plant, is endemic to the Qinghai-Tibetan Plateau. A total of 114 individual of R. tanguticum from 10 geographically separate populations were analyzed using seven pairs simple sequence repeats (SSR) markers. 102 alleles were recorded, with an average of 14.6 alleles per locus (ranging from 13 to 17) and the expected heterozygosity (H_e) ranged from 0.384 to 0.515 (average 0.459). The genetic differentiation between populations was relatively high (F_st = 0.249); the gene flow (N_m = 0.754), however, was limited, which suggested that around 21.18% of the total genetic variations occurred between populations. Our results revealed high levels of genetic variations within and between populations. The endangered status of this species is probably due to harvesting of the wild populations, rather than a lack of the genetic diversity. Anthropologic effects as well as other factors may, together, have shaped the genetic structure of this species.     

Mating system and population genetic structure of Bruguiera gymnorrhiza (Rhizophoraceae), a viviparous mangrove species in China

In order to explain the diversity patterns and develop the conservation strategies, the population genetic structures and the mating systems of Bruguiera gymnorrhiza from the coastlines of south China were investigated in this study. The mating system parameters were analyzed using progeny arrays for allozyme markers. The multilocus outcrossing rates (tm) ranged from 0.845 (Fugong) to 0.267 (Dongzhai harbor). High allozyme variations within the five collected populations were determined and compared with the published data of other plant species with the mixed mating systems. At species level, the percentage of polymorphic loci (P) was 80%, the average number of alleles per locus (A) was 2.440, and the heterozygosity (He) was 0.293. The total gene diversity within each population (H_S = 0.2782) and the coefficient of genetic differentiation (G_ST = 0.0579) among the populations were estimated. On the basis of this population genetic structure, it is suggested that the gene flow (Nm = 3.85) is quite high, which is possibly related to its water-dispersed hypocotyls. It is also suggested that the mating system of this species is of mixed mating.     

Genetic diversity and geographical differentiation of Dipteronia Oliv. (Aceraceae) endemic to China as revealed by AFLP analysis

The genus Dipteronia Oliv. endemic to central and southern China consists of two species, Dipteronia sinensis Oliv. and Dipteronia dyeriana Henry, both of them are rare and endangered. AFLP markers were used to characterize the genetic diversity and geographical differentiation of the genus. Eight out of 32 PstI + 3/MseI + 3 selective primer combinations screened were applied to the analysis on 142 individuals of 17 D. sinensis and 4 D. dyeriana populations, respectively. A total of 324 fragments with 316 polymorphic were amplified. The proportion of polymorphic loci (PPB) was 97.53%. The Nei's gene diversity in D. sinensis and D. dyeriana was 0.3319 and 0.3047, respectively. About 43.6% (G_ST = 0.4356) of the genetic variation occurred among the populations, indicating a relatively high genetic differentiation among the populations. Cluster analysis grouped the 21 populations into two groups according to their species delimitation. The populations of D. sinensis were further divided into three subgroups corresponding to their geographical distributions. Correlation analysis revealed a significant correlation (p < 0.05) between geographical distance and genetic distance of these populations, suggesting that the relatively high genetic differentiation among the populations of D. sinensis might be caused by geographical isolation.     

Genetic diversity of Aegiceras corniculatum (Myrsinaceae) revealed by amplified fragment length polymorphism (AFLP)

Aegiceras corniculatum is a cryptoviviparous mangrove tree distributed in the Indo-West Pacific. The genetic structure of 13 populations of A. corniculatum from South China, Malay Peninsula, Sri Lanka, and North Australia, was assessed by amplified fragment length polymorphism (AFLP) markers. Our results showed a relatively high level of genetic variation at the species level (P = 92%, H_E = 0.294 and H_s = 0.331 ± 0.001). The value of G_ST was 0.698, suggesting significant genetic differentiation among populations. At the population level, however, genetic diversity was low (P = 24%, H_E = 0.086 and H_s = 0.127 ± 0.001). When populations were grouped according to geographic regions, i.e., South China, Malay Peninsula and Sri Lanka, it was inferred from analysis of molecular variance (AMOVA) that about half the total variation (49%) was accounted for differentiation between regions. A UPGMA dendrogram based on genetic distance also revealed five major clades corresponding to geographical regions within the distribution of A. corniculatum, although the precise relationships among the clades were not fully concordant with expected geographical delineations and need further study.     

Genetic relationship and genetic diversity among Typha taxa from East Asia based on AFLP markers

The genetic relationship and diversity among four Typha taxa in East Asia were evaluated using amplified fragment length polymorphism (AFLP) markers. Three AFLP selective primer combinations generated a total of 707 amplification products, of which 704 (99.6%) were polymorphic. The unweighted pair-group method with arithmetic mean (UPGMA) dendrogram and principal component analysis (PCA) plot confirmed the taxonomic status of four separate species. East Asian Typha taxa separated into two groups: the first with Typha angustifolia and the second with T. orientalis, T. laxmanni, and T. latifolia with a high bootstrap value for UPGMA (93%) and a low first score for PCA (25%). The two clusters corresponded with two sections based on the bracteoles in the female flower: section Bracteolatae and section Ebracteolatae. T. angustifolia showed the highest genetic diversity among the four Typha taxa (percentage of polymorphic loci [PPL] = 71%, H_o = 0.157), whereas T. latifolia had the lowest genetic diversity (PPL = 40%, H_o = 0.117). Genetic diversity was related to the presence of the gap between male and female inflorescences. A positive correlation between genetic distance and geographic distance was clearly found in the two species with continuous inflorescences (T. latifolia and T. orientalis). This positive correlation was not observed in the other species with discontinuous spikes (T. angustifolia and T. laxmanni).     

Assessing genetic diversity of populations of topmouth culter (Culter alburnus) in China using AFLP markers

Genetic diversity of five wild populations and a cultured population of topmouth culter (Culter alburnus) was investigated using amplified fragment length polymorphism (AFLP). A total of 373 reproducible bands amplified with seven AFLP primer combinations were obtained from 163 fish. The percentage of polymorphic loci ranged widely from 37.0% to 69.2% within distinct populations. The cultured population appeared to have a lower level of polymorphism (37.0%), gene diversity (0.121 ± 0.188) and Shannon's Information index (0.183 ± 0.263) than the wild populations. Analysis of molecular variance (AMOVA) revealed that average F_ST value overall loci was 0.2671, and the percentage of variation within population (73.29%) was larger than among populations (26.71%) (P < 0.01). The six populations were clustered into two major clades with UPGMA. The results from analysis of population pairwise gene flow indicated moderate gene flow among populations. Our study indicated that the genetic diversity of the cultured population was reduced compared with the wild populations. Geographic isolation, habitat, and artificial selection all may have played important roles in population differentiation. The information may be beneficial to future broodstock selection and defining conservation management for the different populations of topmouth culter.     

Genetic diversity and local population structure in Ambrosina bassii (Araceae,Ambrosineae), a Mediterranean relict species

The effects of habitat fragmentation on the genetic structure of Ambrosina bassii are analyzed. The species, whose reproductive biology is mostly unknown, is the only representative of its genus and tribe and it is endemic to the central Mediterranean area. The selected study area was the island of Sicily, in which wild populations show a wide morphological variability and ecological amplitude. Patterns of within- and among-population genetic diversity in eleven Sicilian populations, occurring in six disjunct areas, were examined by means of allozyme electrophoresis. High levels of genetic diversity were found as shown by the mean expected heterozygosity (H_e = 0.263), the percentage of polymorphic loci (P₉₅ = 65.3), the mean number of alleles per locus (A = 2.0). Genetic differentiation between populations was relatively low (mean F_ST = 0.091 and Nm = 1.98). A very weak correlation exists between genetic distances and geographic distances between populations. Despite its restricted and fragmented geographical range, A. bassii showed (i) high levels of genetic diversity, mainly within populations; (ii) no genetic differentiation between populations and morphotypes.     

Genetic diversity within and among populations of the endangered species Taxus fuana (Taxaceae) from Pakistan and implications for its conservation

The West Himalayan yew, Taxus fuana Nan Li & R.R. Mill (Taxaceae), is an endangered species endemic to the Western Himalayas. An investigation of the genetic diversity of wild populations of T. fuana in Pakistan was undertaken. The genetic diversity and genetic structure was quantified using random amplified polymorphic DNA (RAPD) variation in 219 individuals of the 10 populations. Of the 32 universal primers screened 16 produced highly reproducible, clear RAPD bands. Using these primers, 193 discernible DNA fragments were generated, of which 164 (84.97%) were polymorphic. The statistical results indicated that there was a relatively low genetic diversity within populations (with percentages of polymorphic bands, PPB, ranging from 29.53 to 50.26%, with an average of 38.34% and a Nei's genetic diversity index (H_E) of 0.1165), and a high genetic differentiation among populations (G_ST = 0.5842, Φ_ST = 0.5685) within these populations. The gene flow (N_m) was low with only 0.3558.     

Genetic diversity of the endangered and endemic species Psathyrostachys huashanica natural populations using simple sequence repeats (SSRs) markers

Psathyrostachys huashanica Keng., a species endemic to China, is only distributed in Huashan Mountain in Shaanxi Province. It has been listed as “national first-class protected rare species.” In this study, the microsatellites of barley were used to analyze genetic diversity of P. huashanica populations sampled from three valleys (Huangpu, Xian and Huashan Valleys) in Mt. Huashan. A total of 33 alleles of 11 loci were detected from 266 individuals. The observed average number of alleles (A) is 2.75; the effective number of alleles (Ae) is 1.67. The percentage of polymorphic loci (PPB) is 58.33% in Huangpu Valley, 75% in Xian Valley, 83.33% in Huashan Valley, and the total PPB is 83.33%. About 77.6% of (F_ST = 0.324) genetic diversity was observed within the subpopulations. Genetic differentiation within each subpopulations was higher than that among the subpopulations. Mean genetic distance is 0.17 (range: 0.010–0.401). Correlation analysis detected significant correlation between genetic distance and vertical distance of altitude in Huashan Valley. Differentiation mainly occurred between the higher altitude subpopulations and the lower altitude subpopulations, suggesting that altitude might be the major factor that restricted the gene flow between different altitude subpopulations and resulted in differentiation of subpopulations.     

Genetic differentiation in endangered Gynostemma pentaphyllum (Thunb.) Makino based on ISSR polymorphism and its implications for conservation

Studies were performed to investigate the genetic variation of 14 natural populations of Gynostemma pentaphyllum (Thunb.) Makino, an outcrossing clonal plant species in China, using inter-simple sequence repeat (ISSR) markers. Fourteen selected primers were used to amplify DNA samples from 140 individuals, and totally 194 loci were detected. The percentage of polymorphic bands (PPBs) showed that the genetic diversity was pretty high at the species level (PPB = 96.39%) but quite low at the population level (PPB = 1.03–25.26%). Shannon's information index (I) and Nei's gene diversity (h) displayed a similar trend to PPB. According to the hierarchical analysis of molecular variance (AMOVA) and Nei's analysis of gene diversity, the percentages of genetic variation among populations were 88.66 and 88.94%, respectively, indicating a high level of inter-population genetic differentiation. The low levels of genetic diversity within populations and high genetic differentiation among populations were assumed to result from the limited gene flow, the clonal nature and genetic drift. Based on the genetic data, effective conservation strategies were proposed for conserving this traditional Chinese medicinal herb. Concerning the management of G. pentaphyllum, we suggested that in situ conservation be an important and practical measure for maintaining the genetic diversity and that a possibly maximum number of populations be conserved. Populations EMS and HLT, in which particularly low levels of genetic variation were characterized, should be given the priority for ex situ conservation.     

Genetic diversity and inter-specific relations of western Mediterranean relic Abies taxa as compared to the Iberian A. alba

Several Abies species are currently present in the Mediterranean region and most of them are endemic taxa and tertiary relicts. Using six nuclear microsatellites, we studied the genetic structure and inter-specific relationships among West Mediterranean firs, A. pinsapo (Spain), A. maroccana and A. tazaotana (Morocco). Based on the hypothesis that A. pinsapo could historically exchange genes with A. alba growing in the Pyrenees via secondary contact, we investigated the level of genetic admixture between these species using a Bayesian approach. The studied populations showed moderate genetic diversity (mean H_E = 0.598) and a high level of genetic differentiation (F_ST = 0.225) that was especially pronounced between A. alba and the African firs. All populations experienced a strong bottleneck effect that was likely induced by climatic changes occurring in the West Mediterranean during the last glacial cycle and the Holocene. According to Bayesian clustering, both African taxa grouped together in a single cluster, the two A. pinsapo populations formed a second cluster, and two additional clusters were detected within A. alba. Our results indicate that A. tazaotana is genetically very close to A. maroccana, and hence these two taxa should probably not be considered as separate species. We found no genetic admixture between A. pinsapo and A. alba and only minor between A. pinsapo and the African fir populations suggesting an isolation effect of the Gibraltar Strait. Current limited distributions of firs in the Mediterranean region together with changing climate may lead to further deterioration of the genetic diversity levels. Hence, future efforts should focus on monitoring the demography and genetic threats to existing populations.     

Genetic and morphological contrasts between wild and anthropogenic populations of Agave parryi var. huachucensis in south-eastern Arizona

Background and Aims

At least seven species of Agave, including A. parryi, were cultivated prehistorically in Arizona, serving as important sources of food and fibre. Many relict populations from ancient cultivation remain in the modern landscape, offering a unique opportunity to study pre-Columbian plant manipulation practices. This study examined genetic and morphological variation in six A. p. var. huachucensis populations of unknown origin to compare them with previous work on A. parryi populations of known origin, to infer their cultivation history and to determine whether artificial selection is evident in populations potentially managed by early agriculturalists.

Methods

Six A. p. var. huachucensis and 17 A. parryi populations were sampled, and morphometric, allozyme and microsatellite data were used to compare morphology and genetic structure in purportedly anthropogenic and wild populations, as well as in the two taxa. Analysis of molecular variance and Bayesian clustering were performed to partition variation associated with taxonomic identity and hypothesized evolutionary history, to highlight patterns of similarity among populations and to identify potential wild sources for the planting stock.

Key Results A

p. var. huachucensis and A. parryi populations differed significantly both morphologically and genetically. Like A. parryi, wild A. p. var. huachucensis populations were more genetically diverse than the inferred anthropogenic populations, with greater expected heterozygosity, percentage of polymorphic loci and number of alleles. Inferred anthropogenic populations exhibited many traits indicative of past active cultivation: greater morphological uniformity, fixed heterozygosity for several loci (non-existent in wild populations), fewer multilocus genotypes and strong differentiation among populations.

Conclusions

Where archaeological information is lacking, the genetic signature of many Agave populations in Arizona can be used to infer their evolutionary history and to identify potentially fruitful sites for archaeological investigation of ancient settlements and cultivation practices. The same approach can clearly be adopted for other species in similar situations.     

Evidence for genetic differentiation of Octopus vulgaris (Mollusca, Cephalopoda) fishery populations from the southern coast of Brazil as revealed by microsatellites

Octopus vulgaris is a cephalopod species in several oceans and commonly caught by artisanal and industrial fisheries. In Brazil, O. vulgaris populations are mainly distributed along the southern coast and have been subjected to intensive fishing during recent years. Despite the importance of this marine resource, no genetic study has been carried out to examine genetic differences among populations along the coast of Brazil. In this study, 343 individuals collected by commercial vessels were genotyped at six microsatellite loci to investigate the genetic differences in O. vulgaris populations along the southern coast of Brazil. Genetic structure and levels of differentiation among sampling sites were estimated via a genotype assignment test and F-statistics. Our results indicate that the O. vulgaris stock consists of four genetic populations with an overall significant analogous F_ST (Φ_CT = 0.10710, P < 0.05) value. The genetic diversity was high with an observed heterozygosity of Ho = 0.987. The negative values of F_IS found for most of the loci examined suggested a possible bottleneck process. These findings are important for further steps toward more sustainable octopus fisheries, so that this marine resource can be preserved for long-term utilization.     

Genetic associations of body composition,flexibility and injury risk with ACE,ACTN3 and COL5A1 polymorphisms in Korean ballerinas

[Purpose]

The purpose of this study was to exam the association of body composition, flexibility, and injury risk to genetic polymorphisms including ACE ID, ACTN3 RX, and COL5A1 polymorphisms in ballet dancers in Korea.

[Methods]

For the purpose of this study, elite ballerinas (n = 97) and normal female adults (n = 203) aged 18 to 39 were recruited and these participants were tested for body weight, height, body fat, fat free mass, flexibility, injury risks on the joints and gene polymorphisms (ACE, ACTN3, COL5A1 polymorphism).

[Results]

As results, the ACE DD genotype in ballerinas was associated with higher body fat and percentage of body fat than the ACE II and ID genotypes (p < 0.05). In the study on the ACTN3 polymorphism and ballerinas, the XX genotype in ballerinas had lower body weight and lower fat-free mass than the RR and RX genotype (p < 0.005). Also, the means of sit and reach test for flexibility was lower in the ACTN3 XX genotype of ballerinas than the RR and RX genotype of ballerinas (p < 0.05). Among the sports injuries, the ankle injury of the XX-genotyped ballerinas was in significantly more prevalence than the RR and XX-genotyped ballerinas (p < 0.05). According to the odd ratio analysis, XX-genotyped ballerinas have the injury risk on the ankle about 4.7 (95% CI: 1.6~13.4, p < 0.05) times more than the RR and RX-genotyped ballerinas. Meanwhile, the COL5A1 polymorphism in ballerinas has no association with any factors including flexibility and injury risks.

[Conclusion]

In conclusion, ACE polymorphism and ACTN3 polymorphism were associated with ballerinas'' performance capacity; COL5A1 was not associated with any factors of performance of Ballerinas. The results suggested that the ACE DD genotype is associated with high body fat, the ACTN3 XX genotype is associated with low fat-free mass, low flexibility, and higher risk of ankle-joint injury.     

Genetic diversity and biogeography of the traditional Chinese medicine, Gardenia jasminoides, based on AFLP markers

Gardenia jasminoides Ellis is used in traditional Chinese medicine (TCM) in China. Levels of genetic variation and patterns of population structure within and among eight wild or cultivated populations of G. jasminoides Ellis in China were investigated using amplified fragment length polymorphism (AFLP) markers. Of the 11 primers screened, four produced highly reproducible AFLP bands. Using these primers, 244 discernible DNA fragments were generated with 165 bands (67.6%), were polymorphic, indicating considerable genetic variation at the species level. In contrast, there were relatively low levels of polymorphism at the population level with the percentage of polymorphic bands (PPB) ranging from 36.89% to 59.43%. Genetic diversity within populations ranged from 0.2086 to 0.3108, averaging 0.2392 at the species level. A high level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis (76.59%), Shannon's index analysis (64.8%) and AMOVA analysis (72.75%). No significant statistical differences (analysis of molecular variance [AMOVA], p = 0.0639) in AFLP variation were found between regions. However, the variance among populations and within populations differed significantly (p < 0.001). An indirect estimate of historical levels of gene flow (N_m = 1.7448) was consistent with the high mean genetic identity (mean I = 0.9263) found among populations. There is an association between geographic and genetic distances between populations. Presently gene change exists between populations.     

Inter-simple sequence repeat (ISSR) analysis of genetic variation of Chondrus crispus populations from North Atlantic

ISSR analysis was used to investigate genetic variations of 184 haploid and diploid samples from nine North Atlantic Chondrus crispus Stackhouse populations and one outgroup Yellow Sea Chondrus ocellatus Holmes population. Twenty-two of 50 primers were selected and 163 loci were scored for genetic diversity analysis. Genetic diversity varied among populations, percentage of polymorphic bands (PPB) ranged from 27.0 to 55.8%, H (Nei's genetic diversity) ranged from 0.11 to 0.20 and I (Shannon's information index) ranged from 0.16 to 0.30. Estimators PPB, H and I had similar values in intra-population genetic diversity, regardless of calculation methods. Analysis of molecular variance (AMOVA) apportioned inter-population and intra-population variations for C. crispus, showing more genetic variance (56.5%) occurred in intra-population, and 43.5% variation among nine populations. The Mantel test suggested that genetic differentiation between nine C. crispus populations was closely related with geographic distances (R = 0.78, P = 0.002). Results suggest that, on larger distance scale (ca. >1000 km), ISSR analysis is useful for determining genetic differentiations of C. crispus populations including morphologically inseparable haploid and diploid individuals.