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 of the threatened aquatic plant Ottelia alismoides in the Yangtze River

Ottelia alismoides is a threatened submerged macrophyte in China. Genetic variation and population structure of 11 O. alismoides populations from lakes in mid-lower reaches of the Yangtze River were assessed by inter-simple sequence repeat (ISSR) markers. Eleven primer combinations produced a total of 130 unambiguous bands of which 57 (43.9%) were polymorphic. O. alismoides exhibited a very low level of intra-population genetic diversity (P_p = 13.0%, H_E = 0.042, I = 0.063). The main factors responsible for that were its short life history and high degree of autogamy in the reproductive system of the species. The F statistics calculated by different approaches consistently revealed a high genetic differentiation among populations, contributing >55% of the total gene diversity. The evident population structure of O. alismoides could be due to self-fertilizing reproduction, restricted gene flow and genetic drift. Estimates of gene flow by F_ST and coalescent-based simulation analysis indicated a restricted recurrent gene exchange among populations (Nm = 0.180, M = 0.190). Genetic drift played a more important role than gene flow in the current population genetic structure of O. alismoides because its habitat range was fragmented and highly influenced by environment changes. The results are discussed in relation to both in situ and ex situ conservation efforts of the species. A conservation strategy for conserving all extant populations to maximize genomic representation of the species is recommended.     

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.     

Low genetic variation detected within the widespread mangrove species Nypa fruticans (Palmae) from Southeast Asia

Genetic diversity within and among six natural populations of Nypa fruticans from China, Vietnam, and Thailand was assessed using SSR and ISSR analysis. Our results showed an extremely low level of genetic diversity of N. fruticans (at the species level, P = 11.76% and 2.88%, He = 0.0279 and 0.0113, I = 0.0470 and 0.0167 by SSRs and ISSRs, respectively) across a total of 183 individuals. No genetic variation was detected within any population except for the Thailand population by SSRs (P = 11.76%, He = 0.0417; I = 0.0622). The bottlenecks during glacial epochs, founder effects, and propagation pattern may be responsible for the extremely low level of genetic diversity of N. fruticans.     

Population genetic differentiation of Schisandra chinensis and Schisandra sphenanthera as revealed by ISSR analysis

Schisandra chinensis (Turcz.) Baill. and Schisandra sphenanthera Rehd. et Wils. are well-known Chinese medicinal plants. The population genetic variation of the two species was studied using inter simple sequence repeat (ISSR) molecular markers. High levels of genetic diversity are revealed in both S. chinensis (P = 88.36%, h = 0.2894, I = 0.4396) and S. sphenanthera (P = 84.09%, H = 0.2782, I = 0.4280). However, the population genetic differentiation is significantly different between the two species. The S. sphenanthera harbors as high as 27% of the genetic variation among populations but 73% within populations, whereas in S. chinensis 17% of the genetic variation occurs among populations and 83% within populations. Both significant (P < 0.05) heterozygosity excess and shifted mode of allele frequency distribution are detected in four out of six populations of S. chinensis and one out of five populations of S. sphenanthera, suggesting the occurrence of recent population bottlenecks in the two species. The different patterns of genetic variation in S. chinensis and S. sphenanthera are discussed in relation to their differences in pollination mechanism, geographic distribution and historical events, and the level of gene flow and genetic drift.     

Genetic and phytochemical diversities of Cynomorium songaricum Rupr. in Northwest China indicated by ISSR markers and HPLC-fingerprinting

Cynomorium songaricum Rupr. is a parasitic plant and its most common host is Nitraria tangutorum Bobr. which grows in desert areas. To support the sustainable use and conservation of C. songaricum, genetic and phytochemical studies were undertaken on 16 different populations. Samples were analyzed using inter-simple sequence repeat (ISSR) markers and high performance liquid chromatography (HPLC)-fingerprinting. Total phenolic acids were determined by UV spectrophotometry. At the population level, percentage of polymorphic bands (PPB) ranged from 29.89 to 64.94%, Nei's genetic diversity index (H) ranged from 0.0931 to 0.2055, Shannon's information index (I) ranged from 0.1422 to 0.3140. 16 populations were classified into four chemotypes according to HPLC-fingerprinting analysis. Total phenolic acids contents ranged from 56.79 to 123.50 mg g⁻¹. The results of the genetic and chemical studies are used to suggest a planting and conservation system for C. songaricum.     

Analysis of the genetic structure of Rhodiola rosea (Crassulaceae) using inter-simple sequence repeat (ISSR) polymorphisms

The genetic diversity and differentiation of eleven R. rosea populations from different parts of its wide area of occurrence were studied by ISSR markers. Using eight primers, 252 DNA fragments were generated, and 243 of those DNA fragments were found to be polymorphic, indicating a high genetic variability at the species level (P = 96.4%, h = 0.176, SI = 0.291). Relatively low levels of diversity were determined at the population level (P 30.6-59.1%, h 0.088-0.165, SI 0.137-0.257). AMOVA analysis revealed that the majority of the genetic variation was within populations (65.42%), and the variance among populations was 34.58%. Cluster analysis revealed two groups of R. rosea populations; these groups likely represent distinct evolutionary lines in the species, which are different in genetic structure, evolutionary history and chorological migration routes.     

Ecological genetics of Reaumuria soongorica (Pall.) Maxim. population in the oasis–desert ecotone in Fukang,Xinjiang, and its implications for molecular evolution

Using inter-simple sequence repeat (ISSR) markers, studies were performed to characterize the population genetic diversity and structure of Reaumuria soongorica (Pall.) Maxim. in the oasis–desert ecotone in Fukang, Xinjiang. Eleven primers were screened to amplify DNA sequences from 132 individuals, which corresponded to seven subpopulations. Totally, 176 loci, all of them polymorphic, were detected, and the percentage of polymorphic bands (PPB) was 100%, indicating a relatively high genetic diversity within the R. soongorica population. According to the hierarchical analysis of molecular variances (AMOVA), the analysis of Shannon's diversity and Nei's analysis of gene diversity, the percentages of genetic variation among subpopulations were 15.87%, 16.28% and 14.58%, respectively, which meant that 83.72–85.42% of total genetic variation occurred within subpopulations. Besides, POPGENE analysis also revealed a relatively high gene flow (N_m = 2.9290) among subpopulations. Correlation analysis showed that there existed no significant correlation between the ISSR-based genetic diversity of the seven R. soongorica subpopulations and their ecological factors (mainly in soil) (P > 0.05). However, on the other hand, Mantel test revealed a significant correlation between inter-subpopulation genetic distances and geographic distances (r = 0.637, P < 0.01), indicating that geographic distance was among the important factors affecting the population genetic structure of R. soongorica. Meanwhile, a comparative analysis was performed between the present ISSR-based and the previously published RAPD-based results, and revealed obvious discrepancies, implying the different evolutionary patterns of the genomic regions sampled by ISSR and RAPD markers.     

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 species Kirengeshoma palmata (Saxifragaceae) in China

In order to investigate the levels of genetic diversity of the endangered species Kirengeshoma palmata (Saxifragaceae), four extant populations were sampled and analyzed using inter-simple sequence repeats (ISSR) markers. We expected a low genetic diversity level, but our results revealed a high level of intraspecific genetic diversity, probably resulting from this species being in a refuge during the last glaciation (at population level: P = 63.25%, A_e = 1.47, H_E = 0.26 and H_O = 0.37; at species level: P = 79.00%, A = 1.5538, H_T = 0.2586 and H_sp = 0.3104). A low level of genetic differentiation among populations was detected based on Nei's genetic diversity analysis (16.69%) and AMOVA (19.36%). Populations shared high levels of genetic identity. Insect pollination and seed dispersal by wind may have facilitated extensive gene flow and are likely responsible for this present structure of genetic variation.     

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.     

广东省五节芒遗传多样性的ISSR分析

采用ISSR分子标记技术,对广东省内分布的能源植物五节芒(Miscanthus floridulus)13个野生居群共215个个体的遗传多样性进行了研究.9个ISSR引物共扩增到了81个位点,其中76个是多态性位点.结果表明,广东五节芒的遗传多样性水平很高,物种水平上,多态位点百分率(PPB)为93.83％,Nei's...     

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 Sinojackia dolichocarpa (Styracaceae), a species endangered and endemic to China,detected by inter-simple sequence repeat (ISSR)

Sinojackia dolichocarpa, a species endangered and endemic to China, is distributed only in the regional area of Shimen and Sangzhi in Hunan Province. Inter-simple sequence repeat (ISSR) markers were used to investigate the genetic diversity within and among the four natural populations of S. dolichocarpa. Leaf samples were collected from 84 individuals. Thirteen ISSR primers selected from 80 primers gave rise to 137 discernible DNA bands of which 100 (72.99%) were polymorphic. On average each primer gave rise to 10.5 bands including 7.7 bands with polymorphic profile. At the species level, high genetic diversity was detected (PPB: 72.99%; H_E: 0.2255; Ho: 0.3453). However, relatively low genetic diversity existed within populations. Population in Maozhuhe (MZH) exhibits the greatest level of variability (PPB: 40.38%, H_E: 0.1566, Ho: 0.2330), whereas the population in Jingguanmen (JGM) finds its own variability at the lowest level (PPB: 30.66%; H_E: 0.1078; Ho: 0.1601). A high level of genetic differentiation among populations was revealed by Nei's gene diversity statistics (45.30%), Shannon's information measure (45.24%) and analysis of molecular variance (AMOVA) (52.88%). The main factors responsible for the high level of differentiation among populations are probably related to the selfing reproductive system and the isolation of populations. The strong genetic differentiation among populations indicates that the management for the conservation of genetic variability in S. dolichocarpa should aim to preserve every population.     

ISSR analysis of genetic diversity of the Qinghai-Tibet Plateau endemic Rhodiola chrysanthemifolia (Crassulaceae)

Inter-simple sequence repeat markers (ISSR) were used to estimate genetic diversity within and among 10 populations of Rhodiola chrysanthemifolia along Nianqingtangula Mountains and Brahmaputra, a species endemic to the Qinghai-Tibet Plateau and an endangered medicinal plant. Of the 100 primers screened, 13 produced highly polymorphic DNA fragments. Using these primers, 116 discernible DNA fragments were generated of which 104 (89.7%) were polymorphic, indicating substantial genetic diversity at the species level. Genetic diversity measured by the percentage of polymorphic bands (PPB) at the population level ranged from 21.97% to 48.8%. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly among populations (77.3%), but no regional differentiation was discernible. Variance within populations was only 22.7%. The main factor responsible for this high level of differentiation among populations is probably the historical geographical and genetic isolation of populations in a harsh mountainous environment. Concerning the management of R. chrysanthemifolia, the high genetic differentiation of populations indicates the necessity of conserving the maximum possible number of populations.     

Genetic diversity of wild populations of Rheum tanguticum endemic to China as revealed by ISSR analysis

Rheum tanguticum is an important but endangered traditional Chinese medicine endemic to China. The wild resources have been declining. Establishing the genetic diversity of the species would assist in its conservation and breeding program. Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population genetic structure in 13 wild populations of R. tanguticum from Qinghai Province. Thirteen selected primers produced 329 discernible bands, with 326 (92.94%) being polymorphic, indicating high genetic diversity at the species level. The Nei's gene diversity (He) was estimated to be 0.1724 within populations (range 0.1026–0.2104), and 0.2689 at the species level. Analysis of molecular variance (AMOVA) showed that the genetic variation was found mainly within populations (71.02%), but variance among populations was only 28.98%. In addition, Nei's differentiation coefficients (G_st) was found to be high (0.3585), confirming the relatively high level of genetic differentiation among populations. Mantel test revealed a significant correlation between genetic and geographic distances (r = 0.573, P = 0.002), and the unweighted pair-group method using arithmetic average (UPGMA) clustering and Principal coordinates analysis (PCoA) demonstrated similar results. Meanwhile, the genetic diversity of R. tanguticum positively correlated with altitude and annual mean precipitation, but negatively correlated with latitude and annual mean temperature. This result might be an explanation that the natural distribution of R. tanguticum is limited to alpine cold areas. We propose conservation strategy and breeding program for this plant.     

Genetic diversity of Astragalus nitidiflorus, a critically endangered endemic of SE Spain,and implications for its conservation

Inter-simple sequence repeats (ISSR) markers were used to assess the genetic diversity and population structure in five populations of Astragalus nitidiflorus, a critically endangered species endemic to southeast Spain. Eight primers amplified 78 bands with 40 (51.3%) being polymorphic. Statistical results indicated a low genetic diversity at the population and species level, with percentages of polymorphic bands (PPB) ranging from 28.2 to 37.2% (an average of 31.8%), and means of gene diversity (H_E) of 0.129 and 0.171 respectively. The Shannon’s index (SI) ranged from 0.160 to 0.214 at the population level and was 0.260 at the species level. A low level of genetic differentiation among populations was detected, based on the Shannon’s information index (0.297), the coefficient of genetic differentiation between populations (G_ST = 0.2418) and AMOVA analysis (Φ_ST = 0.255). The estimated gene flow (Nm) was 0.789. The high genetic connectivity found among populations of A. nitidiflorus is an evidence of a recent habitat fragmentation. In addition, a bottleneck event in the past has been revealed, with a subsequent reduction of population size and a loss of genetic variation. Based on these results, the conservation strategy of A. nitidiflorus was proposed.     

PCR-RFLP analysis of mitochondrial DNA cytochrome b gene among Haruan (Channa striatus) in Malaysia

Haruan (Channa striatus) is in great demand in the Malaysian domestic fish market. In the present study, mtDNA cyt b was used to investigate genetic variation of C. striatus among populations in Peninsular Malaysia. The overall population of C. striatus demonstrated a high level of haplotype diversity (h) and a low-to-moderate level of nucleotide diversity (π). Analysis of molecular variance (AMOVA) results showed a significantly different genetic differentiation among 6 populations (F_ST = 0.37566, P = 0.01). Gene flow (Nm) was high and ranged from 0.32469 to infinity (∞). No significant relationship between genetic distance and geographic distance was detected. A UPGMA tree based on the distance matrix of net interpopulation nucleotide divergence (d_A) and haplotype network of mtDNA cyt b revealed that C. striatus is divided into 2 major clades. The neutrality and mismatch distribution tests for all populations suggested that C. striatus in the study areas had undergone population expansion. The estimated time of population expansion in the mtDNA cyt b of C. striatus populations occurred 0.72-6.19 million years ago. Genetic diversity of mtDNA cyt b and population structure among Haruan populations in Peninsular Malaysia will be useful in fisheries management for standardization for Good Agriculture Practices (GAP) in fish-farming technology, as well as providing the basis for Good Manufacturing Practices (GMP).     

O father where art thou? Paternity analyses in a natural population of the haploid–diploid seaweed Chondrus crispus

The link between life history traits and mating systems in diploid organisms has been extensively addressed in the literature, whereas the degree of selfing and/or inbreeding in natural populations of haploid–diploid organisms, in which haploid gametophytes alternate with diploid sporophytes, has been rarely measured. Dioecy has often been used as a proxy for the mating system in these organisms. Yet, dioecy does not prevent the fusion of gametes from male and female gametophytes originating from the same sporophyte. This is likely a common occurrence when spores from the same parent are dispersed in clumps and recruit together. This pattern of clumped spore dispersal has been hypothesized to explain significant heterozygote deficiency in the dioecious haploid–diploid seaweed Chondrus crispus. Fronds and cystocarps (structures in which zygotes are mitotically amplified) were sampled in two 25 m² plots located within a high and a low intertidal zone and genotyped at 5 polymorphic microsatellite loci in order to explore the mating system directly using paternity analyses. Multiple males sired cystocarps on each female, but only one of the 423 paternal genotypes corresponded to a field-sampled gametophyte. Nevertheless, larger kinship coefficients were detected between males siring cystocarps on the same female in comparison with males in the entire population, confirming restricted spermatial and clumped spore dispersal. Such dispersal mechanisms may be a mode of reproductive assurance due to nonmotile gametes associated with putatively reduced effects of inbreeding depression because of the free-living haploid stage in C. crispus.     

SPAR methods revealed high genetic diversity within populations and high gene flow of Vanda coerulea Griff ex Lindl (Blue Vanda), an endangered orchid species

Molecular genetic fingerprints of seven populations of Vanda coerulea comprising of thirty-two genotypes from Northeast India were developed using PCR based markers. Genetic variability in the wild genotypes of V. coerulea was analyzed using two different single primer amplification reactions (SPAR) methods, viz., random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR). A total of 32 genotypes were used to investigate the existing natural genetic diversity at intra-specific level. Two hundred and twenty six (226) amplification products were scored by RAPD and ISSR, both of which collectively showed 58.88% polymorphism with a mean intra-population genetic diversity (Hpop) of 0.119. However, their level of diversity at inter- and intra-population levels was significant, with the percentage of polymorphic loci (Pp) ranging from 17.70% to 45.13%, Shannon's information index (I) from 0.105 to 0.268 and Nei's gene diversity (h) from 0.072 to 0.185 with mean Nei's gene diversity 0.174 and the overall estimate of gene flow being (Nm) 1.165. Analysis of molecular variance (AMOVA) showed 96.07% of variation at intra-population level, whereas 3.93% variation was recorded at inter-population level. Only one major cluster was detected by cluster analysis using the unweighted pair-group method with arithmetic average (UPGMA). Present investigation suggests the efficiency of SPAR methods to estimate the genetic diversity of V. coerulea and can be seen as a starting point for future research on the population and evolutionary genetics of this species.