Phenotype and genetic diversity in potato onion cultivars from three provinces of northeast China

Phenotypic characters and ISSR markers in forty nine potato onion cultivars (Allium cepa L. var. aggregatum G. Don) were investigated. Twenty three phenotypic characters, including sprouting time, plant height, spherical index, and yield, were investigated. Cluster analysis separated forty nine cultivars into five groups (D = 0.52). 143 polymorphic amplified bands, with an average of 8.41 polymorphic bands per primer, were obtained from seventeen ISSR primers. The number of polymorphic bands detected with each primer ranged from 6 to 12, with an average of 8.82. The percentage of polymorphisms was 93.5%. Genetic similarity varied from 0.4969 to 0.8616, the average value of the effective number of alleles, Nei's genetic diversity and Shannon's information index was 1.4716, 0.3072 and 0.4590, respectively. Forty nine varieties were classified into six groups according to the ISSR (D = 0.72). These results showed that two methods combined were accuracy for characterization of the genetic diversity and identification of potato onion cultivars.     

High genetic differentiation revealed by RAPD analysis of narrowly endemic Sinocalycanthus chinensis Cheng et S.Y. Chang,an endangered species of China

Genetic diversity and genetic differentiation of narrowly endemic Sinocalycanthus chinensis Cheng et S.Y. Chang, an endangered species of China, were analyzed using random amplified polymorphic DNA (RAPD) markers. Totally, 165 stable and clearly scored RAPD bands were achieved from 12 primers. The genetic diversity of S. chinensis was high (P% = 68.84; h = 0.2421 ± 0.1978; I = 0.3615 ± 0.2789), whereas that at the population level was relatively low (P% = 14.49; h = 0.0578 ± 0.0167; I = 0.0843 ± 0.0236). High genetic differentiation among populations was detected based on Shannon's information index (0.7668), Nei's gene diversity (0.7613) and AMOVA (0.8183). This might be explained by its survival in refugia during the last glaciation in southeastern China with origin from a widespread continental progenitor. The 10 populations from different geographical sites could be clustered into two groups. Low gene flow due to mixed mating system and anthropogenic activities likely played important roles in shaping the population genetic structure of the species.     

Assessment of the genetic diversity and genetic relationships of Lilium in China using ISSR markers

China is a center of natural distribution and diversity of genus Lilium around the world. In the study, the genetic diversity and genetic relationships of Lilium in China were analyzed by inter-simple sequence Repeat (ISSR) markers. The 6 highly polymorphic ISSR primers were selected to amplify the 20 Lilium species. The results showed that a total of 114 DNA bands were amplified, all of which were polymorphic loci (P = 100%), the effective number of alleles (Ne) was 1.2753, and the difference value between observed number of alleles (Na) and effective number of alleles (Ne) was 0.7247, and the Nei's genetic diversity (H) was 0.2048, and the Shannon's information index (I) was 0.3503. These results indicated that there is significant genetic difference among Lilium species in China. Taking the average genetic similarity coefficient (Gs) 0.5313 as the threshold, the 20 tested Lilium species were clustered into 5 groups, which was not entirely consistent with traditional clustering by morphological traits. The results obtained from this study can provide a reference for the molecular study of Lilium germplasm resources.     

Genetic diversity and structure of the noxious alien grass Praxelis clematidea in southern China

Praxelis clematidea (Asteraceae), a plant species native to South America, is a noxious weed in southern China. We examined the genetic variation and population structure of 12 populations (76 individuals) of P. clematidea from Fujian, Guangdong, and Hainan Provinces in China using inter-simple sequence repeat (ISSR) analysis. From an initial set of 69 ISSR primers, 10 were selected which yielded 80 reproducible bands. Polymorphic bands (P) were 100%, Shannon's information index (I) was 0.4226, and Nei's gene diversity (H) was 0.2791. We infer that the high levels of genetic diversity exhibited by P. clematidea may have contributed to its invasiveness. Gene flow among populations was 2.4930, which has led to homogenization. The coefficient of population differentiation (Gst = 0.1671) indicated low levels of genetic variation among populations and high levels of genetic polymorphism within populations. There was a negative correlation between population elevation and genetic diversity, while there was a significant positive correlation between genetic distance and geographic distance based on a Mantel test (r = 0.5820, P < 0.01). Some populations from different provinces clustered together in principal coordinate and UPGMA analyses indicating that human-mediated events may have contributed to the dispersal of the species.     

Analysis of genetic diversity in Aconitum kongboense L. revealed by AFLP markers

The systematic evaluation of the molecular diversity encompassed in Aconitum kongboense L. inbreds or parental lines offers an efficient means of exploiting the heterosis in A. kongboense as well as for management of biodiversity. An excellent and novel DNA-based molecular, amplified fragment length polymorphisms (AFLPs) markers, was firstly used to analysis the genetic diversity in A. kongboense genotypes. Out of 256 primers screened, a total of ten combinations successfully produced scorable, clear, reproducible and relatively high polymorphism bands, 64.12% of which were polymorphic. The values of number of polymorphic loci (NPL), percentage of polymorphic loci (PPB), observed number of alleles (Na) and effective number of alleles (Ne) were highest in population 3 (NPL = 77, PPB = 68.75%, Na = 1.688 ± 0.466, Ne = 1.412 ± 0.397) and lowest in population 2 (NPL = 57, PPB = 50.89%, Na = 1.509 ± 0.502, Ne = 1.273 ± 0.340). In addition, Jaccard's similarity coefficients among different populations varied from 0.45 to 1.00 with an average of 0.55. The data collected will contribute to identification, rational exploitation and conservation of germplasms of A. kongboense, and potentially useful to aid its breeding.     

Genetic diversity and structure of natural Pinus tabulaeformis populations in North China using amplified fragment length polymorphism (AFLP)

Chinese pine (Pinus tabulaeformis carr.), endemic to China, is a conifer species with extensive and fragmented distribution in North China. In this study, the genetic diversity and structure of 20 natural populations of this species were investigated using amplified fragment length polymorphism (AFLP) markers. A total of 445 fragments were revealed with 8 pairs of primers, 379 (85.17%) of which were polymorphic. A moderate level of genetic diversity was detected at the species level (Shannon's information index I = 0.356, Nei's gene diversity H_E = 0.271) and at the population level (I = 0.219, H_E = 0.206). Most of genetic variation was within populations while a considerable level of genetic differentiation was detected (G_ST = 0.352, Ф_ST = 0.304). The high differentiation could be attributed to the complex and fragmented habitats, and a limited gene flow among populations (N_m = 0.572). The Mantel test indicated that there was significant correlation (r = 0.455, P < 0.001) between Nei's genetic distance and geographical distance among all the populations. The results suggested that proper countermeasures should be taken to prevent the habitat further deterioration and maintain the genetic diversity of this species.     

Genetic diversity of natural populations of Larix principis-rupprechtii in Shanxi Province,China

Prince Rupprecht's Larch (Larix principis-rupprechtii Mayr.) is one of dominant components of middle and high elevation forests in North China. Shanxi Province is well known as “the Hometown of Prince Rupprecht's Larch” in China. In this study, six natural populations of this species across Shanxi were selected to investigate the genetic variation of the species using amplified fragment length polymorphism (AFLP) markers. Results showed that in comparison with some other species of Larix, higher genetic diversity was revealed at the species level for L. principis-rupprechtii (percentage of polymorphic loci PPL = 71.9%, Nei's gene diversity H_E = 0.225, Shannon information index I = 0.341). Most of genetic variation existed within populations (80.5%), while the genetic differentiation among populations was significant (p < 0.001) and higher (G_st = 0.194) than most other species of Larix. The differentiation can be attributed to the limited gene flow (N_m = 1.035) among populations, which could be due to the spatial isolation and habitat fragmentation. The six populations can be divided into three groups based on the Nei's genetic distances between populations (from 0.033 to 0.076). There was no significant correlation (r = 0.268, p > 0.05) between genetic distance and geographic distance among populations. The measures for in-situ or ex-situ conservation should be taken to preserve the genetic diversity of this species.     

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 population structure of the red stingray,Dasyatis akajei inferred by AFLP marker

To examine population genetic diversity and variation of the red stingray Dasyatis akajei, samples from 1 freshwater region and 6 coastal localities within its distribution range were analyzed by using amplified fragment length polymorphism (AFLP) technology. A total of 207 loci were identified by 4 primer combinations from 87 individuals, 174 of which were polymorphic (84.1%). A high level of Nei's gene diversity was observed with the overall value of 0.230 ± 0.179. No significant genealogical clusters associated with sampling sites were revealed on the UPGMA tree. Both analysis of molecular variance (AMOVA, Φ_ST = 0.085, P = 0.00) and pairwise F_ST indicated significant genetic differentiation among four marine samples. However, no particular genetic differentiation was detected between marine and the limited sampling freshwater populations (AMOVA, Φ_CT = 0.056, P > 0.05). Except for the TZ vs. WZ (5.193), the gene flow estimates (N_m) demonstrated the effective immigrants were 1.918-2.976, suggesting low level dispersal between pairwise marine populations. Species-specific habits (demersal and sluggish habits) are probably responsible for the population structuring.     

Genetic variation within the sand-fixation species Caragana microphylla (Leguminosae) in Horqin sandy land detected by inter-simple sequence repeats analysis

Caragana microphylla is the most dominant and constructive shrub species in the Horqin sandy land in the northeast of China. We evaluated it's the level of genetic variation within and among populations sampled from two different populations types in Horqin sandy land by using inter-simple sequence repeat polymorphism (ISSR) molecular markers. The results showed that eight ISSR primers generated 106 bands, of which 87 were polymorphic. At the species level, genetic diversity was relatively high (P = 82.08%, h = 0.2831, I = 0.4233). Genetic variation in natural populations (h = 0.2152, I = 0.3169) was more than that in plantation populations (h = 0.2021, I = 0.3040). Based on Nei's G_ST value, more genetic differentiation among plantation populations was detected (G_ST = 0.7787). Six populations of C. microphylla clustered into two clades. These results have important implications for restoring and managing the degraded ecosystem in arid and semi-arid areas.     

Genetic diversity and structure of the critically endangered tree Dimorphandra wilsonii and of the widespread in the Brazilian Cerrado Dimorphandra mollis: Implications for conservation

We performed a comparative analysis of the genetic diversity and structure of two congeneric tree species, one critically endangered, with only 21 known individuals in the wild, Dimorphandra wilsonii, and the other widely distributed Dimorphandra mollis. Eight populations of D. mollis and all known trees of D. wilsonii, from three areas, were screened for variability with ISSR markers. Percentage of polymorphic bands, Nei's gene diversity and Shannon's index were considerably lower in D. wilsonii (P = 40.0%, h = 0.124 and I = 0.190), as compared to D. mollis (P = 70.4%, h = 0.190 and I = 0.297). Bayesian clustering showed that D. wilsonii individuals are clustered in three populations, which had high differentiation among them. Several measures for their conservation were suggested: protection of all extant populations, ex situ conservation of seeds, production of saplings in nurseries and foundation of new populations in reserve areas.     

Genetic variation and clonal diversity in populations of Nelumbo nucifera (Nelumbonaceae) in central China detected by ISSR markers

To obtain accurate estimates of population structure for purposes of conservation planning for wild lotus (Nelumbo nucifera Gaertn.) in central China, genetic diversity among and within six populations, and clonal diversity within another two populations of the species were analyzed. The genetic diversity was high (percentage of polymorphic bands, PPB = 90.0%; Shannon's information index, I = 0.383 ± 0.234) at the species level, but low within individual study populations (PPB = 35.8%; Shannon's information index I = 0.165 ± 0.241). The mean coefficient of gene differentiation (G_st) was 0.570, indicating that 43.0% of the genetic diversity resided within the population. Analysis of molecular variance (AMOVA) indicated that 50.47% of the genetic diversity among the study populations was attributed to geographical location while 12.3% was attributed to differences in their habitats. An overall value of mean estimated number of gene flow (N_m = 0.377) indicated that there was limited gene flow among the sampled populations. The level of clonal diversity found within the populations was considerably high (Simpson's diversity index, D = 0.985) indicating that clonal diversity contributes to a major extent to the overall genetic variation in the genetic structure of N. nucifera. On the basis of the high G_st and D values detected in this study we recommend that any future conservation plans for this species should be specifically designed to include those representative populations with the highest genetic variation for both in situ conservation and germplasm collection expeditions.     

Genetic diversity of two endemic and endangered Plantago species

Plantago algarbiensis and Plantago almogravensis are two endangered and endemic species from Portugal. Due to the rarity and endangered nature of these species as well as the lack of molecular data, their genetic variation was evaluated using ISSR and RAPD markers. P. algarbiensis species showed higher genetic variability (73.9% of polymorphism) than P. almogravensis (61.2%). The two species revealed a high level of genetic diversity, with a Nei's genetic diversity of 0.1965 and 0.2309 and a Shannon's diversity index of 0.2975 and 0.3520, for P. almogravensis and P. algarbiensis, respectively. A low level of genetic differentiation was observed (Gst = 0.1873) among the species. However, the cluster and PCA analyses, based on genetic similarity, revealed two main, clearly separate clusters, which directly corresponded to the plants isolated from each species. In situ and ex situ measures should be applied in order to preserve both species but, based on these results, P. almogravensis population should be a priority for conservation.     

Wild Panax vietnamensis and Panax stipuleanatus markedly increase the genetic diversity of Panax notoginseng (Araliaceae) revealed by start codon targeted (SCoT) markers and ITS DNA barcode

In order to evaluate whether the two wild species, Panax vietnamensis (from Vietnam) and Panax stipuleanatus (from primeval forest, Yunan Province) could markedly increase the genetic diversity of cultivated Panax notoginseng (Wenshan, Yunnan Province), both start codon targeted (SCoT) markers and internal transcribed spacer (ITS) DNA barcode were firstly employed in this genus. A total of 173 amplification bands were generated by 16 selected SCoT primers, in which 153 (89.5%) were polymorphic. Nei's gene-diversity indicated that the genetic diversity of three species (h = 0.16 and I = 0.27) was obviously higher than that of P. notoginseng (h = 0.09). Similarly, 38 different ITS sites out of 639 (5.9%) were detected among three species, but only one was different within 22 samples of P. notoginseng. Analysis of molecular variance (AMOVA) showed a greater proportion of genetic diversity existed within (61.3%) rather than among (38.7%) groups at genus level. In addition, P. vietnamensis had a closer relationship with P. notoginseng than P. stipuleanatus. These results would be significant for increasing the genetic diversity of P. notoginseng population by hybridization with P. vietnamensis and P. stipuleanatus, thus obtaining more varieties for future cultivar breeding and germplasm resources management.     

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.     

Population structure and genetic diversity of Dysosma versipellis (Berberidaceae), a rare endemic from China

Dysosma versipellis (Berberidaceae) is an endangered and endemic species in China. To provide scientific foundation for formulating conservation strategies, we sampled six extant populations of this species and assessed the levels and patterns of genetic diversity using ISSR markers (11 primers). Of 144 bands detected 57.64% were polymorphic, but on average only 20.72% were polymorphic within populations. Our results revealed a low level of intraspecific genetic diversity (at population level: H_pop = 0.082, H_B = 0.177, SI = 0.1194; at species level: H_pop = 0.207, H_B = 0.378, SI = 0.3069). A high level of genetic differentiations among populations was detected based on Nei's genetic diversity analysis (60%), AMOVA analysis (65%), and Bayesian analysis (53%). The low levels of heterozygosity and high genetic differentiation observed in D. versipellis may be the consequence of low rate of natural recruitment, clonal growth, gene drift, and habitat fragmentation. Based on this, we suggest that in situ conservation be an important and practical measure for maintaining the genetic diversity of this species. Ex situ conservation should sample from different populations across the distribution range of the species to conserve high genetic diversity.     

Effect of cliff connectivity on the genetic population structure of a rock-dwelling land snail species with frequent self-fertilization

To examine the effect of habitat connectivity on the genetic differentiation in the frequently selfing gastropod Chondrina avenacea we assessed RAPD profiles in 12 subpopulations from continuous cliffs and in 12 subpopulations from small, naturally fragmented cliffs in the Swiss Jura mountains. Subpopulations of C. avenacea on continuous cliffs showed a smaller number of polymorphic RAPD loci and a lower gene diversity (Nei's h = 0.049) than subpopulations on fragmented cliffs (h = 0.078). Genetic distance was not correlated with geographical distance between subpopulations. G_ST analysis indicated strong isolation effects. Neither the number of polymorphic loci nor Nei's h was correlated with local population density. Our analysis indicates that the local population structure of C. avenacea is a combined result of frequent self-fertilization, genetic drift and low dispersal. Similar findings have been reported in studies on spatial population structure of plants. We suggest that future management actions (selective tree cutting, and controlled access of sport climbers) should also consider small, isolated limestone cliffs to preserve the genetic diversity of C. avenacea.     

Low genetic diversity and significant structuring in the endangered Mentha cervina populations and its implications for conservation

Eighteen populations of the endangered aromatic and medicinal plant Mentha cervina (Lamiaceae) were sampled across its natural range, in the western half of the Iberian Peninsula, and inter-simple sequence repeats (ISSRs) markers were used to assess genetic diversity and population structure. M. cervina populations exhibited a relatively low genetic diversity (percentage of polymorphic loci PPB = 14.2–58.3%, Nei's genetic diversity H_e = 0.135–0.205, Shannon's information index I = 0.08 − 0.33). However, the genetic diversity at species level was relatively high (PPB = 98.3%; H_e = 0.325; I = 0.23). The results of the analysis of molecular variance indicated very structured populations, with 50% of the variance within populations, 44% among populations and 6% between regions defined by hydrographic basins, in line with the gene differentiation coefficient (G_ST = 0.532). A Mantel test did not find significant correlation between genetic and geographic distance matrices (r = 0.064), indicating that isolation by distance is not shaping the present genetic structure. The levels and patterns of genetic diversity in M. cervina populations were assumed to result largely from a combination of evolutionary history and its unique biological traits, such as breeding system, low capacity of dispersion, small effective size and habitat fragmentation. The high genetic differentiation among populations indicates the necessity of conserving the maximum possible number of populations. The results also provide information to select sites for ex situ conservation. Optimal harvesting strategies, cultivation and tissue culture should also be developed as soon as possible to guarantee sustainable use of the species under study.     

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 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.