Genetic diversity of the endangered Chinese endemic herb Primulina tabacum (Gesneriaceae) revealed by amplified fragment length polymorphism (AFLP)

Primulina tabacum Hance, is a critically endangered perennial endemic to limestone area in South China. Genetic variability within and among four extant populations of this species was assessed using AFLP markers. We expected a low genetic diversity level of this narrowly distributed species, but our results revealed that a high level of genetic diversity remains, both at population level (55.5% of markers polymorphic, H _E = 0.220, I _S = 0.321), and at species level (P = 85.6% of markers polymorphic, H _E = 0.339, I _S = 0.495), probably resulting from its refugial history and/or breeding system. High levels of genetic differentiation among populations was apparent based on Nei’s genetic diversity analysis (G _st=0.350). The restricted gene flow between populations is a potential reason for the high genetic differentiation. The population genetic diversity of P. tabacum revealed here has clear implications for conservation and management. To maintain present levels of genetic diversity, in situ conservation of all populations is necessary.     

Genetic diversity in an endangered alpine plant, Eryngium alpinum L. (Apiaceae), inferred from amplified fragment length polymorphism markers

Eryngium alpinum L. is an endangered species found across the European Alps. In order to obtain base-line data for the conservation of this species, we investigated levels of genetic diversity within and among 14 populations from the French Alps. We used the amplified fragment length polymorphism (AFLP) technique with three primer pairs and scored a total of 62 unambiguous, polymorphic markers in 327 individuals. Because AFLP markers are dominant, within-population genetic structure (e.g. FIS) could not be assessed. Analyses based either on the assumption of random-mating or on complete selfing lead to very similar results. Diversity levels within populations were relatively high (mean Nei's expected heterozygosity = 0.198; mean Shannon index = 0.283), and a positive correlation was detected between both genetic diversity measurements and population size (Spearman rank correlation: P = 0. 005 and P = 0.002, respectively). Moreover, FST values and exact tests of differentiation revealed high differentiation among populations (mean pairwise FST = 0.40), which appeared to be independent of geographical distance (nonsignificant Mantel test). Founder events during postglacial colonizations and/or bottlenecks are proposed to explain this high but random genetic differentiation. By contrast, we detected a pattern of isolation by distance within populations and valleys. Predominant local gene flow by pollen or seed is probably responsible for this pattern. Concerning the management of E. alpinum, the high genetic differentiation leads us to recommend the conservation of a maximum number of populations. This study demonstrates that AFLP markers enable a quick and reliable assessment of intraspecific genetic variability in conservation genetics.     

Genetic diversity of the endangered Chinese endemic plant Monimopetalum chinense revealed by amplified fragment length polymorphism (AFLP)

Monimopetalum chinense Rehd. is an endangered woody vine endemic to eastern China. Using amplified fragment length polymorphism (AFLP) markers, we examined levels of genetic variation within and among eleven populations located across the species’ distribution. Although modest levels of heterozygosity were detected, other measures of genetic diversity registered relatively high levels of variability, both at the species level (P = 91.0%, H_E = 0.232, I_S = 0.365) and at the population level (P = 53.0%, H_E = 0.155, I_S = 0.239). Populations also exhibited high levels of genetic differentiation (Nei’s genetic diversity analysis, G_ST = 0.330), corresponding to isolation-by-distance and hierarchical population structure. These results indicate that, despite low levels of gene flow, populations of M. chinense still harbor substantial amounts of genetic diversity. Management plans for the species should include measures that ensure genetic diversity remains high within and among extant populations.     

Genetic variation in the endangered wild apple (Malus sylvestris (L.) Mill.) in Belgium as revealed by amplified fragment length polymorphism and microsatellite markers

The genetic variation within and between wild apple samples (Malus sylvestris) and cultivated apple trees was investigated with amplified fragment length polymorphisms (AFLP) and microsatellite markers to develop a conservation genetics programme for the endangered wild apple in Belgium. In total, 76 putative wild apples (originating from Belgium and Germany), six presumed hybrids and 39 cultivars were typed at 12 simple sequence repeats (SSR) and 139 amplified fragment length polymorphism (AFLP) loci. Principal co-ordinate analysis and a model-based clustering method classified the apples into three major gene pools: wild Malus sylvestris genotypes, edible cultivars and ornamental cultivars. All presumed hybrids and two individuals (one Belgian, one German) sampled as M. sylvestris were assigned completely to the edible cultivar gene pool, revealing that cultivated genotypes are present in the wild. However, gene flow between wild and cultivated gene pools is shown to be almost absent, with only three genotypes that showed evidence of admixture between the wild and edible cultivar gene pools. Wild apples sampled in Belgium and Germany constitute gene pools that are clearly differentiated from cultivars and although some geographical pattern of genetic differentiation among wild apple populations exists, most variation is concentrated within samples. Concordant conclusions were obtained from AFLP and SSR markers, which showed highly significant correlations in both among-genotypes and among-samples genetic distances.     

Genetic variation of Calycophyllum spruceanum in the Peruvian Amazon Basin, revealed by amplified fragment length polymorphism (AFLP) analysis

An understanding of the level, structure and origin of genetic variation within and among populations of tropical trees is essential for devising optimum management strategies for their sustainable utilization and conservation. Here, amplified fragment length polymorphism (AFLP) analysis was used to partition genetic variation within and among nine populations of the predominantly riverine tree, Calycophyllum spruceanum , sampled across a wide geographical range along river tributaries of the Peruvian Amazon Basin. Analysis of molecular variance ( AMOVA ) employed 65 AFLP markers and revealed most variation among individuals within populations (91%), although variation among populations was highly significant ( P < 0.001). Calculation of genetic distances and nested AMOVA indicated a degree of structuring among populations based on geographical proximity, although clustering did not depend on geographical distance alone. No firm evidence was obtained for unidirectional seed dispersal by water playing an important role in determining genetic structure over the geographical range sampled. Implications of data for optimising genetic management of the species are discussed and areas for further study identified.     

Population genetics of the yellow fever mosquito in Trinidad: comparisons of amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) markers

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a polymerase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in order to estimate population genetic diversity and genetic structure parameters, one must assume that dominant (amplified) alleles are identical in state, recessive (unamplified) alleles are identical in state, AFLP fragments segregate according to Mendelian expectations and that the genotypes of an AFLP locus are in Hardy-Weinberg equilibrium (HWE). The HWE assumption is untestable for natural populations using dominant markers. Restriction fragment length polymorphism (RFLP) markers segregate as codominant alleles, and can therefore be used to test the HWE assumption that is critical for analysing AFLP data. This study examined whether the dominant AFLP markers could provide accurate estimates of genetic variability for the Aedes aegypti mosquito populations of Trinidad, West Indies, by comparing genetic structure parameters using AFLP and RFLP markers. For AFLP markers, we tested a total of five primer combinations and scored 137 putative loci. For RFLP, we examined a total of eight mapped markers that provide a broad coverage of mosquito genome. The estimated average heterozygosity with AFLP markers was similar among the populations (0.39), and the observed average heterozygosity with RFLP markers varied from 0.44 to 0.58. The average FST (standardized among-population genetic variance) estimates were 0.033 for AFLP and 0.063 for RFLP markers. The genotypes at several RFLP loci were not in HWE, suggesting that the assumption critical for analysing AFLP data was invalid for some loci of the mosquito populations in Trinidad. Therefore, the results suggest that, compared with dominant molecular markers, codominant DNA markers provide better estimates of population genetic variability, and offer more statistical power for detecting population genetic structure.     

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.     

Low genetic differentiation among seasonal cohorts in Senecio vulgaris as revealed by amplified fragment length polymorphism analysis

Common groundsel, Senecio vulgaris (Asteraceae), is a highly selfing semelparous ephemeral weed that belongs to the few plant species in central Europe capable of growing, flowering and fruiting all year round. In temperate climates, flowering S. vulgaris cohorts were found to appear up to three times per year. Using amplified fragment length polymorphism (AFLP) molecular markers we examined temporal genetic differentiation among spring, summer and autumn cohorts at each of seven sites located in two regions in Switzerland. Strong genetic differentiation among cohorts may indicate the existence of seasonal races of S. vulgaris, reproductively isolated by nonoverlapping flowering phenologies. Analysis of molecular variance (amova) revealed that < 2.5% of the AFLP variation resided among cohorts within sites, whereas there was significant genetic differentiation among plants from different sites (15.6%) and among individuals within cohorts (81.9%). Significant genetic differentiation was also observed between the two regions. Isolation-by-distance was found on a regional scale, but not on a local scale. Gene flow was estimated to be approximately 15-fold higher among cohorts within sites than among sites. We further found, on average, similar levels of genetic diversity within the three seasonal cohorts. The results of this study demonstrate that season of growth represents a weak barrier for genetic exchange among S. vulgaris populations and does not affect molecular variance. Therefore, there is no evidence for the existence of seasonally specialized races of S. vulgaris. We discuss some implications of the results for the biological control of S. vulgaris using a native rust fungus.     

A population genetic study of the endangered plant species Limonium dufourii (Plumbaginaceae) based on amplified fragment length polymorphism (AFLP)

Limonium dufourii ( Plumbaginaceae ) is a triploid species with obligate apomictic reproduction and is endemic to the East Mediterranean coast of Spain, where it is present in only six populations, most of which have a very low number of individuals. Genetic variation and population structure in this species was studied using amplified fragment length polymorphisms (AFLPs) as markers, using the same individuals as in a previous study with random amplified polymorphic DNA (RAPD). Three different primers provided 252 bands of which 51 were polymorphic among the 152 individuals analysed. Those polymorphic bands were able to define 65 different phenotypes, of which all but two were present in only one population. The comparative analyses of data from AFLPs with those from RAPDs show a high degree of concordance. Additionally, and given the nature of these markers, we propose the estimation of nucleotide divergences from AFLP patterns. Relationships among the different AFLP patterns and the estimates of population genetic parameters obtained with this evolutionary distance are in good agreement with previous results. These analyses show that substantial genetic variability and differentiation exist within and among populations of L. dufourii . Their higher reproducibility and the possibility of obtaining estimates of nucleotide divergence make AFLPs a much better DNA fingerprinting technique.     

扩增片段长度多态性实验技术及在动物遗传多样性研究中的应用

扩增片段长度多态性(AFLP)是一种有效的分子遗传标记方法,具有经济、简便、模板需要量少、重复性高、结果可靠等优点。目前AFLP在动物方面的应用还不是很多,处于初级阶段,主要用于鉴定分类关系、种群遗传多样性分析、遗传连锁图谱构建等方面。     

The use of amplified fragment length polymorphism (AFLP) in the isolation of sex-specific markers

Sex identification is a problem in research and conservation. It can often be solved using a DNA test but this is only an option if a sex-specific marker is available. Such markers can be identified using the amplified fragment length polymorphism (AFLP) technique. This is usually a taxonomic method, as it produces a DNA fingerprint of 50-100 PCR bands. However, if male and female AFLP products are compared, sex-specific markers are confined to the heterogametic sex and can rapidly be identified. Once a marker is found, AFLP can be used to sex organisms directly or the marker can be sequenced and a standard PCR test designed.     

Data from amplified fragment length polymorphism (AFLP) markers show indication of size homoplasy and of a relationship between degree of homoplasy and fragment size

We investigate the distribution of sizes of fragments obtained from the amplified fragment length polymorphism (AFLP) marker technique. We find that empirical distributions obtained in two plant species, Phaseolus lunatus and Lolium perenne, are consistent with the expected distributions obtained from analytical theory and from numerical simulations. Our results indicate that the size distribution is strongly asymmetrical, with a much higher proportion of small than large fragments, that it is not influenced by the number of selective nucleotides nor by genome size but that it may vary with genome-wide GC-content, with a higher proportion of small fragments in cases of lower GC-content when considering the standard AFLP protocol with the enzyme MseI. Results from population samples of the two plant species show that there is a negative relationship between AFLP fragment size and fragment population frequency. Monte Carlo simulations reveal that size homoplasy, arising from pulling together nonhomologous fragments of the same size, generates patterns similar to those observed in P. lunatus and L. perenne because of the asymmetry of the size distribution. We discuss the implications of these results in the context of estimating genetic diversity with AFLP markers.     

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.     

Trypanosoma brucei gambiense: study of population genetic structure of Central African stocks using amplified fragment length polymorphism (AFLP)

To understand the maintenance and resurgence of historical Human African Trypanosomiasis (HAT) foci, AFLP was used to genotype 100 Central African Trypanosoma brucei s.l. stocks. This technique confirmed the high genetic stability of T. b. gambiense group 1 stocks and the micro genetic variability within Central African T. b. gambiense stocks. It revealed several T. b. gambiense genotypes and allowed the identification of minor and major genotypes in HAT foci. The coexistence of these genotypes in the same focus suggests that clustering of stocks according to HAT focus does not provide the true genetic picture of trypanosome circulating within the disease focus because the minor genotypes are generally underestimated. The presence of minor and major genotypes in HAT foci may explain the persistence and the resurgence of Central African sleeping sickness foci.     

Trypanosoma evansi: genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar ('type A') whereas 2 isolates differed substantially ('type B'). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades.     

Genetic structure and gene flow in a metapopulation of an endangered plant species,Silene tatarica

We investigated the distribution of genetic variation within and between seven subpopulations in a riparian population of Silene tatarica in northern Finland by using amplified fragment length polymorphism (AFLP) markers. A Bayesian approach-based clustering program indicated that the marker data contained not only one panmictic population, but consisted of seven clusters, and that each original sample site seems to consist of a distinct subpopulation. A coalescent-based simulation approach shows recurrent gene flow between subpopulations. Relative high FST values indicated a clear subpopulation differentiation. However, amova analysis and UPGMA-dendrogram did not suggest any hierarchical regional structuring among the subpopulations. There was no correlation between geographical and genetic distances among the subpopulations, nor any correlation between the subpopulation census size and amount of genetic variation. Estimates of gene flow suggested a low level of gene flow between the subpopulations, and the assignment tests proposed a few long-distance bidirectional dispersal events between the subpopulations. No apparent difference was found in within-subpopulation genetic diversity among upper, middle and lower regions along the river. Relative high amounts of linkage disequilibrium at subpopulation level indicated recent population bottlenecks or admixture, and at metapopulation levels a high subpopulation turnover rate. The overall pattern of genetic variation within and between subpopulations also suggested a 'classical' metapopulation structure of the species suggested by the ecological surveys.     

Amplified fragment length polymorphism (AFLP) analysis of genetic variation in Moringa oleifera Lam

Moringa oleifera is an important multipurpose tree introduced to Africa from India at the turn of this century. Despite limited knowledge of the levels of genetic diversity and relatedness of introduced populations, their utilization as a source of seed for planting is widespread. In order to facilitate reasoned scientific decisions on its management and conservation and prepare for a selective breeding programme, genetic analysis of seven populations was performed using amplified fragment length polymorphism (AFLP) markers. The four pairs of AFLP primers ( Pst I/ Mse I) generated a total of 236 amplification products of which 157 (66.5%) were polymorphic between or within populations. Analysis of molecular variance ( AMOVA ) revealed significant differences between regions and populations, even though outcrossing perennial plants are expected to maintain most variation within populations. A phenetic tree illustrating relationships between populations suggested at least two sources of germplasm introductions to Kenya. The high levels of population differentiation detected suggest that provenance source is an important factor in the conservation and exploitation of M. oleifera genetic resources.     

Genetic distance between Nasutitermes takasagoensis (Isoptera: Termitidae) populations in Taiwan and the Yaeyama Islands, analyzed by amplified fragment length polymorphism markers

Dispersal ability and degree of inbreeding in a population can indirectly be assessed using genetic markers. It has been suggested that winged termites are not able to fly distances greater than several hundred meters. Using the amplified fragment length polymorphism (AFLP) technique, we previously analyzed the population substructure of Nasutitermes takasagoensis in the Yaeyama Islands, southern Japan. We found that there is moderate subpopulation structure based on the AFLP data. Moreover, the relatively low G_st and genetic distances among insular populations suggest that there is a possibility that winged termites are able to fly over distances of several kilometers. To learn more about this, in the present study, we compared new samples collected from the Hengchuen Peninsula in southern Taiwan with samples previously collected from the Yaeyama Islands. Using the samples collected in Taiwan, genetic distance was calculated against samples from six of the Yaeyama Islands. Genetic diversity, average heterozygosity, percentage of polymorphic loci, G_st and gene flow (Nm) were calculated. A total of 155 AFLP fragments were obtained for three primer combinations used, with 8 (5%) polymorphic bands. Genetic distance between the populations of Taiwan and the rest of the islands was greater than the genetic distance among the Yaeyama Islands. Although previous studies showed that average Nm among the Yaeyama Islands was 2.47, all of the observed Nm between Taiwan and the Yaeyama Islands were below 1. These findings suggest that the geographic distance between Taiwan and the Yaeyama Islands is large enough to prevent gene flow between them.     

Origin of Spanish invasion by the zebra mussel, Dreissena polymorpha (Pallas, 1771) revealed by amplified fragment length polymorphism (AFLP) fingerprinting

The zebra mussel, Dreissena polymorpha is an aquatic nuisance invasive species originally native to the Ponto-Caspian region where it is found in lakes and delta areas of large rivers draining into the Black and Caspian seas. The dispersal of D. polymorpha began at the end of the 18th century, at a time when shipping trade become increasingly important and many canals were built for linking different navigable river systems in Europe. Over the past 200 years, zebra mussels spread to most of the lakes, rivers and waterways in Europe by a combination of natural and anthropogenic dispersal mechanisms. D. polymorpha invaded Spain around 2001, being found for the first time in the Riba-roja reservoir at the lower part of the Ebro River, North-East Spain. The relatively late invasion of Spain was most likely caused by the presence of the Pyrenees, which isolated the Iberian Peninsula from the rest of the European continent, and acted as a barrier to the dispersal of D. polymorpha. In recent studies, molecular genetic methods have successfully been used to determine phylo-geographic relationships, which may reflect invasion corridors and can help retrace source populations. Zebra mussels from populations in Great Britain, The Netherlands, Belgium, France, Germany, Spain, Italy, Romania and North America were analyzed using PCR based amplified fragment length polymorphism (AFLP)-fingerprinting to determine the source population of D. polymorpha in Spain. The phylogenetic analyses and pair-wise genetic distances revealed that the recent invasion of zebra mussels in Spain is most likely from France.     

Hybrid status of kuwini,Mangifera odorata Griff. (Anacardiaceae) verified by amplified fragment length polymorphism

Mangifera odorata Griff. (Anacardiaceae), was suggested to be a hybrid between M. indica L. and M. foetida Lour. due to morphological intermediacy. Results from this study show that M. indica and M. foetida produced unique amplified fragment length polymorphism (AFLP) profiles. Mangifera odorata did not produce any unique bands. All the M. odorata samples additively inherit bands specific to M. indica and M. foetida, which strongly suggested the hybrid origin. Three major clusters were produced in the phenogram. All samples of M. indica, M. foetida and M. odorata segregated distinctly. Mangifera odorata was closer to M. foetida than to M. indica, indicating that backcrossing with M. foetida might have taken place. AFLP analysis therefore verified the hybrid status of M. odorata.