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 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.     

Use of amplified fragment length polymorphism markers to assess genetic diversity of Lolium species from Portugal

We evaluated the use of amplified fragment length polymorphism (AFLP) markers to distinguish genotypes, populations and species of Lolium. Accessions of two species Lolium perenne and Lolium multiflorum and their hybrid Lolium x hybridum, collected by the Institute of Grass and Environmental Research in 1995 from locations across Portugal, were used. The genetic variation within and between populations from the extremes of latitude and altitude was determined and assessed. Three primer pair combinations generated 765 polymorphic bands. Principal coordinate analysis of similarities between 127 plants showed high dimensionality in the data. Axes 1-3 were associated primarily with species differences, axes 4-14 with population differences within species and axis 15 onwards with within population differences. UPGMA analysis confirmed the groupings. The three populations of L. perenne formed a discrete cluster widely separated from all other populations. There were two distinct groups of L. x hybridum, of which one was similar to and overlapped with L. multiflorum and the second formed a distinct cluster. Analyses of individual bands showed that every inter- and intraspecific contrast involved a different sets of bands, again confirming the high dimensionality of the data. No single band was strictly diagnostic of any population or species. Nevertheless, the UPGMA analysis showed little or no overlap between populations. Thus, despite the high ratio of within-to-between population genetic variance, the full AFLP banding pattern of each genotype is a relatively reliable fingerprint diagnostic of its parent population. The high dimensionality implies that many different factors contribute to the differences observed. This adds to the potential value of the methodology, since it implies that there is a reasonably high likelihood of finding bands relevant to a given environmental gradient or other factor influencing the distribution of genetic diversity.     

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.     

Genetic relationships among wild and cultivated blackberries (Rubus caucasicus L.) based on amplified fragment length polymorphism markers

Abstract

Rubus is a large genus of flowering plants in the rose family, Rosaceae, subfamily Rosoideae. The blackberries, as well as various other Rubus species with mounding or rambling growth habits, are often called brambles. Little information is available on the genetic diversity of wild-grown blackberries. The objective of this study was to determine the genetic relationships among nine promising (high-yield capacity, free of pest and diseases, better fruit traits) wild blackberry (Rubus caucasicus L.) selections and the well-known cultivar, “Chester” by using amplified fragment length polymorphism (AFLP) markers. Genotypes were evaluated with three selective primer-enzyme combinations, producing a total of 223 AFLP fragments with 53% polymorphism ratio. Clustering of genotypes using unweighted pair-group method of arithmetic average cluster analysis clearly separated groups of wild blackberry genotypes while the variety “Chester” was clustered independently. Wild selections represented a distinct germplasm source on the basis of the estimated genetic distance among them. Genetic diversity data from this study will be helpful in using and exploiting the wild genetic material for breeding purposes as well as for further research.     

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.     

Accurate gene diversity estimates from amplified fragment length polymorphism (AFLP) markers

Three procedures for the estimation of null allele frequencies and gene diversity from dominant multilocus data were empirically tested in natural populations of the outcrossing angiosperm Persoonia mollis (Proteaceae). The three procedures were the square root transform of the null homozygote frequency, the Lynch & Milligan procedure, and the Bayesian method. Genotypes for each of 116 polymorphic loci generated by amplified fragment length polymorphism (AFLP) were inferred from segregation patterns in progeny arrays. Therefore, for the plus phenotype (band present), heterozygotes were distinguished from homozygotes. In contrast to previous studies, all three procedures produced very similar mean estimates of heterozygosity, which were in turn accurate estimators of the direct value (HO = 0.28). A second population of P. mollis displayed markedly lower levels of heterozygosity (HO = 0.20) but approximately twice as many polymorphic loci (284). These AFLP results show that biases in estimates of average null allele frequency and heterozygosity are largely eliminated in highly polymorphic dominant marker data sets displaying a J-shaped beta distribution with a high percentage of loci containing more than three null homozygotes and relatively few loci with no null homozygotes. This distribution may be typical of outcrossing angiosperms.     

Population structure and genetic diversity of Huperzia serrata (Huperziaceae) based on amplified fragment length polymorphism (AFLP) markers

The levels and pattern of the genetic variation within and among natural populations of Huperzia serrata were investigated using amplified fragment length polymorphism markers. Seven primer combinations used in the study amplified 615 discernible bands with 532 (86.5%) being polymorphic, indicating a considerable high level of genetic diversity at the species level. AMOVA analysis revealed a low level of genetic differentiation among the ten populations. The UPGMA cluster of all samples showed that individuals from the same population occasionally failed to cluster in one distinct group. A Mantel test showed no significant correlation between genetic distance and geographical distance (r = 0.278, P = 0.891), suggesting that the gene flow was not restricted geographically. A number of factors that might affect the genetic profiles of H. serrata included clonal growth, selective effect of niche and outcrossing, as well as the effective wind-dispersal of spores.     

AFLP标记的特点及其在昆虫学研究中的应用

扩增片段长度多态性（AFLP）是一种新兴的很有效的分子遗传标记方法, 它通过对基因组DNA限制性内切酶酶切片段进行选择性扩增而揭示多态性,具有快速、经济简便、不需要预先知道模板DNA的信息、模板需要量少、重复性高、结果可靠及具有很高的信息含量等优点。AFLP也具有缺点,主要是标记是显性的,同其他显性标记一样,不能区分杂合体和纯合体,因而不能更好地估算种群遗传的变异,对种群遗传结构的分析不能提供更多的统计信息;AFLP技术较复杂,而且经常使用放射性同位素,对模板DNA质量要求也较高。为了克服AFLP的这些缺点,人们又在其基础上发展了其他相关技术,例如AFRP、SAMPL、DALP和TE-AFLP等。目前AFLP在昆虫方面的应用还不是很多,处于初级阶段,主要应用在生态型鉴定、种群遗传分析、连锁图谱构建等方面,相信随着其技术的发展完善,必将会越来越多地应用于昆虫学的研究中。     

Cleaved AFLP (cAFLP), a modified amplified fragment length polymorphism analysis for cotton

In certain plant species including cotton (Gossypium hirsutum L. or Gossypium barbadense L.), the level of amplified fragment length polymorphism (AFLP) is relatively low, limiting its utilization in the development of genome-wide linkage maps. We propose the use of frequent restriction enzymes in combination with AFLP to cleave the AFLP fragments, called cleaved AFLP analysis (cAFLP). Using four Upland cotton genotypes (G. hirsutum) and three Pima cotton (G. barbadense), we demonstrated that cAFLP generated 67% and 132% more polymorphic markers than AFLP in Upland and Pima cotton, respectively. This resulted in 15.5 and 25.5 polymorphic cAFLP markers per AFLP primer combination, as compared to 9.1 and 11.0 polymorphic AFLP. The cAFLP-based genetic similarity (GS) is generally lower than the AFLP-based GS, even though both marker systems are overall congruent. In some cases, cAFLP can better resolve genetic relationships between genotypes, rendering a higher discriminatory power. Given the high-resolution power of capillary-based DNA sequencing system, we further propose that AFLP and cAFLP amplicons from the same primer combination can be pooled as one sample before electrophoresis. The combination produced an average of 18.5 and 31.0 polymorphic markers per primer pair in Upland and Pima cotton, respectively. Using several restriction enzyme combinations before pre-selective amplification in combination with various frequent 4 bp-cutters or 6 bp-cutters after selective amplification, the pooled AFLP and cAFLP will provide unlimited number of polymorphic markers for genome-wide mapping and fingerprinting.     

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.     

Assessment of genetic diversity among and within Achillea species using amplified fragment length polymorphism (AFLP)

Amplified fragment length polymorphism (AFLP) markers were used to assess the genetic diversity of 57 Achillea accessions belonging to five species, A. millefolium, A. filipendulina, A. tenuifolia, A. santolina and A. biebersteinii. Nine AFLP primer combinations were used, which produced 301 polymorphic bands. In most species, a high level of genetic variation was detected among the genotypes. The Jaccard's similarity indices (J), based on AFLP profiles, were subjected to UPGMA cluster analysis. Application of Mantel's test for cophenetic correlation to the cluster analysis indicated the high fitness of the accessions to a group (r = 0.918). The dendrogram generated revealed five major groups corresponding to five species. The principle coordinate analysis (PCoA) data confirmed the results of the clustering. Among the species, A. teunifolia and A. santolina showed the greatest and the least genetic diversity, respectively. A. filipendulina accessions were acquired primarily from the same ecological regions of western Iran. Accessions belonging to A. biebersteinii originated from the Isfahan province and were separated from other species at the root of the dendrogram. The results of the clustering method, based on AFLP markers, corresponded closely with the geographical origins of the genotypes. The results of the present study could contribute to a better understanding and management of conservation and exploitation of the Achillea germplasm.     

Genetic diversity in European pigs utilizing amplified fragment length polymorphism markers

The use of DNA markers to evaluate genetic diversity is an important component of the management of animal genetic resources. The Food and Agriculture Organisation of the United Nations (FAO) has published a list of recommended microsatellite markers for such studies; however, other markers are potential alternatives. This paper describes results obtained with a set of amplified fragment length polymorphism (AFLP) markers as part of a genetic diversity study of European pig breeds that also utilized microsatellite markers. Data from 148 AFLP markers genotyped across samples from 58 European and one Chinese breed were analysed. The results were compared with previous analyses of data from 50 microsatellite markers genotyped on the same animals. The AFLP markers had an average within-breed heterozygosity of 0.124 but there was wide variation, with individual markers being monomorphic in 3-98% of the populations. The biallelic and dominant nature of AFLP markers creates a challenge for their use in genetic diversity studies as each individual marker contains limited information and AFLPs only provide indirect estimates of the allelic frequencies that are needed to estimate genetic distances. Nonetheless, AFLP marker-based characterization of genetic distances was consistent with expectations based on breed and regional distributions and produced a similar pattern to that obtained with microsatellites. Thus, data from AFLP markers can be combined with microsatellite data for measuring genetic diversity.     

Comparison of genetic diversity of the invasive weed Rubus alceifolius poir. (Rosaceae) in its native range and in areas of introduction, using amplified fragment length polymorphism (AFLP) markers

Theory predicts that colonization of new areas will be associated with population bottlenecks that reduce within-population genetic diversity and increase genetic differentiation among populations. This should be especially true for weedy plant species, which are often characterized by self-compatible breeding systems and vegetative propagation. To test this prediction, and to evaluate alternative scenarios for the history of introduction, the genetic diversity of Rubus alceifolius was studied with amplified fragment length polymorphism (AFLP) markers in its native range in southeast Asia and in several areas where this plant has been introduced and is now a serious weed (Indian Ocean islands, Australia). In its native range, R. alceifolius showed great genetic variability within populations and among geographically close populations (populations sampled ranging from northern Vietnam to Java). In Madagascar, genetic variability was somewhat lower than in its native range, but still considerable. Each population sampled in the other Indian Ocean islands (Mayotte, La Réunion, Mauritius) was characterized by a single different genotype of R. alceifolius for the markers studied, and closely related to individuals from Madagascar. Queensland populations also included only a single genotype, identical to that found in Mauritius. These results suggest that R. alceifolius was first introduced into Madagascar, perhaps on multiple occasions, and that Madagascan individuals were the immediate source of plants that colonized other areas of introduction. Successive nested founder events appear to have resulted in cumulative reduction in genetic diversity. Possible explanations for the monoclonality of R. alceifolius in many areas of introduction are discussed.     

NaCl对黄孢原毛平革菌(Phanerochaete chrysosporium)的损伤效应

染料中含有大最NaCl是影响黄孢原毛平革菌脱色效率的重要因素。为研究NaCl对黄孢原毛平革菌处理功能的影响,分别采用孢子和菌丝球与不同浓度的NaCl混合培养10d,以观察孢子生长和菌丝球的损伤效应,并利用透射电子显微镜和AFLP法对其进行细胞结构分析与DNA扩增,通过分析不同浓度NaCl对其生长及微观结构的影响、NaCl浓度与DNA相似性关系以及构建UPGMA相似性树状图等方法,评价NaCl对P.chrysosporium结构与功能的损伤效应。结果显示,3％NaCl对黄孢原毛平革菌影响较小,细胞结构保持完整,异常细胞量为14．2％,DNA变异率小,与空白的相似度达90％以上,表明黄孢原毛平革菌在3％的浓度范围内结构功能基本不受影响;5％NaCl使DNA相似度下降为71．4％,下降幅度最为显著,并且细胞内含物松散和出现胞浆空泡化趋势,异常细胞占有量为71．1％,说明3％～5％的浓度范围最易对P．chrysosporium的结构与功能产生不良影响;NaCl浓度≥8％可对黄孢原毛平革菌产生严重损伤,细胞变形严重,空泡化,DNA相似度降至67％以下,异常细胞量约90％,表明此浓度范围可使黄孢原毛平革菌基本丧失了原有的结构与功能。     

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。     

Estimating genetic diversity of Arabidopsis thaliana ecotypes with amplified fragment length polymorphisms (AFLP)

The extensive natural variation of Arabidopsis thaliana ecotypes is being increasingly exploited as a source of variants of genes which control (agronomically) important traits. We have subjected 19 different Arabidopsis thaliana ecotypes to an analysis using the anplified fragment length polymorphism (AFLP) technique in order to estimate their genetic diversity. The genetic diversity was estimated applying the method of Nei and Li (1979) and a modified version of it and using 471 informative polymorphisms. The data obtained revealed that within this small set of ecotypes a group of three ecotypes and a further single ecotype exhibit considerable genetic diversity in comparison to the others. These ecotypes clustered at positions significantly separated from the bulk of the ecotypes in the generated similarity plots. The analysis demonstrated the usefulness of the AFLP method for determinating intraspecies genetic diversity as exemplified with Arabidopsis thaliana ecotypes. Results are discussed and compared with data obtained with other methods. Received: 18 June 1999 / Accepted: 28 July 1999     

Clonal structure in a dwarf bamboo (Sasa senanensis) population inferred from amplified fragment length polymorphism (AFLP) fingerprints

Amplified fragment length polymorphism (AFLP) fingerprints were used to reveal clonal structure of a dense population of dwarf bamboo, Sasa senanensis, in a 10-ha study plot at Sugadaira Montane Research Center, University of Tsukuba, Nagano, Japan. We generated AFLP fingerprints for 51 leaf samples, collected at 50 m intervals, using three selective primer pairs. A total of 135-166 fragments were detected per sample, and 22 different fingerprints were identified based on 24-83 differing fragments. Our results demonstrate that the S. senanensis population in our plot consists of at least 22 clones and that the largest single clone occurs over a distance of about 300 m. Furthermore, the clone distribution pattern implies a relationship between site quality and clonal structure.     

Amplified fragment length polymorphism versus random amplified polymorphic DNA markers: clonal diversity in Saxifraga cernua

Random amplified polymorphic DNA (RAPD) markers are sensitive to changes in reaction conditions and may express polymorphisms of nongenetic origin. Taxa with variable chromosome numbers are particularly challenging cases, as differences in DNA content may also influence marker reproducibility. We addressed these problems by comparing RAPD and amplified fragment length polymorphism (AFLP) analyses of clonal identity and relationships in a chromosomally variable arctic plant, the polyploid Saxifraga cernua, which has been thought to be monoclonal over large geographical distances. Fifty-seven plants from four Greenland populations were analysed using a conservative scoring approach. In total, 26 AFLP and 32 RAPD multilocus phenotypes (putative clones) were identified, of which 21 were identical and each of the remaining five AFLP clones was split into two to three very similar RAPD clones. This minor difference can be explained by sampling error and stochastic variation. The pattern observed in Greenland corroborates our previous results from Svalbard, suggesting that rare sexual events in S. cernua are sufficient to maintain high levels of clonal diversity even at small spatial scales. We conclude that although AFLP analysis is superior in terms of efficiency, RAPDs may still be used as reliable markers in small low-tech laboratories.