Amplified fragment length polymorphism (AFLP) markers reveal extra-pair parentage in a bird species: the bluethroat (Luscinia svecica)

We tested the use of amplified fragment length polymorphism (AFLP) to assess the frequency of extra-pair parentage in a bluethroat (Luscinia svecica namnetum) population. Thirty-six families totalling 162 nestlings were analysed. Using a combination of three primer pairs, we reached an exclusion probability of 93% for the population. This probability can reach 99% considering families independently. We revealed that extra-pair fertilizations are very common: 63.8% of all broods contain at least one extra-pair young, totalling 41.9% of all young analysed. However, with the technique and the three primer pairs used it was not possible to attribute the parentage exclusions to extra-pair paternity, maternity or both. As brood parasitism has never been reported in this species, it seems likely that the exclusions are due to extra-pair males. This study shows that dominant AFLP markers can be useful for studying the mating system of taxa for which no microsatellite primers are available. This technique allows the approximate estimation of parentage exclusions despite the fact that it is not possible to know which parent has to be excluded.     

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。     

Amplified fragment length polymorphism (AFLP) as suitable markers to study orobanche cumana genetic diversity

Orobanche cumana Wallr., an obligate root parasite of sunflower can cause severe damage to this crop. The genetic diversity obtained with random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) on two Orobanche populations were compared. Nei and Li distance matrices obtained with both methods among the two populations were correlated significantly according to Mantel's test and could partition the populations. The sampling variance of genetic distances within and among populations estimated using bootstrap procedure were not significantly different between the two techniques. The principal difference between the two techniques is that AFLP markers gave a higher degree of resolution for discriminating closely related germplasm than RAPD.     

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。     

Amplified fragment length polymorphism (AFLP): a review of the procedure and its applications

Amplified fragment length polymorphism (AFLP) is a novel molecular fingerprinting technique that can be applied to DNAs of any source or complexity. Total genomic DNA is digested using two restriction enzymes. Double-stranded nucleotide adapters are ligated to the DNA fragments to serve as primer binding sites for PCR amplification. Primers complementary to the adapter and restriction site sequence, with additional nucleotides at the 3′-end, are used as selective agents to amplify a subset of ligated fragments. Polymorphisms are identified by the presence or absence of DNA fragments following analysis on polyacrylamide gels. This technique has been extensively used with plant DNA for the development of high-resolution genetic maps and for the positional cloning of genes of interest. However, its application is rapidly expanding in bacteria and higher eukaryotes for determining genetic relationships and for epidemiological typing. This review describes the AFLP procedure, and recent, novel applications in the molecular fingerprinting of DNA from both eukaryotic and prokaryotic organisms. Received 19 December 1997/ Accepted in revised form 3 June 1998     

Spatial ecological and genetic structure of a mixed population of sexual diploid and apomictic triploid dandelions

Ecological differentiation is widely seen as an important factor enabling the stable coexistence of closely related plants of different ploidy levels. We studied ecological and genetic differentiation between co-occurring sexual diploid and apomictic triploid Taraxacum section Ruderalia by analysing spatial patterns both in the distribution of cytotypes and in the distribution of genetic variation within and between the cytotypes. A significant relationship between ploidy level and elevation was found. This mode of ecological differentiation however, was not sufficient to explain the significant spatial structure in the distribution of diploids and triploids within the population. Strong congruence was found between the spatial genetic patterns within the diploids and within the triploids. We argue that this congruence is an indication of gene flow between neighbouring plants of different ploidy levels.     

Amplified fragment length polymorphism (AFLP) reveals introgression in weedy Onopordum thistles: hybridization and invasion

Onopordum L. (Compositae) is an extremely diverse genus of thistles, which includes several species that have become serious pasture weeds in several regions of the world. We present a comparison of the genetic diversity in invasive forms of Onopordum from Australia with several known native European species. A total of 108 polymorphic genetic markers was generated using amplified fragment length polymorphism (AFLP) fingerprinting. Non-metric multidimensional scaling (NMDS) revealed that Australia contained O. acanthium, O. illyricum and a full range of genetic intermediates between these species. Intermediates largely comprised segregating fragments diagnostic for European O. acanthium and O. illyricum with a low frequency of fragments that were diagnostic for other species never recorded in Australia. The current genetic patterns in Australia may be best explained by a combination of processes, both in the native and in the alien range. These include multiple introductions of seed, including hybrid material, and the continuous dispersal in Australia, leading to an increase in the contact among hybridizing taxa. Such processes appear to have produced more widespread hybridization and introgression in Australian Onopordum than is found in Europe.     

Amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes

An agarose gel based single enzyme AFLP method using EcoR1 digestion of Listeria monocytogenes DNA was developed for epidemiological typing. The method was evaluated with 84 L. monocytogenes cultures, and results were compared with those obtained with serotyping, phage-typing and cadmium and arsenic resistance typing. The AFLP method was reproducible and 14 different banding patterns comprising between five and eight DNA fragments were produced. All except two of the AFLP patterns were serorype specific. Different AFLP patterns were recognised within serovar 4b (four patterns), 1/2a (two patterns), 1/2b (six patterns): single patterns were obtained from cultures of serovars 1/2c, 3a, 3b and 3c. There were associations with AFLP results and those from phage-typing and cadmium and arsenic resistance typing, although each method showed some independence. This preliminary evaluation suggests that this AFLP method will be useful for epidemiological typing of L. monocytogenes.     

Amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes

AFLP analysis using four selective primers was performed on a set of 33 Listeria monocytogenes including strains from patients and foods implicated in outbreaks, human sporadic cases or foods. Strains were tested belonging to serovars 1/2a, 1/2b, 1/2c, 3b, and 4b. Using one of the primers, the AFLP technique generated 20 different sized DNA fragments. The 33 cultures segregated into 14 different patterns, each comprising 7-12 different fragments. Although the method was not sufficiently discriminatory for epidemiological typing, AFLP analysis reconfirmed the observation that L. monocytogenes comprises two major genetic groups: group 1 includes strains of serovars 1/2a and 1/2c, while group 2 serovars 1/2b, 3b and 4b.     

Development and characterization of nine new microsatellite markers in Taraxacum (Asteraceae)

This study aims at developing and characterizing new microsatellite primer pairs in Taraxacum officinale auct. to produce polymorphic markers for genetical and evolutionary studies on apomixis in this sexual‐apomictic complex. A total of 24 diploid plants were tested for allelic polymorphism and heterozygosity. Out of nine loci three deviated significantly from the Hardy–Weinberg equilibrium expectations, probably due to the presence of null‐alleles. Successful cross‐species amplification was obtained for all markers in 29 Taraxacum microspecies from five sections.     

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.     

Amplified fragment length polymorphism (AFLP) fingerprinting of symbiotic fungi cultured by the fungus-growing ant Cyphomyrmex minutus

A PCR-based fingerprinting technique based on amplified fragment length polymorphisms (AFLP) is used to screen symbiotic fungi of the fungus-growing ant Cyphomyrmex minutus for genetic differences. AFLP fingerprints reveal several fungal ‘types’ that (a) represent distinct clones propagated vegetatively by the ant, or (b) correspond to free-living fungi that may be acquired by the ant. Fungal types identified by AFLP fingerprints correspond to vegetative-compatibility groups established previously, suggesting that vegetative compatibility can be used as a crude indicator of genetic differences between fungi of C. minutus.     

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.     

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.     

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.     

Amplified fragment length polymorphism analysis of the population structure and genetic diversity of Phoebe zhennan (Lauraceae), a native species to China

Phoebe zhennan S. Lee et F. N. Wei (Lauraceae), is the main source of Gold Phoebe, a rare and extremely valuable wood in China. However it has undergone a dramatic decline. In this study, we used 12 amplified fragment length polymorphism primer combinations to assay 92 accessions, which were highly representative of the entire P. zhennan germplasm. It revealed that P. zhennan consisted of three genetic populations, named as SCZ (central Sichuan), CQH (eastern Sichuan, Chongqin, Hubei and Hunan) and YG (Yunnan and Guizhou), probably owing to natural selection caused by topography differences. The CQH population further diverged into two geographical sub-populations: CD-CQ (SCD and west region of Chongqin) and HB-HN (eastern side of Chongqin, Hubei and Hunan). The loci were moderately polymorphic (40.4%). The genetic distance between SCZ and YG was the highest, between CD-CQ and HB-HN the lowest. Pairwise fixation indices (Ф_PT) between any inferred populations were significant. This rare species exhibited low genetic diversity; therefore, the results provided significant data related to the conservation and management of P. zhennan. That is, with this genetic information, land managers are equipped with better tools allowing them to more effectively protect this species and its limited genetic diversity.     

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.     

Amplified fragment length polymorphisms (AFLP) reveal details of polyploid evolution in Dactylorhiza (Orchidaceae)

The utility of the PCR-based AFLP technique (polymerase chain reaction; amplified fragment length polymorphisms) was explored in elucidating details of polyploid evolution in the Eurasian orchid genus Dactylorhiza. We emphasized Swedish taxa but also included some material from the British Isles and elsewhere in Europe. Three different sets of primers, amplifying different subsets of restriction fragments, independently revealed similar patterns for relationships among the Dactylorhiza samples investigated. The AFLP data support the general picture of polyploid evolution in Dactylorhiza, i.e., that allotetraploid derivatives have arisen repeatedly as a result of hybridization beween the two parental groups D. incarnata s.l. (sensu lato; diploid marsh orchids) and the D. maculata group (spotted orchids). Within the incarnata s.l. group, morphologically defined varieties were interdigitated. The D. maculata group consisted of two distinct subgroups, one containing autotetraploid D. maculata subsp. maculata and the other containing diploid D. maculata subsp. fuchsii. Allotetraploids showed a high degree of additivity for the putative parental genomes, and relationships among them were partly correlated to morphologically based entities, but also to geographic distribution. Thus, allotetraploid taxa from the British Isles clustered together, rather than with morphologically similar plants from other areas.     

Amplified fragment length polymorphism (AFLP) suggests old and recent immigration into the Alps by the arctic-alpine annual Comastoma tenellum (Gentianaceae)

Aim This study aims to elucidate the phylogeography of the arctic‐alpine annual Comastoma tenellum (Rottb.) Toyok. (Gentianaceae) and to unravel the history of its immigration into the Alps. Location Although samples from Alaska and Central Asia were also included, our study focusses on Europe, especially on the Alps. Methods We applied amplified fragment length polymorphism (AFLP) fingerprinting on 37 populations (162 individuals) of C. tenellum and analysed the results phenetically. Results As C. tenellum is mainly inbreeding, there is typically little to no intrapopulational genetic variation. Two populations from Alaska and Altai are strongly separated from all other accessions. The majority of the populations from the Alps group together with high bootstrap support. They fall into an unsupported Alps I group (northwards of Gran Paradiso) and a well‐supported Alps II group (south‐western Alps). The remaining European populations form a weakly‐supported branch constituting accessions from the Carpathians, Scandinavia and two populations from the Eastern Alps. Main conclusions Comastoma tenellum reached the Alps at least twice. The first immigration event resulted in a lineage that is clearly separated from the other European accessions. The immigration must have occurred well before the last glaciation because this lineage shows further phylogeographical structuring into two groups (Alps II in the south‐western Alps and Alps I in the rest of the Alps). This pattern is presumably due to isolation in different glacial refugia. In addition to the old immigration event, the species reached the Alps in recent times either from Scandinavia or from the Carpathians via long‐distance dispersal. These immigrations resulted in (at least) two populations that are spatially small and poor in individuals.     

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。