Using Amplified Fragment Length Polymorphisms (AFLP) to Study Genetic Variability in Several Freshwater Rotifer Species

We have used amplified fragment length polymorphisms (AFLP) to investigate the potential of this technique as a tool to measure genetic variability in eight species of freshwater rotifers: Brachionus calyciflorus, Lecane bulla, L. luna, L. quadridentata, Plationus patulus, Philodina acuticornis odiosa, Rotaria neptunia, and R. rotatoria. We used nine combinations of oligonucleotides. We observed a total of 806 amplified bands, 798 polymorphic and 8 monomorphic. The data were analyzed using cluster analysis with UPGMA, first within each set of oligonucleotide combination and finally using all nine combinations. Our best dendrogram clearly separated monogononts from digononts, and grouped the species of monogononts in the two genera. However, it grouped R. neptunia with P. acuticornis odiosa rather than with R. rotatoria. These results are discussed in view of recent works in the literature measuring genetic variability and discussing the phylogeny of the Rotifera.     

AFLP分子标记技术的改进

对AFLP技术的限制性内切酶组合进行了改进。以两个低频酶组合代替传统的高频酶与低频酶组合进行分子标记筛选,结果表明,采用低频酶组合(EcoRⅠ PstⅠ)产生的条带比传统酶切组合(EcoRⅠ MseⅠ和PstⅠ MseⅠ)要少且分布不均匀,多集中在150~800bp之间,条带信号强度要大,多态性明显得到提高,分子标记筛选成功率也得到了提高,而且其成本更低的特点有利于AFLP技术在我国的进一步推广普及。     

Molecular taxonomy of Vicia ser. Vicia based on Amplified Fragment Length Polymorphisms

 Amplified Fragment Length Polymorphisms (AFLPs) were used to study the taxonomic relationships in Vicia ser. Vicia. 109 accessions representing the taxa and distribution in the series were evaluated using two primer combinations in an attempt to clarify its taxonomy. The study concludes that four species exist in the series. Vicia incisa should be considered as a separate taxon and does not belong to the Vicia sativa aggregate. Within the series Vicia pyrenaica appears to be the most closely related to the Vicia sativa aggregate. Within the aggregate 6 taxa can be recognised. They are very closely related and for none of the taxa in the aggregate is there a taxon-specific absence or presence of AFLP bands. We consider these taxa to be subspecies. Several accessions belonging to the aggregate were found which could not be placed within one of the taxa and are most likely the products of mutations or hybridisation between taxa. Received March 12, 2001 Accepted July 12, 2001     

Genetic diversity of Dioscorea dumetorum (Kunth) Pax using Amplified Fragment Length Polymorphisms (AFLP) and cpDNA

We have utilized Amplified Fragment Length Polymorphisms (AFLP) in conjunction with chloroplast DNA (cpDNA) sequence data to study the genetic diversity in 53 accessions of Dioscorea dumetorum from six countries in West and Central Africa. Our results provide a comparison of the two marker systems with regards to their applicability to differentiate intraspecific genotypes and the grouping of the accessions based on localities of collection. A total of 1052 AFLP fragments (of which 94.1% were polymorphic) produced from twelve primer combinations indicate a relatively high level of polymorphism among the accessions. Three major genetic groups that do not strictly follow a geographic distribution pattern were identified using Neighbour-joining and the principal coordinate (PCo) analyses. Accessions from Togo showed higher numbers of private fragments and the highest percentage polymorphism (59.4%). The detection of highest genetic diversity in accessions from Nigeria and Togo and their relationship to other accessions suggest that these countries are the centre of origin and diversity of D. dumetorum. The moderately high genetic diversity (average of 61%) is suggesting great influence on the D. dumetorum germplasms through exchange and transfer of cultivars among local farmers in the sub-region. In contrast, DNA sequence data from the psbA-trnH and the rpoB-trnC chloroplast regions revealed no variation among accessions from the different localities and clearly differentiated by AFLP patterns. The results demonstrate the usefulness of the AFLP marker in generating high polymorphism in the D. dumetorum accessions from West and Central Africa and hence may be used for agronomic purposes.     

Utility of Amplified Fragment Length Polymorphisms (AFLP) to analyse genetic structures within the Alexandrium tamarense species complex

Phylogenetic analyses of the Alexandrium tamarense species complex using ribosomal RNA sequences show a differentiation of ribotypes/clades into geographic areas and not into the three morphotypes/species A. tamarense, A. fundyense and A. catenella. Different parts of the rRNA operon have proven informative in revealing the existence and the relationships of these geographic clades, whereas even internal transcribed spacer (ITS) regions lack the resolution required to gain a deeper insight into the population structure of the species complex. Here, the utility of the DNA fingerprinting technique Amplified Fragment Length Polymorphism (AFLP) as a possible tool for such purposes was tested. A mixed sampling strategy was used in order to assess the amount of variation of AFLP banding patterns at the level of populations and geographic clades. We also describe optimized methods to achieve a good reproducibility. Our results suggest that AFLPs can provide useful information at the population level using clonal samples from a certain bloom, whereas the amount of variation that we found is too high to allow for meaningful comparisons of a few strains collected from different localities at different time points even though they belong to one geographic clade.     

Amplified Fragment Length Polymorphism (AFLP) as a source of genetic markers for red algae

A recent PCR-based fingerprinting technique, amplified fragment length polymorphism (AFLP), was successfully applied to the red alga Chondrus crispus Stackh. This is apparently the first account to describe the application of AFLP methodology to an alga. Six isolates of C. crispus were analyzed by AFLP. A total of twenty-five primer pairs were screened and six primer pairs were selected for further investigation. Both conservative and variable markers were identified within and between populations; some markers were unique to individuals. As such, AFLP should prove useful as a source of genetic markers in algae for applications as diverse as genome mapping to population genetic investigations. This revised version was published online in June 2006 with corrections to the Cover Date.     

用扩增片段的长度多态性分子标记探讨盾叶薯蓣的遗传多样性

用扩增片段的长度多态性(amplified fragment length polymorphism,AFLP)标记分析研究了中国5个盾叶薯蓣居群30个个体的遗传多样性。筛选出9对AFLP引物,从中检测到14698条清晰可见的条带,其中多态性带12628条,多态性比率85.92%。Shannon信息指数(I)为0.3656±0.1721,物种水平的Nei基因多样性(H)为0.2322±0.2200。遗传变异分析表明,物种水平的遗传分化系数Gst为0.4827,说明其群体间存在一定的遗传分化,居群间的基因流Nm为0.5358,居群间遗传交换较小。聚类分析结果显示5个居群盾叶薯蓣有较为丰富的遗传变异,且与地理分布有相关性。     

Comparative analysis of genetic diversity in the mangrove species Avicennia marina (Forsk.) Vierh. (Avicenniaceae) detected by AFLPs and SSRs

Avicennia marina is an important mangrove species with a wide geographical and climatic distribution which suggests that large amounts of genetic diversity are available for conservation and breeding programs. In this study we compare the informativeness of AFLPs and SSRs for assessing genetic diversity within and among individuals, populations and subspecies of A. marina in Australia. Our comparison utilized three SSR loci and three AFLP primer sets that were known to be polymorphic, and could be run in a single analysis on a capillary electrophoresis system, using different- colored fluorescent dyes. A total of 120 individuals representing six populations and three subspecies were sampled. At the locus level, SSRs were considerably more variable than AFLPs, with a total of 52 alleles and an average heterozygosity of 0.78. Average heterozygosity for AFLPs was 0.193, but all of the 918 bands scored were polymorphic. Thus, AFLPs were considerably more efficient at revealing polymorphic loci than SSRs despite lower average heterozygosities. SSRs detected more genetic differentiation between populations (19 vs 9%) and subspecies (35 vs 11%) than AFLPs. Principal co-ordinate analysis revealed congruent patterns of genetic relationships at the individual, population and subspecific levels for both data sets. Mantel testing confirmed congruence between AFLP and SSR genetic distances among, but not within, population comparisons, indicating that the markers were segregating independently but that evolutionary groups (populations and subspecies) were similar. Three genetic criteria of importance for defining priorities for ex situ collections or in situ conservation programs (number of alleles, number of locally common alleles and number of private alleles) were correlated between the AFLP and SSR data sets. The congruence between AFLP and SSR data sets suggest that either method, or a combination, is applicable to expanded genetic studies of mangroves. The codominant nature of SSRs makes them ideal for further population-based investigations, such as mating-system analyses, for which the dominant AFLP markers are less well suited. AFLPs may be particularly useful for monitoring propagation programs and identifying duplicates within collections, since a single PCR assay can reveal many loci at once. Received: 3 October 2000 / Accepted: 19 February 2001     

Identification and Mapping of Amplified Fragment Length Polymorphism Markers Linked to Shell Color in Bay Scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F₁ families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.     

AFLPs support deep relationships among darters (Percidae: Etheostomatinae) consistent with morphological hypotheses

Recent attention has focused on the efficacy of amplified fragment length polymorphisms (AFLPs) for resolving deep evolutionary relationships. Here we show that AFLPs provide resolution of deep relationships within the family Percidae that are more consistent with previous morphological hypotheses than are relationships proposed by previous molecular analyses. Despite in silico predictions, we were able to resolve relatively ancient divergences, estimated at >25 MA. We show that the most distantly related species share the fewest fragments, but suggest that large data sets and extensive taxon sampling are sufficient to overcome this obstacle of the AFLP technique for deep divergences. We compare genetic distances estimated from mitochondrial DNA with those from AFLPs and contrast traditional PAUP^* Nei–Li AFLP genetic distances with a recently proposed method utilizing the Dice equation with constraining nucleotides.     

Amplified Fragment Length Polymorphism (AFLP) reveals no genetic divergence of the Eastern Alpine endemic Oxytropis campestris subsp. tiroliensis (Fabaceae) from widespread subsp. campestris

Applying Amplified Fragment Length Polymorphism, we explored genetic differences between widespread Oxytropis campestris subsp. campestris and O. campestris subsp. tiroliensis, a presumed glacial relict restricted to a small area along the main chain of the Eastern Alps. We could not find genetic differences between the two taxa. Neither do the morphological characters given in the literature discriminate between them. Therefore Oxytropis campestris subsp. tiroliensis is unlikely a glacial relict that survived Pleistocene glaciations on nunataks, but rather a genetically insignificantly differentiated phenotype that arose in the course of postglacial recolonisation. There is no phylogeographical structure in O. campestris s.l. in the Alps most probably due to the fact that the taxon did not survive the cold stages of the Pleistocene in the interior of the Alps but immigrated to that region at a later date.     

Genetic variation in nine Shorea species (Dipterocarpaceae) in Indonesia revealed by AFLPs

Shorea is the largest and most important genus of the Dipterocarpaceae. The genetic diversity and structure of nine Shorea species from two different locations, namely Nanjak Makmur in Sumatra and Sumalindo in Borneo, were evaluated using amplified fragment length polymorphism (AFLP) markers. A total of 274 trees were investigated at 85 polymorphic AFLP loci. Levels of genetic diversity of these species ranged from  = 0.100 for S. acuminata to  = 0.165 for S. blumutensis. The population of rare species S. blumutensis possessed the highest genetic diversity suggesting that geographically restricted species can have levels of genetic variation comparable to closely related widespread common congeners. Analyses of molecular variance revealed that the genetic variation was mainly found among species in both locations (57.7% in Sumatra; 56.3% in Borneo). The unweighted pairgroup method using arithmetic averages dendrogram of all samples revealed an almost complete separation of species. Thus, AFLP markers proved appropriate for phylogenetic studies of Shorea species. Specific markers have been detected showing high-frequency differences among species and between regions within species. Sequence information of these markers can be used to develop specific polymerase chain reaction markers for wood identification. The possibility of interspecific hybridization was discussed.     

Establishment of the Amplified Fragment Length Polymorphism (AFLP) technique for genotyping of pollen beetle (Meligethes aeneus) - a noxious insect pest on oilseed rape (Brassica napus)

In order to conduct studies concerning genetic variability of pollen beetles (Meligethes aeneus), a genotyping protocol was established. No genome information is available for pollen beetles so the amplified fragment length polymorphism (AFLP) technique was chosen since it does not depend on any prior sequence information of the samples and also is a sensitive and robust technique. However, several modifications were needed in order to adapt the method for analysis of pollen beetles. Basic modifications included (i) alterations of DNA purification, (ii) use of two six-cutter restriction enzymes, (iii) and modified PCR conditions. This protocol resulted in a favourable number of fragments of an appropriate size range for standard gel analysis by a DNA sequencer applicable to a single insect and even body parts enabling different assays to be conducted on a single specimen. Pollen beetles from different areas of Sweden were analysed to verify the reproducibility and efficacy of the protocol as well as for phenetic analysis. The high reproducibility of the modified AFLP protocol allows it to be used as a reliable tool for genotype analysis of pollen beetles.     

Archaeal Diversity in Deep-Sea Sediments Estimated by Means of Different Terminal-Restriction Fragment Length Polymorphisms (T-RFLP) Protocols

Despite the increasing recognition of the quantitative importance of Archaea in all marine systems, the protocols for a rapid estimate of Archaeal diversity patterns in deep-sea sediments have been only poorly tested yet. Sediment samples from 11 deep-sea sites (from 79°N to 36°N, at depths comprised from 469 to 5,571 m) were used to compare the performance of two different primer sets (ARCH21f/ARCH958r and ARCH109f/ARCH 915r) and three restriction enzymes (AluI, Rsa I, and HaeIII) for the fingerprinting analysis of Archaeal diversity using terminal-restriction fragment length polymorphisms (T-RFLP). In silico and experimental analyses indicated that different combinations of primer sets and restriction enzymes provided different values of benthic Archaeal ribotype richness and different Archaeal assemblage compositions. The use of the ARCH109f/ARCH 915r primer set in combination with AluI provided the best results (a number of ribotypes up to four folds higher than other combinations), suggesting that this primer set should be used in future studies dealing with the analysis of the patterns of Archaeal diversity in deep-sea sediments. Multivariate multiple regression analysis revealed that, whatever the T-RFLP protocol utilized, latitude and temperature explained most of the variance in benthic Archaeal ribotype richness, while water depth had a negligible role.     

Polymerase Chain Reaction-Restriction Fragment Length Polymorphisms (PCR-RFLP) and Electrophoretic Assays for the Mouse Obese (Lepob) Mutation

Three polymerase chain reaction-based assays for the mouse Lep^ob mutation are described: Dde I site created by the C←T transversion characterizing Lep^ob enables positive detection of the mutant allele; positive detection of the wild-type Lep allele is achieved by the use of primer sequence which introduces an A←C substitution, creating an Msp I site in the normal allele; and an electrophoretic assay which positively identifies the hetero-zygote.     

Intergeneric and Interspecific Relationships in Araceae tribe Caladieae and Development of Molecular Markers using Amplified Fragment Length Polymorphism (AFLP)

Since amplified fragment length polymorphism (AFLP) analysishas proved useful in distinguishing cultivars of Caladium, itwas used to assess the status of species of Caladium vs. Xanthosoma,both in tribe the Caladieae, and to reassess the position ofHapaline in the same tribe. AFLP analysis using three primercombinations was carried out on four species of Caladium(C.bicolor, C. humboldtii, C. lindenii and C. schomburgkii). Resultsshowed that AFLP can distinguish between the different speciesby their unique and different banding patterns. AFLP analysisconfirmed that C. humboldtii is a species distinct from C. bicolorand that C. lindenii is a true Caladium species and does notbelong to Xanthosoma. UPGMA cluster analysis showed that C.bicolor and C. schomburgkii are most similar and that C. humboldtiiis closer to the C. bicolor / C. schomburgkii cluster comparedwith C. lindenii. Genetic relationships between Caladium, Xanthosoma,Hapaline, Alocasia and Protarum were also examined by AFLP analysisusing eight primer combinations. Several useful molecular markerswere specific either to Caladium orXanthosoma , so that AFLPcan be used to distinguish species of these two genera. Geneticanalysis of the genera examined confirms that the Caladieaeand Colocasieae tribes are distinct and that Hapaline fallswithin the tribe Caladieae and that Protarum is most distantfrom all the genera examined. Copyright 2000 Annals of BotanyCompany Araceae, Caladium, Xanthosoma, Hapaline, Alocasia, Protarum, AFLP DNA fingerprinting, diversity, AFLP markers     

中国食用向日葵种质资源遗传变异的RAPD及AFLP分析

本研究采用RAPD和AFLP方法对23个中国不同地区的食用向日葵(Helianthus annuus L.)骨干品种进行了遗传变异分析,同时对两种标记系统进行了比较.26个RAPD引物产生了总计192条DNA条带,大小分布于0.26 kb～1.98 kb之间,其中165条(86.12%)具有多态性,每条引物产生DNA条带的平均数为7.38.8对AFLP引物组合共产生了576条带,分布于100 bp～500 bp之间,其中的341条具有多态性,多态百分率为76.00%,每对引物组合产生DNA条带的平均数为72.RAPD方法检测到的每位点有效等位基因数(1.76)大于AFLP(1.65),AFLP标记位点的平均多态性信息量(PIC)(0.38)低于RAPD标记位点的PIC(0.41),但AFLP标记具有很高的多态性检测效率(Ai=38.52).用RAPD标记分析23个食用向日葵材料的亲缘关系,Nei氏相似性系数分布在47.84%～82.06%,平均相似性系数为0.649 5, 而采用AFLP的Nei氏相似性系数分布在54.15%～83.52%,平均相似性系数为0.688 4.RAPD数据的标准差为0.13,而AFLP数据的标准差为0.08.因此,采用RAPD和AFLP方法分析食用向日葵遗传变异,RAPD标记具有较低相似性系数和较高方差而AFLP则相反.源于两种不同标记的遗传相似性矩阵的相关系数为0.51,说明采用RAPD和AFLP系统分析食用向日葵遗传变异得到的结果有一定的相关性.无论采用RAPD还是AFLP标记进行聚类分析,都将23个不同基因型的食用向日葵材料分成了三个类群.     

AFLPs are incompatible with RAPD and morphological data in Pennisetum purpureum (Napier grass)

Accessions of Pennisetum purpureum in agricultural research centres in sub-Saharan Africa were genotyped using Amplified Fragment Length Polymorphisms (AFLPs) using primer combinations MluI/MseI. Cluster analyses of resulting bands suggested that the samples could be grouped into five large and five small-sized clusters. Principal coordinate analysis revealed two major clusters being well separated, with bulk of the samples forming one, poorly structured cluster. Our analyses failed to reveal the regional groupings found in previous studies using a subset of our sample universe and based on RAPDs as well as morphological and agronomical data. The inability of the AFLP data to clearly demarcate accessions is ascribed to either the presence of high levels of genetic uniformity, or non-optimal primers and/or bad germplasm management practices. The re-registration of all accessions, based on DNA barcoding, is suggested as a means to resolve the lingering problems regarding the identity of accessions.     

Amplified Fragment Length Microsatellites (AFLM) might be used to develop microsatellite markers in organisms with limited amounts of DNA applied to Arbuscular Mycorrhizal (AM) fungi

Developing microsatellite markers for organisms with limited amounts of DNA can be difficult because sequence information is needed. To overcome this problem in the arbuscular mycorrhizal (AM) fungi Glomus etunicatum and Gigaspora gigantea, global amplification of the genomes of each species was performed with linker-adaptor-PCR from single spores. Amplified fragments were enriched for microsatellite motifs with 5'-biotinylated oligonucleotides and recovered by magnetic streptavidin beads. The recovered fragments were reamplified and separated on denaturing polyacrylamide gels, and 16 selected bands were excised, cloned and sequenced. Seven microsatellite motifs were detected from six clones (efficiency rate of 43.8%). Primers were designed for all putative microsatellite loci and most were successfully amplified from three single-spore preparations and from pools of five, 10 and 20 spores after global amplification. This approach, termed amplified fragment-length micosatellites (AFLM), might aid investigations of organisms that cannot or are not readily cultured in vitro and where DNA is a limiting factor for genetic studies. However, the technique also can be used to isolate microsatellite loci in any organism.     

Restriction Fragment Length Polymorphism (RFLP) for protein disulfide isomerase (PDI) gene sequences in Triticum and Aegilops species

RFLP variation revealed by protein disulfide isomerase (PDI) coding gene sequences was assessed in 170 accessions belonging to 23 species of Triticum and Aegilops. PDI restriction fragments were highly conserved within each species and confirmed that plant PDI is encoded either by single-copy sequences or by small gene families. The wheat PDI probe hybridized to single EcoRI or HindIII fragments in different diploid species and to one or two fragments per genome in polyploids. Four Aegilops species in the Sitopsis section showed complex patterns and high levels of intraspecific variation, whereas Ae. searsii possessed single monomorphic fragments. T. urartu and Ae. squarrosa showed fragments with the same mobility as those in the A and D genomes of Triticum polyploid species, respectively, whereas differences were observed between the hybridization patterns of T. monococcum and T. boeoticum and that of the A genome. The single fragment detected in Ae. squarrosa was also conserved in most accessions of polyploid Aegilops species carrying the D genome. The five species of the Sitopsis section showed variation for the PDI hybridization fragments and differed from those of the B and G genomes of emmer and timopheevi groups of wheat, although one of the Ae. speltoides EcoRI fragments was similar to those located on the 4B and 4G chromosomes. The similarity between the EcoRI fragment located on the 1B chromosome of common and emmer wheats and one with a lower hybridization intensity in Ae. longissima, Ae. bicornis and Ae. sharonensis support the hypothesis of a polyphyletic origin of the B genome. Received: 25 June 1999 / Accepted: 14 September 1999