Amplified Fragment Length Polymorphism Fragments Research Articles

Female‐only sex‐linked amplified fragment length polymorphism markers support ZW/ZZ sex determination in the giant freshwater prawn Macrobrachium rosenbergii

Sex determination mechanisms in many crustacean species are complex and poorly documented. In the giant freshwater prawn, Macrobrachium rosenbergii, a ZW/ZZ sex determination system was previously proposed based on sex ratio data obtained by crosses of sex-reversed females (neomales). To provide molecular evidence for the proposed system, novel sex-linked molecular markers were isolated in this species. Amplified fragment length polymorphism (AFLP) using 64 primer combinations was employed to screen prawn genomes for DNA markers linked with sex loci. Approximately 8400 legible fragments were produced, 13 of which were uniquely identified in female prawns with no indication of corresponding male-specific markers. These AFLP fragments were reamplified, cloned and sequenced, producing two reliable female-specific sequence characterized amplified region (SCAR) markers. Additional individuals from two unrelated geographic populations were used to verify these findings, confirming female-specific amplification of single bands. Detection of internal polymorphic sites was conducted by designing new primer pairs based on these internal fragments. The internal SCAR fragments also displayed specificity in females, indicating high levels of variation between female and male specimens. The distinctive feature of female-linked SCAR markers can be applied for rapid detection of prawn gender. These sex-specific SCAR markers and sex-associated AFLP candidates unique to female specimens support a sex determination system consistent with female heterogamety (ZW) and male homogamety (ZZ).

Identification of Amplified Fragment Length Polymorphism (AFLP) Markers Tightly Associated with Drought Stress Gene in Male Sterile and Fertile Salvia miltiorrhiza Bunge.

Consistent grain yield in drought environment has attracted wide attention due to global climate change. However, the important drought-related traits/genes in crops have been rarely reported. Many near-isogenic lines (NILs) of male sterile and fertile Salvia miltiorrhiza have been obtained in our previous work through testcross and backcross in continuous field experiments conducted in 2006–2009. Both segregating sterile and fertile populations were subjected to bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) with 384 and 170 primer combinations, respectively. One out of 14 AFLP markers (E9/M3246) was identified in treated fertile population as tightly linked to the drought stress gene with a recombination frequency of 6.98% and at a distance of 7.02 cM. One of 15 other markers (E2/M5357) was identified in a treated sterile population that is closely associated with the drought stress gene. It had a recombination frequency of 4.65% and at a distance of 4.66 cM. Interestingly, the E9/M3246 fragment was found to be identical to another AFLP fragment E11/M4208 that was tightly linked to the male sterile gene of S. miltiorrhiza with 95% identity and e-value 4 × 10−93. Blastn analysis suggested that the drought stress gene sequence showed higher identity with nucleotides in Arabidopsis chromosome 1–5.

Genomic distribution of AFLP markers relative to gene locations for different eukaryotic species

BackgroundAmplified fragment length polymorphism (AFLP) markers are frequently used for a wide range of studies, such as genome-wide mapping, population genetic diversity estimation, hybridization and introgression studies, phylogenetic analyses, and detection of signatures of selection. An important issue to be addressed for some of these fields is the distribution of the markers across the genome, particularly in relation to gene sequences.ResultsUsing in-silico restriction fragment analysis of the genomes of nine eukaryotic species we characterise the distribution of AFLP fragments across the genome and, particularly, in relation to gene locations. First, we identify the physical position of markers across the chromosomes of all species. An observed accumulation of fragments around (peri) centromeric regions in some species is produced by repeated sequences, and this accumulation disappears when AFLP bands rather than fragments are considered. Second, we calculate the percentage of AFLP markers positioned within gene sequences. For the typical EcoRI/MseI enzyme pair, this ranges between 28 and 87% and is usually larger than that expected by chance because of the higher GC content of gene sequences relative to intergenic ones. In agreement with this, the use of enzyme pairs with GC-rich restriction sites substantially increases the above percentages. For example, using the enzyme system SacI/HpaII, 86% of AFLP markers are located within gene sequences in A. thaliana, and 100% of markers in Plasmodium falciparun. We further find that for a typical trait controlled by 50 genes of average size, if 1000 AFLPs are used in a study, the number of those within 1 kb distance from any of the genes would be only about 1–2, and only about 50% of the genes would have markers within that distance.ConclusionsThe high coverage of AFLP markers across the genomes and the high proportion of markers within or close to gene sequences make them suitable for genome scans and detecting large islands of differentiation in the genome. However, for specific traits, the percentage of AFLP markers close to genes can be rather small. Therefore, genome scans directed towards the search of markers closely linked to selected loci can be a difficult task in many instances.

Identification of two SCAR markers co-segregated with the dominant Ms and recessive ms alleles in onion (Allium cepa L.)

Cytoplasmic genetic male-sterility is used to produce hybrid onion (Allium cepa L.) seeds worldwide. In this paper, we present the results of research aimed toward identifying PCR-based markers linked to the Ms locus through amplified fragment length polymorphism (AFLP). After screening 512 AFLP primer combinations, only one AFLP fragment was identified as being flanking linked to the dominant Ms allele. Subsequently, the AFLP marker was converted into a sequence-characterized amplified region (SCAR) marker, designated as DNF-566, co-segregated with the dominant Ms allele in first backcross (BC1) segregated populations. Furthermore, we designed another molecular marker (RNS-357) co-segregated with the ms allele to identify different genotypes (i.e., MsMs, Msms, or msms). Both markers could be used for evaluating onion lines with different genetic backgrounds (including male-sterile lines, maintainer lines, male-fertile lines, and commercial based F1 hybrid cultivars). The results of this study indicate that maintainer plants could be directly selected by using these 2 SCAR markers in the onion breeding process, and this may contribute significantly toward breeding onion F1 hybrid cultivars.

Influence of parameter settings in automated scoring of AFLPs on population genetic analysis

The use of procedures for the automated scoring of amplified fragment length polymorphisms (AFLP) fragments has recently increased. Corresponding software does not only automatically score the presence or absence of AFLP fragments, but also allows an evaluation of how different settings of scoring parameters influence subsequent population genetic analyses. In this study, we used the automated scoring package rawgeno to evaluate how five scoring parameters influence the number of polymorphic bins and estimates of pairwise genetic differentiation between populations (F(st)). Steps were implemented in r to automatically run the scoring process in rawgeno for a set of different parameter combinations. While we found the scoring parameters minimum bin width and minimum number of samples per bin to have only weak influence on pairwise F(st) values, maximum bin width and bin reproducibility had much stronger effects. The minimum average bin fluorescence scoring parameter affected F(st) values in an only moderate way. At a range of scoring parameters around the default settings of rawgeno, the number of polymorphic bins as well as pairwise F(st) values stayed rather constant. This study thus shows the particularities of AFLP scoring, be it either manual or automatical, can have profound effects on subsequent population genetic analysis.

Assessment of genetic relationships among Spring Dendrobium cultivars and varietal materials using amplified fragment length polymorphism (AFLP) analysis

Spring Dendrobiums have been largely produced as one of the top grade ornamental pot plants due to their various flower colors, multitudinous flowers and graceful flower shape as well as their long period of florescence. Genetic relationships among Spring Dendrobium cultivars, however, have not been documented. This study analyzed the genetic relatedness of 30 commonly grown cultivars or varietal materials using amplified fragment length polymorphism (AFLP) markers with near-infrared fluorescence-labeled primers. Eight EcoRI + 3 bases/MseI + 3 bases primer set combinations were used in this investigation. Each selected primer set generated 113 to 158 scorable fragments. A total of 1102 AFLP fragments were detected, of which 778 were polymorphic (70.6%). An unweighted pair-group method of the arithmetic averages (UPGMA), principal coordinate analysis (PCOA), and bootstrap analysis were used to analyze the genetic relationships. The 30 cultivars were separated into five clusters. Cluster I contains 6 cultivars that are either from Senlan No.1 or Senlan No. 6 with Jaccardfs similarity coefficients ranging from 0.70 to 0.80. All of these 6 cultivars came from Taiwan, and were derived from somaclonal variants or sports. Just 3 cultivars were positioned in cluster II ranging from 0.71 to 0.76, and also originated from Taiwan. Cluster III included 13 cultivars, Jaccardfs similarity coefficients varied from 0.69 to 0.84. Seven cultivars from Senlan No. 15 or eSnowboy Romancef were situated in cluster IV with Jaccardfs similarity coefficients ranging from 0.69 to 0.82. Only eSantana Canaryf was positioned in cluster ‡X with Jaccardfs similarity coefficient at 0.62. This study established the genetic relationships of these commonly cultivated Spring Dendrobiums, and raised a concern over genetic vulnerability of cultivars in this study because of their close genetic similarities. Key words : Spring Dendrobium , amplified fragment length polymorphism (AFLP), genetic relationship, cultivars and varietal materials.

Isolation and Characterization of Male-Specific DNA Markers in the Rock Bream Oplegnathus fasciatus

Sex-specific DNA markers applicable were very useful for elucidating the sex-determination mechanism and sex control in fishes. In the present study, amplified fragment-length polymorphism (AFLP) approach with 144 primer combinations was employed to identify sex-specific markers in the rock bream. Four male-specific AFLP fragments were identified which were designated as Opl286, Opl237, Opl422, and Opl228. Further sequence analysis of the sex markers' genomic region revealed subtle differences between the males and females. We identified four male-specific single-nucleotide polymorphisms (SNPs) and a deletion of 8bp in marker Opl286, six male-specific SNPs in marker Opl237, three male-specific SNPs in marker Opl422, and eight male-specific SNPs and 1bp inversions in marker Opl228. Specific primers were designed based on the nucleotide variation in the sequences to develop a simple polymerase chain reaction method for identifying the genetic sex of rock bream. As a result, three out of the four male-specific markers were converted into SNP markers. The male-specific AFLP markers and AFLP-derived SNP markers were tested in 100 individuals collected from three locations around the coast of Zhoushan, yielding reproducible sex identification. These male-specific DNA markers are a useful tool for the identification of the sex-determining locus in rock bream and for guiding artificial breeding programs.

Identification of sugar apple cultivars Nua and Nang using AFLP and SCAR markers

Amplified fragment length polymorphism (AFLP) technique was used to identify sugar apple ( Annona squamosa L.) cultivars Nua and Nang. The total of 64 Eco RI- Mse I primer-pair combinations were screened and all of them could produce DNA fingerprinting. Eight AFLP markers were clearly polymorphic among the 2 cultivars which were capable to distinguish the sugar apple varieties between Nua and Nang. Four polymorphic AFLP fragments were subsequently cloned and sequenced for sequence characterized amplified region (SCAR) primer design. The primers were tested with individual DNA samples and the result showed the polymorphisms that were identified from the SCAR markers with specific to only sugar apple variety Nua.

Molecular markers linked to resistance to the gall mite in blackcurrant

With 1 figure and 3 tables AbstractGall mite (Cecidophyopsis ribis Westw.) is one of the most damaging pests of blackcurrants (Ribes nigrum L.) and is a vector of blackcurrant reversion virus. Ce and P genes provide resistance of blackcurrant to gall mite. A linkage map around the resistance locus controlled by predicted P gene was constructed. Forty‐three amplified fragment length polymorphism (AFLP) and 19 microsatellite polymorphic markers obtained from analysis of the progeny obtained in cross with P gene donor ‘Dainiai’ were mapped. The obtained consensus map covers 691.196 cM, with an average marker spacing of 14.706 cM. AFLP fragment CTA‐ACC‐107 was closely linked to resistance to blackcurrant gall mite and was detected in the sixth linkage group. Screening of cultivars and hybrids with known resistance to gall mite confirmed that this dominant 107‐bp AFLP marker is linked to gall mite resistance in a comprehensive range of available Ribes germplasm with different genetic background and it may be used for early diagnosis of resistant to gall mite hybrids.

Identification of a male-specific amplified fragment length polymorphism (AFLP) marker in Broussonetia papyrifera

The present study exhibits amplified fragment length polymorphism (AFLP) molecular marker for sex identification in Broussonetia papyrifera . Based on nine selective amplification primer combinations, 230 bands were produced and the E -AGG/M-CAA combination was found to be a male-specific AFLP marker. Subsequently, this male-specific AFLP fragment was sequenced and converted into a sequence tagged site (STS) marker. Based on STS sequence, two primers, MADB-1 and MADB-2 (Male-Associated DNA from B. papyrifera ), were designed to verify the specificity of the fragment. The results indicate that common homology sequence is existed in both male and female plants while one of the bands amplified via MADB-2 primer was solely present in male individuals at high annealing temperature up to 66°C. Finally, MADB-2 primer was introduced to amplify another 16 plants and it revealed that this primer could be used as a convenient, efficient, reliable, and low-cost molecular marker for sex identification in B. papyrifera . Keywords: Broussonetia papyrifera , AFLP, STS, male-specific marker

Construction of a high-resolution linkage map of Rfd1, a restorer-of-fertility locus for cytoplasmic male sterility conferred by DCGMS cytoplasm in radish (Raphanus sativus L.) using synteny between radish and Arabidopsis genomes

Cytoplasmic male sterility caused by Dongbu cytoplasmic and genic male-sterility (DCGMS) cytoplasm and its nuclear restorer-of-fertility locus (Rfd1) with a linked molecular marker (A137) have been reported in radish (Raphanus sativus L.). To construct a linkage map of the Rfd1 locus, linked amplified fragment length polymorphism (AFLP) markers were screened using bulked segregant analysis. A 220-bp linked AFLP fragment sequence from radish showed homology with an Arabidopsis coding sequence. Using this Arabidopsis gene sequence, a simple PCR marker (A220) was developed. The A137 and A220 markers flanked the Rfd1 locus. Two homologous Arabidopsis genes with both marker sequences were positioned on Arabidopsis chromosome-3 with an interval of 2.4 Mb. To integrate the Rfd1 locus into a previously reported expressed sequence tag (EST)-simple sequence repeat (SSR) linkage map, the radish EST sequences located in three syntenic blocks within the 2.4-Mb interval were used to develop single nucleotide polymorphism (SNP) markers for tagging each block. The SNP marker in linkage group-2 co-segregated with male fertility in an F(2) population. Using radish ESTs positioned in linkage group-2, five intron length polymorphism (ILP) markers and one cleaved amplified polymorphic sequence (CAPS) marker were developed and used to construct a linkage map of the Rfd1 locus. Two closely linked markers delimited the Rfd1 locus within a 985-kb interval of Arabidopsis chromosome-3. Synteny between the radish and Arabidopsis genomes in the 985-kb interval were used to develop three ILP and three CAPS markers. Two ILP markers further delimited the Rfd1 locus to a 220-kb interval of Arabidopsis chromosome-3.

Genetic Linkage Map and Diversity Analysis of Tahitian Vanilla (Vanilla ×tahitensis, Orchidaceae)

ABSTRACTThe first vanilla genetic linkage map was constructed by analyzing the progeny of an interspecific cross between Vanilla ×tahitensis J. W. Moore (pro sp.) and Vanilla pompona Schiede. The two‐way pseudotestcross mapping strategy was used and a total of 225 amplified fragment length polymorphism (AFLP) markers were mapped on 18 linkage groups covering 1035.85 cM. Thirteen tahitian vanilla morphotypes, one Vanilla planifolia Andrews and recent hybrids were compared by measuring seven morphological traits and by genotyping, with 539 AFLP fragments. The morphotypes studied showed relatively homogenous stem traits but differed in their bean and leaf traits as in their AFLP pattern. Genetic variability and AFLP genotyping comparison suggest a diversification in French Polynesia based on sexual reproduction and are consistent with the hypothesis of a single introduction event. These results are the first step for vanilla breeding program and will be also useful for tahitian vanilla resource preservation.

Genotyping selection for resistance against tomato yellow leaf curl virus (TYLCV) conferred by Ty-1 and Ty-3 genes in tomato

The tomato yellow leaf curl virus (TYLCV), transmitted by whitefly, causes major disease losses to tomato crops in tropical and subtropical regions of the world. Several genes conferring resistance to TYLCV, mainly Ty-1 and Ty-3 genes, have been introgressed to cultivated tomato (Solanum lycopersicum) from the wild relative species Solanum chilense. By combining bulked segregant analysis and amplified fragment length polymorphisms (AFLP), several AFLP markers closely linked to Ty-1 and Ty-3 genes were identified from the resistant breeding line TZ841-4. Cloning and sequencing of the selected AFLP fragments allowed us to develop codominant cleaved amplified polymorphic sequence and dominant sequence characterized amplified region markers closely linked to Ty-1. In addition, Ty-3-linked allelic-specific markers have been discriminated by a quantitative real-time PCR protocol. Taken together, these markers constitute useful tools for marker-assisted selection breeding programs to improve genetic resistance to TYLCV, and also to initiate map-based cloning approaches to isolate the resistance genes.

English

  The genetic diversity organization and evaluation of relationships within Tunisian date-palm cultivars were assessed using the random amplified microsatellite polymorphism (RAMPO) and amplified fragment length polymorphism (AFLP) methods. 18 combinations of random/ISSR primers and six AFLP primers combinations were tested with DNAs isolated from 40 date-palm cultivars. Our results show that using both markers systems, the Tunisian date-palm germplasm is characterized by a large and continuous genetic diversity. In addition, due to the greater number of markers per assay, the AFLP technique seems to be more informative than the RAMPO method. In fact, 186 and 428 polymorphic bands were detected using RAMPO and AFLP primers, respectively. Moreover, AFLP markers were found most polymorphic with the highest average PIC value (0.7) and marker index (50.54). In addition, independent as well as combined analyses of the cluster analyses of the RAMPO and AFLP fragments showed that cultivars are clustered independently from the sex of trees or else their geographical origin. On the other hand, based on Pearson and Spearman correlation between RAMPO and AFLP distance, matrices were positive and highly significant. This result indicates good congruence between these two molecular markers. The opportunity of the designed methods is discussed with the molecular characterization of genotypes in order to enhance the conservation and the improvement of the local date-palm germplasm.   Key words: Phoenix dactylifera, genetic diversity, random amplified microsatellite polymorphism (RAMPO), amplified fragment length polymorphism (AFLP),Tunisian.

Genetic characterization of asparagus doubled haploids collection and wild relatives

The genus Asparagus is very large consisting of around 150 species found as herbaceous perennials, tender woody shrubs and vines. The cultivated species ( Asparagus officinalis L., diploid) is a highly prized vegetable, grown in different environments ranging from cool temperate zones to deserts, Mediterranean climates and tropical areas. As a consequence, Asparagus breeders have developed different cultivars that differ for their morpho-agronomic traits, habit and ploidic status (few triploid and tetraploid cultivars are used). Several breeding methods are used for developing cultivars, among which a well developed in vitro anther culture technique produces homozygous clones useful for F 1 hybrids constitution. A fluorescent based AFLP (amplified fragment length polymorphism) technique were applied with the aim to assess genetic diversity among a collection of 173 doubled haploid (DH) androgenetic clones, five Asparagus wild species and interspecific hybrids obtained among the cultivated species and two wild relatives. The average number of AFLP fragments generated per primer set was 105, varying in size from 50 to 550 bp. A total of 1054 AFLP fragments were detected, 20% of which were polymorphic. Genetic similarity based on DNA polymorphisms, showed that a few number of AFLP primer combinations are able to distinguish the cultivated DH clones from the wild species. Indeed, from one DH clone for each anther donors and the wild species were used to construct a dendrogram using Dice's coefficient and the unweighted pair group method with the arithmetic mean (UPGMA). Genetic distances among all DH clones were calculated using the C.S. Chord distance; and a neighbour-joining (NJ) consensus tree was constructed in order to support the breeder for parental genotype choice for asparagus hybrid constitution. These results represent the first study on the genetic relationships among several DH clones from a large collection useful for supporting breeding programs as well as among cultivated DH clones group, wild species and interspecific hybrids.

English

Amplified fragment length polymorphisms (AFLP) were used to evaluate the genetic diversity between 18 date palm (Phoenix dactylifera L.) varieties (11 females and 7 males) collected from the center of Iraq. Six primer pairs were applied to detect polymorphism between varieties. A total of 83 polymorphic AFLP fragments were detected with an average of 13.8 polymorphic fragments/primer combination. Genetic distance was estimated using Jaccard’s genetic similarity index and was ranged from 0.07 to 0.75. Unweighted pair group method with arithmetic mean UPGMA ordered date palm varieties into two main clusters independently of their origin and sex. The first cluster consisted of three sub-clusters. The first one consisted of five female varieties and one male, while the second sub-clusters consisted of five male varieties. The third one consisted of five varieties; four were females and one male. The second main cluster consisted of the remaining two female varieties. Moreover, all primer combinations contributed to the discrimination of date palm varieties, suggesting the efficiency of AFLP method in assessing genetic diversity in date palm. A large range of genetic diversity characterized Iraqi date palm germplasm.   Key words: Genetic diversity, amplified fragment length polymorphisms (AFLP) polymorphisms, molecular characterization, Phoenix dactylifera L.

AFLP fingerprinting suggests an allopolyploid origin of two members of the Leucanthemum vulgare aggregate (Compositae, Anthemideae) in central Europe

In order to clarify the origin of the wide-spread tetraploid Leucanthemum ircutianum and the hexaploid L. adustum, both found in the alpine and dealpine parts of central Europe together with the diploid L. vulgare, we carried out a genetic analysis based on the distribution of amplified fragment length polymorphism (AFLP) fragments in populations of these three taxa, both in the northwestern Alps (Allgäu region, south Germany and adjacent parts of Austria) and the Franconian Jura (vicinity of Regensburg, Bavaria, Germany). Statistical analyses based on AFLP fragments (principal co-ordinate analysis, PCoA; analysis of molecular variance, AMOVA; Bayesian analysis of population structure, BAPS) suggest that neither of the polyploids is of autopolyploid origin based on L. vulgare, but rather are allopolyploids with the second (maternal) diploid parent still unknown.

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

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.

Origin and distribution of autopolyploids via apomixis in the alpine species Ranunculus kuepferi (Ranunculaceae)

AbstractThe evolution of polyploids is strongly influenced by their mode of origin. Autopolyploidy is often hampered by disturbances of chromosome pairing and segregation at meiosis, while allopolyploids exhibit fewer meiotic problems and potentially have selective advantages because of hybridity. Apomixis, the asexual reproduction via seed in flowering plants, can overcome problems of meiotic reduction, favouring range expansions of polyploids. We elucidate the origin and genetic structure of apomictic cytotypes of the alpine species Ranunculus kuepferi by analyzing five microsatellite loci (SSRs) and Amplified Fragment Length Polymorphisms (AFLPs) on eight diploid and twelve polyploid populations from across the range of the species. Multiple allelism appears in three SSR loci in all polyploid cytotypes and is likely a result of facultative unbalanced meiotic events, as it is typical for autopolyploids. Low frequencies of private AFLP fragments (3%) and SSR alleles (4%) in tetraploids suggest that they evolved from diploid populations without the contribution of another divergent parental gene pool. A Neighbor‐joining tree of AFLPs revealed a low genetic divergence of cytotypes and at least three independent origins of tetraploid populations. Autopolyploidy is in R. kuepferi likely evolutionarily young and still has signatures of a facultative unbalanced meiosis. The shift to apomixis avoided consequences of meiotic problems and therefore enhanced rapid establishment of autotetraploids. The lack of hybridity in polyploids suggests that apomixis is successful even without genomic novelty. The distributional success of polyploid cytotypes is probably more due to the benefits of apomictic reproduction than to genetic consequences of polyploidization.

Genomospecies identification and phylogenomic relevance of AFLP analysis of isolated and non-isolated strains of Frankia spp.

Amplified fragment length polymorphism (AFLP) was tested as an alternative to the DNA–DNA hybridization technique (DDH) to delineate genomospecies and the phylogenetic structure within the genus Frankia. Forty Frankia strains, including representatives of seven DDH genomospecies, were typed in order to infer current genome mispairing (CGM) and evolutionary genomic distance (EGD). The constructed phylogeny revealed the presence of three main clusters corresponding to the previously identified host-infecting groups. In all instances, strains previously assigned to the same genomospecies were grouped in coherent clusters. A highly significant correlation was found between DDH values and CGM computed from AFLP data. The species definition threshold was found to range from 0.071 to 0.098 mismatches per site, according to host-infecting groups, presumably as a result of large genome size differences. Genomic distances allowed new Frankia strains to be assigned to nine genomospecies previously determined by DDH. The applicability of AFLP for the characterization of uncultured endophytic strains was tested on experimentally inoculated plants and then applied to Alnus incana and A. viridis field nodules hosting culture refractory spore-positive (Sp+, that sporulate in planta) strains. Only 1.3% of all AFLP fragments were shown to be generated by the contaminant plant DNA and did not interfere with accurate genomospecies identification of strains. When applied to field nodules, the procedure revealed that Alnus Sp+ strains were bona fide members of the Alnus-Myrica host infecting group. They displayed significant genomic divergence from genomospecies G1 of Alnus infecting strains (i.e. Frankia alni) and thus may belong to another subspecies or genomospecies.