COLD ACCLIMATION OF TWO STRAINS OF THE CYANOBACTERIUM, SPIRULINA PLATENSIS

Cephaleuros virescens is a pantropical subaerial green alga with no known long-range dispersal mechanisms. Sexual reproduction is relatively rare and may involve intragametangial fusion of identical, mitotically produced gametes. This situation may be a consequence of adaptation to the subaerial habitat. Genetic variation among populations of C. virescens may be very low and might be positively correlated to the distance (hence, time) separating populations. Thus, assessing the global biogeography of C. virescens requires analysis of what might be low levels of variation. Because C. virescens occurs on literally hundreds of different host species, the question of host-races must also be considered. Preliminary analysis of local populations of C. virescens , originally obtained as field collections from three different host species and subsequently raised in culture, is the first step in addressing the biogeography of this alga. We are using the AFLP plant mapping protocol by PE Applied Biosystems to detect genetic variability in the three isolates of C. virescens. AFLP is a PCR-based DNA fingerprinting technique that detects the presence or absence of restriction fragments rather than fragment length differences. Because the number of restriction fragments that can be detected with the AFLP technique is "virtually unlimited," it is a very powerful tool for assessing the degree of relatedness or variability among cultivars or isolates. AFLP techniques have been used successfully to distinguish morphologically identical bacteria, determine relatedness among soybean accessions, reveal genetic variability within bee samples, and identifyfall armyworm strains and hybrids.     

Microsatellite Markers Reveal Genetic Variation within Sclerotinia sclerotiorum Populations in Irrigated Dry Bean Crops in Brazil

Microsatellites are powerful markers to infer population genetic parameters. We used 10 microsatellite loci to characterize the genetic diversity and structure of 79 samples of Sclerotinia sclerotiorum isolated from four Brazilian dry bean populations and observed that eight of them were polymorphic within populations. We identified 102 different haplotypes ranging from 6 to 18 per locus. Analyses based on genetic diversity and fixation indices indicated variability among and within populations of 28.79% (F_ST = 28793) and 71.21%, respectively. To examine genetic relatedness among S. sclerotiorum isolates, we used internal spacer (ITS1‐5.8S‐ITS2) restriction fragment length polymorphism (PCR‐RFLP) and sequencing analysis. PCR‐RFLP analysis of these regions failed to show any genetic differences among isolates. However, we detected variability within the sequence, which does not support the hypothesis of clonal populations within each population. High variability within and among populations may indicate the introduction of new genotypes in the areas analysed, in addition to the occurrence of clonal and sexual reproduction in the populations of S. sclerotiorum in the Brazilian Cerrado.     

Poa annua L. in Antarctic: searching for the source of introduction

The level of polymorphism, genetic variability and relatedness of a population of Poa annua L. from South Shetlands Islands was studied and compared with results obtained for populations from two potential sources of introduction (Argentina—Ushuaia and Poland—Dziekanów Leśny) using the amplified fragment length polymorphism (AFLP) approach. Five primer pairs used for AFLP profiling amplified 226 scoreable DNA fragments that were used for Clustral and Factorial analyses. The level of molecular variability among all individuals from all the analysed populations reaches 30%. Clustral and Factorial analyses show that all populations formed clear-cut uniform groups according to their locations. However, population from King George Island show high variability. High genetic diversity may be related with escalated human activity at the area of Arctowski Station, favouring introductions of P. annua from many different sources and by many different vectors.     

Characterization of Kenyan Isolates of Fusarium udum from Pigeonpea [Cajanus cajan (L.) Millsp.] by Cultural Characteristics, Aggressiveness and AFLP Analysis

Isolates of Fusarium udum from pigeonpea (Cajanus cajan) plants with wilt symptoms were collected from various districts in Kenya and were characterized using cultural characteristics, aggressiveness and amplified fragment length polymorphism (AFLP). The 56 isolates of F. udum showed a high level of variability in aerial mycelia growth, pigmentation and radial mycelia growth (colony diameter) on potato dextrose agar. The aggressiveness of 17 isolates of F. udum on seven pigeonpea varieties varied and five aggressive groups were observed in the present study. There were no relationships among cultural characteristics and aggressiveness. AFLP analysis of the 56 isolates was tested for genetic variability using seven primer combinations. A total of 326 fragments was generated of which 121 were polymorphic. Ten AFLP groups were identified among the Kenyan isolates and, although they were not genetically distinct, six AFLP subgroups were genetically distinct. AFLP had no relationship with cultural characteristics, aggressiveness and geographical origin of the isolates. This is the first report on the study of genetic variability of F. udum using DNA analysis.     

Assessment and comparison of AFLP and SSR based molecular genetic diversity in Indian isolates of Ascochyta rabiei, a causal agent of Ascochyta blight in chickpea (Cicer arietinum L.)

Ascochyta blight (AB), caused by Ascochyta rabiei (Pass.) Labr. (anamorph), is the most damaging disease of chickpea (Cicer arietinum L.) and is a serious biotic stress constraint for chickpea production. To understand the molecular diversity in A. rabiei populations of India, a total of 64 isolates collected from AB-infected chickpea plants from different agroclimatic regions in the North Western Plain Zone (NWPZ) of India were analyzed with 11 AFLP (amplified fragment length polymorphism) and 20 SSR (simple sequence repeat) markers. A total of 9 polymorphic AFLP primer pairs provided a total of 317 fragments, of which 130 were polymorphic and showed an average PIC value 0.28. Of the SSR markers, 12 showed polymorphism and provided a total of 29 alleles with an average PIC value 0.35. To the best of our knowledge, this is the first report on a comparison of AFLP and SSR diversity estimates in A. rabiei populations. The dendrogram developed based on AFLP and SSR data separately, as well as on the combined marker dataset, grouped the majority of AB isolates as per geographic regions. Model based population structure analysis revealed four distinct populations with varying levels of ancestral admixtures among 64 isolates studied. Interestingly, several AFLP primer combinations and SSR markers showed the locus/allele specific to AB isolates of certain regions, e.g., Hisar, Sriganganagar, Gurdaspur, and Sundarnagar. Genetic variability present in AB isolates of the NWPZ of India suggests the continuous monitoring of changes in A. rabiei population to anticipate the breakdown of AB resistance in chickpea cultivars grown in India.     

The biogeography of kin discrimination across microbial neighbourhoods

The spatial distribution of potential interactants is critical to social evolution in all cooperative organisms. Yet the biogeography of microbial kin discrimination at the scales most relevant to social interactions is poorly understood. Here we resolve the microbiogeography of social identity and genetic relatedness in local populations of the model cooperative bacterium Myxococcus xanthus at small spatial scales, across which the potential for dispersal is high. Using two criteria of relatedness—colony‐merger compatibility during cooperative motility and DNA‐sequence similarity at highly polymorphic loci—we find that relatedness decreases greatly with spatial distance even across the smallest scale transition. Both social relatedness and genetic relatedness are maximal within individual fruiting bodies at the micrometre scale but are much lower already across adjacent fruiting bodies at the millimetre scale. Genetic relatedness was found to be yet lower among centimetre‐scale samples, whereas social allotype relatedness decreased further only at the metre scale, at and beyond which the probability of social or genetic identity among randomly sampled isolates is effectively zero. Thus, in M. xanthus, high‐relatedness patches form a rich mosaic of diverse social allotypes across fruiting body neighbourhoods at the millimetre scale and beyond. Individuals that migrate even short distances across adjacent groups will frequently encounter allotypic conspecifics and territorial kin discrimination may profoundly influence the spatial dynamics of local migration. Finally, we also found that the phylogenetic scope of intraspecific biogeographic analysis can affect the detection of spatial structure, as some patterns evident in clade‐specific analysis were masked by simultaneous analysis of all strains.     

DNA-fingerprinting (AFLP and RFLP) for genotypic identification in species of the <Emphasis Type="Italic">Pleurotus eryngii</Emphasis> complex

Wild populations of edible species are important source of genetic variability for cultivated lines that can undergo a drastic loss of diversity resulting from man’s selection. The development of tools aimed at the clear-cut and safe identification and assessment of genetic variability of the wild and cultivated strains is thus a fundamental goal of molecular genetic research. In this study, we used two polymerase chain reaction (PCR)-based fingerprinting methods—amplified fragment length polymorphism (AFLP) and restriction fragment length polymorphism (RFLP) of laccase and manganese peroxidase genes—to assess genetic differences among strains and independently evolving lineages belonging to the Pleurotus eryngii complex. Both laccase RFLP and AFLP have been proved to distinguish unambiguously the three taxa studied: Pleurotus ferulae, P. eryngii, and P. eryngii var. nebrodensis. AFLP also showed enough sensitivity to detect polymorphisms among the strains, proving to be an efficient DNA fingerprinting tool in studies of strain assignment. The divergent RFLP laccase and manganese peroxidase patterns are also discussed in relation to the role played by these genes in the interaction between these fungi and their host plants.     

AFLP technology for DNA fingerprinting

The AFLP technique is a powerful DNA fingerprinting technology applicable to any organism without the need for prior sequence knowledge. The protocol involves the selective PCR amplification of restriction fragments of a total digest of genomic DNA, typically obtained with a mix of two restriction enzymes. Two limited sets of AFLP primers are sufficient to generate a large number of different primer combinations (PCs), each of which will yield unique fingerprints. Visualization of AFLP fingerprints after gel electrophoresis of AFLP products is described using either a conventional autoradiography platform or an automated LI-COR system. The AFLP technology has been used predominantly for assessing the degree of variability among plant cultivars, establishing linkage groups in crosses and saturating genomic regions with markers for gene landing efforts. AFLP fragments may also be used as physical markers to determine the overlap and positions of genomic clones and to integrate genetic and physical maps. Crucial characteristics of the AFLP technology are its robustness, reliability and quantitative nature. This latter feature has been exploited for co-dominant scoring of AFLP markers in sample collections such as F2 or back-cross populations using appropriate AFLP scoring software. This protocol can be completed in 2-3 d.     

Development of Pathogenicity and AFLP to Characterize Fusarium oxysporum f.sp. momordicae Isolates from Bitter Gourd in China

Bitter gourd (Momordica charantia L.) cultivated in China is regarded as an important vegetable crop and is of considerable economic importance. However, it is susceptible to fusarium wilt, which causes heavy economic losses. Forty‐eight isolates were isolated from diseased bitter gourd plants that displayed typical fusarium wilt symptoms. Based on the morphological features, the rDNA internal transcribed space (ITS) sequences, pathogenicity and host biotypes, all of the isolates tested were pathogenic to the susceptible bitter gourd plants species (cv. ‘Guinongke No. 2’) and were identified as Fusarium oxysporum f. sp. momordicae (FOM). Our results classified different isolates as slightly, moderately or highly virulent. Among the isolates tested, 43 isolates slightly infected bottle gourd (Lagenaria siceraria var. clavata), whereas they did not infect other species from the family Cucurbitaceae. Genetic diversity among 48 isolates was characterized using amplified fragment length polymorphism (AFLP) analysis. The number of bands amplified by each primer pairs ranged from 41 to 66, with sizes ranging from 200 to 500 bp. A total of 366 bands were observed, out of which 363 were polymorphic (99.14%). The Nei's genetic identity of the six geographical populations varied from 0.7362 to 0.9707. The mean Nei's gene diversity index (H = 0.2644) and the mean Shannon's information index (I = 0.4071) at species level were higher than ones at populations level, indicated that the variation within populations was greater than that among populations. The Nei's GST (0.5103) and gene flow (Nm = 0.4923) revealed that genetic differentiation was mainly among populations and few gene exchanges. The dendrogram obtained from AFLP marker showed that there was a good correlation between isolates from different geographical locations and their pathogenicity. The AFLP marker effectively distinguished the high virulent isolates from the less virulent isolates. The highly virulent isolates were distinctly separated in different clusters, which indicated a significantly high correlation with the geographical origin in the AFLP dendrogram. The pathogenicity and molecular marker analysis confirmed the presence of variation in virulence as well as genetic diversity among the FOM isolates studied.     

Genetic diversity of <Emphasis Type="Italic">Rhynchosporium secalis</Emphasis> in Tunisia as revealed by pathotype,AFLP, and microsatellite analyses

Genetic variability among 122 Rhynchosporium secalis isolates collected from barley in three regions of Tunisia was investigated using host differentials, amplified fragment length polymorphism (AFLP), and microsatellite markers. The isolates were collected from a widely grown scald-susceptible barley cultivar Rihane and a range of local landrace cultivars in geographically distinct regions with different agroclimatic conditions. Pathotypic diversity (the proportion of unique pathotypes) was high in R. secalis populations from the high (100% diversity), moderate (95%), and low (100%) rainfall areas of Tunisia, and from both Rihane (which is the sole variety grown in the high rainfall region) and local landraces (which predominate in the low rainfall area). This may reflect a general adaptability for aggressiveness and suggests that the widely grown cultivar Rihane has exerted little or no selection pressure on the pathogen population since its release in 1983. Genotypic diversity (GD), defined as the probability that two individuals taken at random had different genotypes, was high for populations from Rihane, local landraces, and different agro-ecological zones (GD = 0.96–0.99). There was low genetic differentiation among pathogen populations from different host populations (G _ST ≤ 0.08, θ ≤ 0.12) and agro-ecological zones (G _ST ≤ 0.05, θ ≤ 0.04), which may be partly explained by gene flow due to the movement of infected stubble around the country. There was no correlation (r = 0.06, P = 0.39) between virulence phenotype and AFLP haplotype. A phenetic tree revealed groups with low bootstrap values that did not reflect the grouping of isolates based on host, pathotype, or agro-ecological region. The implications of these findings for R. secalis evolutionary potential and scald-resistance breeding in Tunisia are discussed.     

Variability of Colletotrichum kahawae in Relation to Other Colletotrichum Species from Tropical Perennial Crops and the Development of Diagnostic Techniques

Twenty‐six isolates of Colletotrichum kahawae, the causal agent of coffee berry disease, from coffee in Africa, and 25 isolates, mostly of Colletotrichum gloeosporioides, from coffee and other tropical perennial crops, were examined for the ability to metabolize citrate and tartrate and their molecular genetic variability was assessed using restriction fragment length polymorphisms (RFLP) and variable number tandem repeats (VNTR). Twenty‐four isolates of C. kahawae were also assessed using amplified fragment length polymorphisms (AFLP). Vegetative compatibility within a collection of nine isolates, including two of C. gloeosporioides was also assessed. All isolates of C. kahawae from across Africa failed to metabolize citrate or tartrate, but all other isolates metabolized one or both. Colletotrichum kahawae isolates also showed minimal variability using the molecular techniques with two isolates from Cameroon showing slightly different banding patterns in RFLP analysis. All other isolates had variable VNTR and RFLP banding patterns. AFLP analysis failed to detect variability within 12 isolates from Kenya, but did detect differences between isolates from other countries. Five isolates from Kenya were vegetatively compatible but differed from two from Cameroon and from two C. gloeosporioides isolates. Results demonstrate some geographic variability within C. kahawae isolates, although this is small, probably due to the relatively young age of C. kahawae populations. The biochemical and molecular techniques used showed clear differences from other Colletotrichum isolates, and can be used to distinguish the species. Lack of citrate and tartrate metabolism provides a readily applicable diagnostic method.     

Evaluation of RAPD, ISSR and AFLP Markers for Characterization of the Loose Smut Fungus Ustilago tritici

Random Amplified Polymorphic DNA (RAPD), inter simple sequence repeat (ISSR) and Amplified Fragment Length Polymorphism (AFLP) profiling were evaluated for assessing the extent of genetic variation among the isolates of Ustilago tritici (Pers.) Rostr., which causes the loose smut disease of wheat.Thirty random decamer primers, six random primer pairs, four SSR primers such as (GACA)₄, (GATA)₄, (CAA)₅ and (GTG)₅ and nine combinations of AFLP selective primers were used to characterize nine isolates of the fungus. These isolates were collected from infected earheads of seven commercial wheat cultivars grown at eight different locations in Haryana, which is a major wheat growing state in the North-West Plain Zone of India. The RAPD and ISSR primers generated 21 0 scorable amplified fragments, all of which were monomorphic among the isolates.The AFLP primer combinations generated 239 fragments out of which 193 were polymorphic. All the isolates could be precisely differentiated from each other employing AFLP and grouped into two distinct clusters.The molecular classification partly corresponded with geographic distribution and host origin of the isolates. AFLP profiling was found superior to RAPD and ISSR and can be effectively utilized for further characterization of loose smut pathogen.     

Detection of Genetic Variability in Pearl Millet Downy Mildew (Sclerospora graminicola) by AFLP

Downy mildew, caused by Sclerospora graminicola, is an economically important disease of pearl millet in the semiarid regions of Asia and Africa. Amplified restriction fragment length polymorphism (AFLP) was used to detect the extent of genomic variation among 19 fungal isolates from different cultivars of pearl millet grown in various regions of India. Fourteen AFLP primer combinations produced 184 polymorphic bands. An unweighted pair-group method of averages cluster analysis represented by dendrogram and principal coordinate analysis separated the mildew collections into four distinct groups. Isolates having characteristic opposite mating abilities, geographic relatedness, virulence, common host cultivars, and changes through asexual generations reflected heterogeneity of the pathogen. The use of AFLP to detect genetic variation is particularly important in selecting mildew isolates to screen breeding material for identification of resistant millet and monitoring changes in S. graminicola in relation to changes in host for effective disease management.     

An assessment of the AFLP method for investigating population structure in the red alga Chondrus crispus Stackhouse (Gigartinales, Florideophyceae)

The appropriateness of the Amplified Fragment Length Polymorphism (AFLP) technique for investigating Chondrus crispus Stackhouse populations in the Maritime Provinces of Canada was assessed. The AFLP procedure was first subjected to reproducibility testing and three shortcomings were noted: 1) failure to reproduce band intensity between replicate runs for the same individual and primer pair; 2) failure of some bands to replicate; 3) lack of reproducibility for complete replicate runs for some individuals and primer pairs. In the last-mentioned case, the lack of reproducibility resulted in characteristic electropherograms indicative of weak reactions. These weak runs can be attributed to poor restriction digest/ligation reactions and/or substandard PCR, these failures ultimately resulting from low and inconsistent DNA quality. We recommend that reproducibility testing should be completed routinely in studies using the AFLP technique. In the current work, only fragments and individuals that gave reproducible results were used in subsequent analyses. The AFLP method resulted in highly variable markers within and between the populations of C. crispus included in this investigation, which prevented successful resolution of population structure. This situation could result from a lack of suitability for AFLP markers in population genetic studies, and/or too extensive genetic variation for C. crispus populations to be discerned by the AFLP technique. These two possible explanations are discussed. This revised version was published online in August 2006 with corrections to the Cover Date.     

PHENOTYPIC VARIATION AND GENOTYPIC DIVERSITY IN A PLANKTONIC POPULATION OF THE TOXIGENIC MARINE DINOFLAGELLATE ALEXANDRIUM TAMARENSE (DINOPHYCEAE)1

Multiple clonal isolates from a geographic population of Alexandrium tamarense (M. Lebour) Balech from the North Sea exhibited high genotypic and phenotypic variation. Genetic heterogeneity was such that no clonal lineage was repeatedly sampled according to genotypic markers specified by amplified fragment length polymorphism (AFLP) and microsatellites. Subsampling of genotypic data from both markers showed that ordination of individuals by pair‐wise genetic dissimilarity indices was more reliable by AFLP (482 biallelic loci) than by microsatellites (18 loci). However, resulting patterns of pair‐wise genetic similarities from both markers were significantly correlated (Mantel test P < 0.005). The composition of neurotoxins associated with paralytic shellfish poisoning (PSP) was also highly diverse among these isolates and allowed clustering of toxin phenotypes based on prevalence of individual toxins. Correlation analysis of pair‐wise relatedness of individual clones according to PSP‐toxin profiles and both genotypic characters failed to yield close associations. The expression of allelochemical properties against the cryptophyte Rhodomonas salina (Wis?ouch) D. R. A. Hill et Wetherbee and the predatory dinoflagellate Oxyrrhis marina Dujard. manifested population‐wide variation of responses in the target species, from no visible effect to complete lysis of target cells. Whereas the high genotypic variation indicates high potential for adaptability of the population, we interpret the wide phenotypic variation as evidence for lack of strong selective pressure on respective phenotypic traits at the time the population was sampled. Population markers as applied here may elucidate the ecological significance of respective traits when followed under variable environmental conditions, thereby revealing how variation is maintained within populations.     

Genetic diversity of durum wheat as determined by AFLP in fluorescence

The DNA of fifteen Italian cultivars of durum wheat (Triticum turgidum L. ssp. durum) were analyzed by in fluorescence amplified fragment length polymorphism (fAFLP) in order to obtain the characteristic fingerprintings of genotypes and assess their genetic relatedness. Among 64 combinations of fluorescence labelled primers, three different combinations were chosen as producing a total of 6630 AFLP fragments, 2277 (34.3 %) of them being polymorphic. By using this fAFLP methodology a DNA fingerprinting of each durum wheat cultivar was generated for genotype identification. Analysis of the genetic relationships show the low variability among durum wheat cultivars.     

Identification, Mapping, and In Vitro Translation of Campoletis sonorensis Virus mRNAs from Parasitized Heliothis virescens Larvae

Expression of Campoletis sonorensis virus (CsV) in parasitized Heliothis virescens larvae was investigated by Northern blot analysis of poly(A)⁺ mRNAs isolated from H. virescens larvae at various times after parasitization by C. sonorensis. At least 12 CsV mRNAs were detected in parasitized H. virescens larvae. Injection of nonparasitized H. virescens larvae with purified CsV resulted in a pattern of viral mRNAs similar to that observed in naturally parasitized larvae. With CsV DNA restriction fragments which contained expressed sequences, individual CsV mRNAs were mapped to the superhelical DNAs of the viral genome. Two gene-specific probes, which consisted of cloned S1 nuclease-protected restriction fragments, each hybridized to several CsV superhelical DNAs, suggesting that some CsV genes may be shared on several superhelical DNAs. Cloned restriction fragments containing sequences which flank the expressed sequences also hybridized to numerous CsV superhelical DNAs. Some CsV proteins were identified by in vitro translation of hybrid-selected CsV mRNAs.     

Genome Characteristics,Phylogeny and Varying Host Specificity of Polish Kra and Ros Isolates of Tomato torrado virus

The genome sequences of two Polish Kra and Ros isolates of Tomato torrado virus (ToTV) were determined and compared with data of previously described ToTV isolates and other Torradovirus members. Whole‐genome sequence comparisons revealed 97.0–99.6% nucleotide sequence identities and close relatedness, with other known ToTV isolates. The high homology between Kra, Ros and Wal'03 ToTVs is likely responsible for the similar symptoms observed on infected plants. However, the symptoms differed in intensity and various host specificity. We report that Kra ToTV caused a milder expression of symptoms on Solanum tuberosum than Wal'03. We hypothesize this may be a result of the significant variability observed within the 3′‐UTR of RNA1 of Kra as well as of Ros ToTV isolates. In the light of this fact, potato may be considered an indicator plant for distinguishing Kra and Wal'03 ToTV isolates.     

Host association drives genetic divergence in the bed bug,Cimex lectularius

Genetic differentiation may exist among sympatric populations of a species due to long‐term associations with alternative hosts (i.e. host‐associated differentiation). While host‐associated differentiation has been documented in several phytophagus insects, there are far fewer cases known in animal parasites. The bed bug, Cimex lectularius, a wingless insect, represents a potential model organism for elucidating the processes involved in host‐associated differentiation in animal parasites with relatively limited mobility. In conjunction with the expansion of modern humans from Africa into Eurasia, it has been speculated that bed bugs extended their host range from bats to humans in their shared cave domiciles throughout Eurasia. C. lectularius that associate with humans have a cosmopolitan distribution, whereas those associated with bats occur across Europe, often in human‐built structures. We assessed genetic structure and gene flow within and among populations collected in association with each host using mtDNA, microsatellite loci and knock‐down resistance gene variants. Both nuclear and mitochondrial data support a lack of significant contemporary gene flow between host‐specific populations. Within locations human‐associated bed bug populations exhibit limited genetic diversity and elevated levels of inbreeding, likely due to human‐mediated movement, infrequent additional introduction events per infestation, and pest control. In contrast, populations within bat roosts exhibit higher genetic diversity and lower levels of relatedness, suggesting populations are stable with temporal fluctuations due to host dispersal and bug mortality. In concert with previously published evidence of morphological and behavioural differentiation, the genetic data presented here suggest C. lectularius is currently undergoing lineage divergence through host association.     

Optimization of AFLP for extremely large genomes over 70 Gb

Here, we present an improved amplified fragment length polymorphism (AFLP) protocol using restriction enzymes (AscI and SbfI) that recognize 8‐base pair sequences to provide alternative optimization suitable for species with a genome size over 70 Gb. This cost‐effective optimization massively reduces the number of amplified fragments using only +3 selective bases per primer during selective amplification. We demonstrate the effects of the number of fragments and genome size on the appearance of nonidentical comigrating fragments (size homoplasy), which has a negative impact on the informative value of AFLP genotypes. We also present various reaction conditions and their effects on reproducibility and the band intensity of the extremely large genome of Viscum album. The reproducibility of this octo‐cutter protocol was calculated using several species with genome sizes ranging from 1 Gb (Carex panicea) to 76 Gb (V. album). The improved protocol also succeeded in detecting high intraspecific variability in species with large genomes (V. album, Galanthus nivalis and Pinus pumila).