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

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

Identification of amplified restriction fragment polymorphism (AFLP) markers tightly linked to the tomato Cf-9 gene for resistance to Cladosporium fulvum

Using the technique of amplified restriction fragment polymorphism (AFLP) analysis, and bulked segregant pools from F₂ progeny of the cross Lycopersicon esculentum (Cf9)× L. pennellii , approximately 42 000 AFLP loci for tight linkage to the tomato Cf-9 gene for resistance to Cladosporium fulvum have been screened. Analysis of F₂ recombinants identified three markers which co-segregated with Cf-9 . The Cf-9 gene has recently been isolated by transposon tagging using the maize transposon Dissociation ( Ds ). Analysis of plasmid clones containing Cf-9 shows that two of these markers are located on opposite sides of the gene separated by 15.5 kbp of intervening DNA. AFLP analysis provides a rapid and efficient technique for detecting large numbers of DNA markers and should expedite plant gene isolation by positional cloning and the construction of high-density molecular linkage maps of plant genomes.     

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.     

Identification of Phoenix dactylifera L. varieties based on amplified fragment length polymorphism (AFLP) markers

The amplified fragment length polymorphism (AFLP) technique was applied to identify palm varieties. Fluorescence labelled primers were used in selective amplifications and the amplified fragments were detected on capillary gel electrophoresis using an automated DNA sequencer with the analysis fragment option. This is a rapid and efficient technique for detecting a large number of DNA markers on the date palm. Phoenix dactylifera L. varieties Bou-Fegous, Medjool, and E-528 from Estación Phoenix (Elche), Spain, were analysed, yielding a total of 310 AFLP fragments derived from five primer combinations. The process for regenerating the date palm cultivars from in vitro tissue culture should yield individuals phenotypically and genetically identical to the explant they are derived from. The AFLP markers obtained were successfully used for comparing and identifying vitroplants of palm.     

RAPD markers linked to the Vf gene for scab resistance in apple

Scab (Venturia inaequalis) is one of the most harmful diseases of apple, significantly affecting world apple production. The identification and early selection of resistant genotypes by molecular markers would greatly improve breeding strategies. Bulked segregant analysis was chosen for the identification of RAPD markers linked to the Vf scab resistant gene. Five different RAPD markers, derived from the wild species Malus floribunda. 821, were identified, and their genetic distance from Vf gene was estimated. The markers OPAM19₂₂₀₀ and OPAL07₅₈₀ were found to be very closely linked to the Vf gene. This result was indirectly confirmed by the analysis of resistant genotypes collected from various breeding programmes. Except for cv Murray, which carries the Vm gene, all these resistant genotypes showed the markers OPAM19₂₂₀₀ and OPAL07₅₈₀.     

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.     

Cleaved amplified polymorphic sequence and amplified fragment length polymorphism markers linked to the fertility restorer gene in chili pepper (Capsicum annuum L.)

Cytoplasmic male sterility (CMS) in plants, which is due to failure to produce functional pollen, is a maternally inherited trait. Specific nuclear genes that sup-press CMS, termed fertility restorer (Rf) genes, have been identified in several plants. In this study, Rf-linked molecular markers in pepper (Capsicum annuum L.) were detected by bulked segregant analysis of eight amplified fragment length polymorphisms (AFLPs). Only AFRF8 was successfully converted to a cleaved amplified polymorphic sequence (CAPS) marker. This was named AFRF8CAPS and genotype determination using it agreed with that obtained with the original AFRF8. A linkage map with a total size of 54.1 cM was constructed with AFRF8CAPS and the seven AFLP markers using the Kosambi function. The AFRF8CAPS marker was shown to be closest to Rf with a genetic distance of 1.8 cM. These markers will be useful for fast and reliable detection of restorer lines during F(1) hybrid seed production and breeding programs in pepper.     

Saturation mapping of the apple scab resistance gene Vf using AFLP markers

Using the amplified fragment length polymorphism (AFLP) technique combined with a ”narrow-down” bulk segregant strategy enabled us to quickly identify 15 tightly linked AFLP markers to the Vf gene that confers resistance to the apple scab disease. High-resolution mapping placed all 15 AFLP markers within an interval of 0.6 cM around the Vf region; 7 of them were found to be inseparable from the Vf gene, 1 was localized left of, and the remaining 7 located right of the Vf gene. In addition, eight previously identified RAPD markers were also mapped, but only three, including M18, AM19, and AL07, were localized within this short interval, and none co-segregated with the Vf gene. The saturation of the Vf region with AFLP markers will accelerate both marker-assisted selection and map-based cloning. The advantages of this ”narrow-down” strategy, estimation of physical distances among AFLP markers, and their potential application are also discussed. Received: 22 December 1999 / Accepted: 25 March 2000     

Determination of phylogenetic relationships between some wild wheat species using amplified fragment length polymorphism (AFLP) markers

This study reports the molecular characterization, polymorphism, and phylogenetic relationships of Triticum aestivum , T. dicoccoides , T. urartu , and T. monococcum ssp. boeoticum , obtained from different locations in Anatolia, using 33 primer combinations to generate amplified fragment length polymorphism (AFLP) patterns in 31 individual plant samples. The objectives of this work were to estimate the phylogenetic relationships between these species and to investigate the genetic distance as a result of ecological and climatic factors. The origin of the A genome of polyploid wheats is also discussed. Eight hundred and seventy-five AFLP fragments had polymorphic loci, 133 of which were unique to T. monococcum ssp. boeoticum , 66 were unique to T. urartu , and 141 were unique to T. dicoccoides . Analysis using the program POPGENE showed polymorphism levels of T. monococcum ssp. boeoticum , T. urartu , and T. dicoccoides of 42.63, 32.34, and 27.71%, respectively. No correlation between genetic distance and ecological or climatic factors was recorded in this study. Our results support the hypothesis that T. urartu is a diploid ancestor of T. dicoccoides and T. aestivum .  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 67–72.     

DNA fingerprinting of Cryptosporidium parvum isolates using amplified fragment length polymorphism (AFLP)

The genetic variability of 10 Cryptosporidium parvum isolates of human and animal origin was investigated using amplified fragment length polymorphism (AFLP). Analysis of fluorescent dye-labeled amplified products was carried out using an ABI PRISMS 377 DNA sequencer and ABI PRISMS GeneScan software. One-hundred and twelve primer combinations were evaluated using a single C. parvum isolate. The patterns generated were highly reproducible. For subsequent study, a subset of 9 primer pairs that yielded 30-90 DNA fragments after the polymerase chain reaction, within the size range of 50-500 bp, was used to screen the 10 C. parvum isolates, including 7 bovine, 1 equine, and 2 of human origin. The animal isolates produced identical fingerprint patterns with every primer combination tested. Of the 2 human isolates tested, 1 of the isolates, passaged in calves, generated the same AFLP DNA banding patterns as the animal isolates, whereas the other isolate, obtained directly from human feces, produced unique patterns. Polymorphism, detected by comparison of the fingerprint patterns of the latter human isolate with the common pattern shared by all other isolates, ranged from 17 to 35% for the 9 primer pairs. The results show that AFLP is a useful method for differentiating C. parvum isolates into 2 distinct genotypes.     

A genetic map of melon (Cucumis melo L.) based on amplified fragment length polymorphism (AFLP) markers

 Genetic maps facilitate the study of genome structure and evolution, and the identification of monogenic traits or Mendelian components of quantitative traits. We evaluated 228 RAPD, microsatellite and AFLP markers for linkage analysis in melon (Cucumis melo L.) varieties MR-1 (resistant to Fusarium wilt, powdery and downy mildews) and Ananas Yokneum (AY; susceptible to these diseases) and constructed a detailed genetic map. The mapping population consisted of 66 backcross progenies derived from AY×(MR-1×AY). Despite a relatively low level of polymorphism in the species, AFLP markers were found to be more efficient in mapping the melon genome than RAPD or microsatellite markers. The map contains 197 AFLPs, six RAPDs and one microsatellite marker assigned to 14 major and six minor linkage groups, and covers 1942 cM with the average distance between adjacent markers of approximately 10 cM. The maximum distance allowed between markers is 27.5 cM. About 11% of the intervals (20 out of 173) are over 20 cM (but less than 27.5 cM). The map has immediate utility for identifying markers linked to disease resistance genes that are suitable for marker-assisted breeding. The use of microsatellite markers for integration with other maps is also discussed. Received: 12 March 1997 / Accepted: 20 May 1997     

Using amplified fragment length polymorphism (AFLP) to unravel species relationships and delimitations in Minthostachys (Labiatae)

Minthostachys (Benth.) Spach (Labiatae) is a South American genus of aromatic shrubs frequently collected as a condiment, for the preparation of tea, or for medicinal purposes. Notoriously difficult species delimitation, conflicting taxonomic treatments of the past, and the lack of a revision with modern methods have hampered the understanding of this ecologically and economically interesting group. The amplified fragment length polymorphism (AFLP) study presented in this paper supplements field and morphological studies within the framework of a taxonomic and systematic revision. AFLP is found to be unsuitable for the reconstruction of the phylogeny of Minthostachys . Although, in some cases, morphologically well-defined species are also genetically distinct, extensive gene flow seems to occur between strikingly different species growing in the immediate vicinity and even between Minthostachys and Clinopodium ( Xenopoma ) vanum . Samples from the most complicated species, M. mollis , are genetically very heterogeneous and mostly fall into two clusters according to their geographical origin, exhibiting a high discrepancy with the pattern of morphological variation.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 153 , 9–19.     

An improved amplified fragment length polymorphism (AFLP) protocol for discrimination of Listeria isolates

Nine restriction enzyme combinations and 108 different primer combinations were initially tested for suitability for amplified fragment length polymorphism (AFLP) analysis of Listeria monocytogenes; the combination of HindIII and HpyCH4IV showed consistently strong signals on gels, amplified an adequate number of DNA fragments and detected polymorphism among closely related strains based on AscI macrorestriction profiles. AFLP also distinguished between L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri, L. welshimeri and L. grayi species. All Listeria species showed species-specific clusters, with less than 33% similarity between different species. A total of 34 L. monocytogenes strains were characterised by using both AFLP and pulsed-field gel electrophoresis (PFGE). The results of AFLP analysis of L. monocytogenes strains were in concordance with those obtained by PFGE. Both methods identified 29 different genotypes of L. monocytogenes and had a high discrimination index (> 0.999). By combining the results of AFLP and PFGE, subtype discrimination was further improved. Numerical analysis of both AFLP and PFGE profiles yielded three genomic groups of L. monocytogenes strains. AFLP was found to be faster and less labour-intensive than PFGE. We conclude that the AFLP protocol is a highly discriminatory, reproducible and valuable tool in characterisation of Listeria strains and may also be suitable for Listeria species identification.     

Cost-effective fluorescent amplified fragment length polymorphism (AFLP) analyses using a three primer system

The amplified fragment length polymorphism (AFLP) technique is a widely used multi-purpose DNA fingerprinting tool. The ability to size-separate fluorescently labelled AFLP fragments on a capillary electrophoresis instrument has provided a means for high-throughput genome screening, an approach particularly useful in studying the molecular ecology of nonmodel organisms. While the 'per-marker-generated' costs for AFLP are low, fluorescently labelled oligonucleotides remain costly. We present a cost-effective method for fluorescently end-labelling AFLPs that should make this tool more readily accessible for laboratories with limited budgets. Both standard fluorescent AFLPs and the end-labelled alternatives presented here are repeatable and produce similar numbers of fragments when scored using both manual and automated scoring methods. While it is not recommended to combine data using the two approaches, the results of the methods are qualitatively comparable, indicating that AFLP end-labelling is a robust alternative to standard methods of AFLP genotyping. For researchers commencing a new AFLP project, the AFLP end-labelling method outlined here is easily implemented, as it does not require major changes to PCR protocols and can significantly reduce the costs of AFLP studies.     

Data from amplified fragment length polymorphism (AFLP) markers show indication of size homoplasy and of a relationship between degree of homoplasy and fragment size

We investigate the distribution of sizes of fragments obtained from the amplified fragment length polymorphism (AFLP) marker technique. We find that empirical distributions obtained in two plant species, Phaseolus lunatus and Lolium perenne, are consistent with the expected distributions obtained from analytical theory and from numerical simulations. Our results indicate that the size distribution is strongly asymmetrical, with a much higher proportion of small than large fragments, that it is not influenced by the number of selective nucleotides nor by genome size but that it may vary with genome-wide GC-content, with a higher proportion of small fragments in cases of lower GC-content when considering the standard AFLP protocol with the enzyme MseI. Results from population samples of the two plant species show that there is a negative relationship between AFLP fragment size and fragment population frequency. Monte Carlo simulations reveal that size homoplasy, arising from pulling together nonhomologous fragments of the same size, generates patterns similar to those observed in P. lunatus and L. perenne because of the asymmetry of the size distribution. We discuss the implications of these results in the context of estimating genetic diversity with AFLP markers.     

Identification of amplified restriction fragment length polymorphism markers linked to genes controlling boar sperm viability following cryopreservation

This study investigated two hypotheses: 1) that consistent between-boar variation in frozen semen quality exists and is genetically determined, and 2) molecular markers linked to genes controlling semen freezability can be identified using amplified restriction fragment length polymorphism (AFLP) technology. Five ejaculates were collected from each of 129 boars. Semen was diluted into a commercial freezing buffer (700 mOsm/kg, 3% glycerol) and five straws (0.5 ml) per ejaculate were cryopreserved (to -5 degrees C at 6 degrees C/min, then -5 degrees C to -80 degrees C at 40 degrees C/min). Semen was assessed for percentage of motile cells, motility characteristics (computer-aided semen analysis; CASA), plasma membrane integrity (SYBR-14 positive), and acrosome integrity (positive for fluorescein-labeled peanut agglutinin; PNA). Consistent between-boar variability was detected for postthaw sperm motility (P < 0.01), membrane integrity (P < 0.01), acrosome integrity (P < 0.01), and all CASA characteristics (P < 0.05). There was no significant difference between ejaculates (P > 0.05) or straws (P > 0.05) for any viability assessment. Multivariate pattern analysis of the viability data set highlighted three groups of boars producing spermatozoa with poor, average, and good postthaw recovery (42, 63, and 24 boars, respectively). DNA from Large White boars (n = 22) previously classified as good and poor freezers was screened for AFLP markers. Twenty-eight polymerase chain reaction primer combinations generated 2182 restriction fragment bands, of which 421 were polymorphic. Sixteen candidate genetic markers (P < 0.005) were identified by comparing the AFLP profile with semen freezability using logistic regression analysis. These findings support the hypothesis that there is a genetic basis for variation in postthaw semen quality between individuals, and that AFLP technology may be able to identify molecular markers linked to genes influencing this variation.     

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.     

Genotyping and genetic diversity of Arcobacter butzleri by amplified fragment length polymorphism (AFLP) analysis

AIMS: To investigate the potential of amplified fragment length polymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. METHODS AND RESULTS: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and North American origin from human infections, chickens, turkeys, ducks, sheep and poultry abbatoir effluent were studied by use of a protocol that involved stringent PCR amplification of fragments derived from digestion of genomic DNA with restriction enzymes BglII and Csp6I. The mean similarity value of duplicate profiles of 10 isolates was 91.15%, indicating the method to be reproducible. Numerical analysis of all 73 isolates distinguished 51 subtypes at the 91% similarity level, of which 39 comprised single strains. The remaining 34 isolates were distributed among 12 subtypes, each of which contained strains homogeneous with respect to their respective source of isolation. However, contemporaneous strains from the same source could also be distinguished. CONCLUSIONS: AFLP profiling is an effective method for typing the genetically diverse organism A. butzleri. SIGNIFICANCE AND IMPACT OF THE STUDY: The study represents a comprehensive analysis of the genetic diversity of A. butzleri by use of isolates from six countries spanning three continents and also shows that several distinct A. butzleri genotypes may be found in a given environment. AFLP profiling appears to have considerable potential for molecular epidemiological studies of this ubiquitous emerging pathogen that is implicated as a causative agent of both human and animal disease.     

Genomic variations of Mycoplasma capricolum subsp. capripneumoniae detected by amplified fragment length polymorphism (AFLP) analysis

The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment length polymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size range of 40-500 bp. The similarity between individual AFLP profiles, calculated by Jaccard's coefficient, ranged from 0.92 to 1.0. On the basis of the polymorphisms detected, the analysed strains can explicitly be grouped into two major clusters, equivalent to two evolutionary lines of the organism found by 16S rDNA analysis. The present data support previous observations regarding genetic homogeneity of M. capricolum subsp. capripneumoniae, and confirm the two evolutionary lines of descent found by analysis of 16S rRNA genes.