A new and versatile method for the successful conversion of AFLP-TM markers into simple single locus markers

Genetic markers can efficiently be obtained by using amplified fragment length polymorphism (AFLP) fingerprinting because no prior information on DNA sequence is required. However, the conversion of AFLP markers from complex fingerprints into simple single locus assays is perceived as problematic because DNA sequence information is required for the design of new locus-specific PCR primers. In addition, single locus polymorphism (SNP) information is required to design an allele-specific assay. This paper describes a new and versatile method for the conversion of AFLP markers into simple assays. The protocol presented in this paper offers solutions for frequently occurring pitfalls and describes a procedure for the identification of the SNP responsible for the AFLP. By following this approach, a high success rate for the conversion of AFLP markers into locus-specific markers was obtained

<i>Trypanosoma evansi</i>: Genetic variability detected using amplified restriction fragment length polymorphism (AFLP) and random amplified polymorphic DNA (RAPD) analysis of Kenyan isolates

We compared two methods to generate polymorphic markers to investigate the population genetics of Trypanosoma evansi; random amplified polymorphic DNA (RAPD) and amplified restriction fragment length polymorphism (AFLP) analyses. AFLP accessed many more polymorphisms than RAPD. Cluster analysis of the AFLP data showed that 12 T.evansi isolates were very similar (‘type A’) whereas 2 isolates differed substantially (‘type B’). Type A isolates have been generally regarded as genetically identical but AFLP analysis was able to identify multiple differences between them and split the type A T. evansi isolates into two distinct clades

Molecular variation of Trypanosoma brucei subspecies as revealed by AFLP fingerprinting

Genetic analysis of Trypanosoma spp. depends on the detection of variation between strains. We have used the amplified fragment length polymorphism (AFLP) technique to develop a convenient and reliable method for genetic characterization of Trypanosome (sub)species. AFLP accesses multiple independent sites within the genome and would allow a better definition of the relatedness of different Trypanosome (sub)species. Nine isolates (3 from each T. brucei subspecies) were tested with 40 AFLP primer combinations to identify the most appropriate pairs of restriction endonucleases and selective primers. Primers based on the recognition sequences of EcoRI and BglII were chosen and used to analyse 31 T. brucei isolates. Similarity levels calculated with the Pearson correlation coefficient ranged from 15 to 98%, and clusters were determined using the unweighted pair-group method using arithmetic averages (UPGMA). At the intraspecific level, AFLP fingerprints were grouped by numerical analysis in 2 main clusters, allowing a clear separation of T. b. gambiense (cluster I) from T. b. brucei and T. b. rhodesiense isolates (cluster II). Interspecies evaluation of this customized approach produced heterogeneous AFLP patterns, with unique genetic markers, except for T. evansi and T. equiperdum, which showed identical patterns and clustered together

Distribution of genetic diversity in wild European populations of prickly lettuce (Lactuca serriola): implications for plant genetic resources management

Genetic variation in Lactuca serriola, the closest wild relative of cultivated lettuce, was studied across Europe from the Czech Republic to the United Kingdom, using three molecular marker systems, simple sequence repeat (SSR, microsatellites), AFLP and nucleotide-binding site (NBS) profiling. The ‘functional’ marker system NBS profiling, targeting disease resistance genes of the NBS/LRR family, did not show marked differences in genetic diversity parameters to the other systems. The autogamy of the species resulted in low observed heterozygosity and high population differentiation. Intra-population variation ranged from complete homogeneity to nearly complete heterogeneity. The highest genetic diversity was found in central Europe. The SSR results were compared to SSR variation screened earlier in the lettuce collection of the Centre for Genetic Resources, the Netherlands (CGN). In the UK, practically only a single SSR genotype was found. This genotype together with a few other common SSR genotypes comprised a large part of the plants sampled on the continent. Among the ten most frequent SSR genotypes observed, eight were already present in the CGN collection. Overall, the CGN collection appears to already have a fair representation of genetic variation from NW Europe. The results are discussed in relation to sampling strategies for improving genebank collections of crop wild relatives

Genetic exchange in <i>Trypanosoma brucei</i>: evidence for mating prior to metacyclic stage development

It is well established that genetic exchange occurs between Trypanosoma brucei parasites when two stocks are used to infect tsetse flies under laboratory conditions and a number of such crosses have been undertaken. Both cross and self-fertilisation can take place and, with the products of mating being the equivalent of F1 progeny in a Mendelian system and. Recently, analysis of a large collection of independent progeny using a series of polymorphic micro and minisatellite markers, has formally demonstrated that the allelic segregation at loci on each of the 11-megabase chromosomes conforms to ratios predicted for a classical diploid genetic system involving meiosis as well as independent assortment of markers on different chromosomes. Further extensive analysis of these F1 progeny, using a large panel of micro and minisatellite markers, has led to the construction of a genetic map of one parasite stock A. MacLeod, A. Tweedie and S. McLellan et al., The genetic map of Trypanosoma brucei, Nucleic Acids Res 33 (2005), pp. 6688–6693. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (10)

The utility of NBS profiling for plant systematics: a first study in tuber-bearing Solanum species

Systematic relationships are important criteria for researchers and breeders to select materials. We evaluated a novel molecular technique, nucleotide binding site (NBS) profiling, for its potential in phylogeny reconstruction. NBS profiling produces multiple markers in resistance genes and their analogs (RGAs). Potato (Solanum tuberosum L.) is a crop with a large secondary genepool, which contains many important traits that can be exploited in breeding programs. In this study we used a set of over 100 genebank accessions, representing 49 tuber-bearing wild and cultivated Solanum species. NBS profiling was compared to amplified fragment length polymorphism (AFLP). Cladistic and phenetic analyses showed that the two techniques had similar resolving power and delivered trees with a similar topology. However, the different statistical tests used to demonstrate congruency of the trees were inconclusive. Visual inspection of the trees showed that, especially at the lower level, many accessions grouped together in the same way in both trees; at the higher level, when looking at the more basal nodes, only a few groups were well supported. Again this was similar for both techniques. The observation that higher level groups were poorly supported might be due to the nature of the material and the way the species evolved. The similarity of the NBS and AFLP results indicate that the role of disease resistance in speciation is limite

Are current ecological restoration practices capturing natural levels of genetic diversity? A New Zealand case study using AFLP and ISSR data from mahoe (Melicytus ramiflorus)

Sourcing plant species of local provenance (eco-sourcing) has become standard practice in plant community restoration projects. Along with established ecological restoration practices, knowledge of genetic variation in existing and restored forest fragments is important for ensuring the maintenance of natural levels of genetic variation and connectivity (gene flow) among populations. The application of restoration genetics often employs anonymous ‘fingerprinting’ markers in combination with limited sample sizes due to financial constraints. Here, we used two such marker systems, AFLPs and ISSRs, to estimate population-level genetic variation of a frequently used species in restoration projects in New Zealand, māhoe (Melicytus ramiflorus, Violaceae). We examined two rural and two urban forest fragments, as potential local source populations, to determine whether the māhoe population at the recently (re)constructed ecosystem at Waiwhakareke Natural Heritage Park (WNHP), Hamilton, New Zealand reflects the genetic variation observed in these four potential source populations. Both marker systems produced similar results and indicated, even with small population sizes, that levels of genetic variation at WNHP were comparable to in situ populations. However, the AFLPs did provide finer resolution of the population genetic structure than ISSRs. ISSRs, which are less expensive and technically less demanding to generate than AFLPs, may be sufficient for restoration projects where only a broad level of genotypic resolution is required. We recommend the use of AFLPs when species with a high conservation status are being used due to the greater resolution of this technique

On the identity of broad-shelled mussels (Mollusca, Bivalvia, Mytilus) from the Dutch delta region

Late Quaternary (Eemian) deposits of the Netherlands contain shells that resemble those of living Mytilus galloprovincialis. Similar broad-shelled mytilids also occur in estuaries of the southwestern Netherlands together with slender individuals typical of M. edulis. We sampled living mussels along a depth gradient in the Oosterschelde to a) investigate whether a relation exists between shell shape and depth, b) test if the broadshelled specimens might represent M. galloprovincialis (or a hybrid with M. edulis) and c) assess by inference if the Quaternary specimens might be attributed to M. galloprovincialis as well. In order to do so, we compared genetic (length polymorphism of Me 15/16, COIII sequences and AFLPs) and shellmorphological characteristics (juvenile L/W ratios and socalled Verduin parameters) of the same specimens. The obtained dataset indicates that all studied mussels from the Oosterschelde should be attributed to M. edulis, including those with broad shell outlines. No correlation of shell-morphology and depth-distribution was found. The worn and generally damaged state of the Eemian specimens precluded measurement of the Verduin parameters, while juvenile L/W ratios turned out not to be diagnostic. Therefore the shell characters examined in this study are insufficient to demonstrate the possible presence of M. galloprovincialis shells in Quaternary deposits of the Netherlands.

Genetic Diversity, Phylogenetics and Molecular Systematics of Guizotia Cass. (Asteraceae)

The genus Guizotia belongs to the tribe Heliantheae in the family Asteraceae. It has been placed under different subtribes. The genus has its center of origin, distribution and genetic diversity in Ethiopia, where G. abyssinica (niger) has been domesticated. Amplified Fragment Length Polymorphism (AFLP), Random Amplified Polymorphic DNA (RAPD) and DNA sequencing were applied to study the genetic diversity, phylogenetics, and molecular systematics of this genus. A large number of niger populations, representing all regions in Ethiopia where this crop is grown, was investigated using AFLP and RAPD molecular marker techniques. The extent of genetic variation in niger is distributed throughout its growing regions, regardless of the extent and altitude of cultivation. Despite the fact that most of the variation was within populations, significant population differentiation was obtained (AMOVA; P < 0.001) in all guizotias. It is concluded that both G. abyssinica and its wild and/or weedy relatives have wide genetic bases that need to be conserved and utilized for the improvement of G. abyssinica. Further collection of niger germplasm and exploration and conservation of highly localized guizotias are recommended. Most of the diagnostic markers generated from AFLPs and RAPDs in this study were specific to G. arborescens and G. zavattarii. Phylogenetic analyses of the genus Guizotia were undertaken based on molecular sequence data from the internal transcribed spacers (ITS) and five chloroplast DNA regions. The analyses revealed a close phylogenetic relationship between G. abyssinica and G. scabra ssp. schimperi and support the previous suggestion that the latter is the progenitor of the former. According to this study, G. scabra ssp. scabra, G. scabra ssp. schimperi, and the Chelelu and Ketcha populations are best viewed at present as separate species within the genus Guizotia. Those perennial guizotias with highly localized geographic distribution appears to have evolved first during the evolutionary history of the genus. This study supports the placement of the genus Guizotia within the subtribe Milleriinae. It is suggested that the present species composition of Guizotia and the subtribal placement of the genus need to be redefined

Phylogenetic relationships in Betula (Betulaceae) based on AFLP markers

The genus Betula comprises various species in boreal and temperate climate zones of the Northern Hemisphere. The taxonomy of Betula is controversial and complicated by parallel evolution of morphological traits, polyploidization events, and extensive hybridization and introgression among species. Multilocus molecular data from AFLPs were used to provide phylogenetic information. A large number of polymorphic markers (321 variable bands) were produced in 107 Betula accessions from 23 species and 11 hybrids. The AFLP results were largely congruent with the results from previously examined nuclear DNA markers. Four distinct subgenera were identified within the genus Betula. These subgenera were partly in disagreement with the traditional (but disputed) division of the genus. In addition, the results indicated several groups of conspecific taxa. The majority of the species fell within subgenus Betula and shared a high degree of similarity with B. pendula. All hybrids were associated with this group, and the AFLP data contained signals on putative parents for some of the interspecific hybrids. Subgenus Chamaebetula and part of the Neurobetula species should be merged with Betula. The subgenera Betulenta, Betulaster, and the remaining part of Neurobetula are distinct and well supported. Although our results indicate that four major taxonomic groups can be recognized within the genus Betula, the relationship between them remains unclear. This may be due to the occurrence of hybridization and introgression, which would have a homogenizing effect on the relationships between species. Naturally occurring Betula species of hybrid origin may explain the low bootstrap values within the Betula clade