Identification and isolation of Mu-flanking fragments from maize

Transposable elements have been utilized as mutagens to create mutant libraries for functional genomics.Isolation of genomic seg-ments flanking the insertion Mutator (Mu) is a key step in insertion mutagenesis studies.Herein,we adopted a modified AFLP method to identify and isolate Mu-flanking fragments from maize.The method consists of the following steps: 1) double-digestion of genomic DNA with Bgl ⅡMsp Ⅰ and ligation of digested fragments to the Bgl Ⅱ- and Msp Ⅰ-adaptors; 2) enrichment of a subset of Bgl Ⅱ/Msp Ⅰ fragments followed by selective amplification of the Mu-flanking fragments; 3) simultaneous display of AFLP bands derived from the flanking re-gions for both insert and native Mu transposons; 4) identification and isolation of AFLP bands resulting from Mu insertions by comparing the banding profiles between Mu-induced mutants and their parental lines; and 5) confirmation of flanking fragments related to these Mu insertions.Using this approach,we have isolated flanking fragment(s) resulting from Mu insertion for every Mu-indueed mutant,and one such fragment,M196-FF,is found to contain a partial sequence of the DNA topoisomerase Ⅰ gene Topl.Moreover,the modified AFLP method including all restriction enzymes,adaptors and primers has been optimized in this study.The modified AFLP method has been proved to be simple and efficient in the isolation of Mu-flanking fragments and will find its usefulness in the functional genomics of maize.     

A modified AFLP with fluorescence-labelled primers and automated DNA sequencer detection for efficient fingerprinting analysis in plants

A modified AFLP (amplified fragment length polymorphism) technique is described. Fluorescence-labelled primers were used in the selective amplifications. The amplified fragments were detected on denaturing polyacrylamide gels using an automated ALF DNA sequencer with the fragment option. The modified AFLP technique avoids the use of isotopes or silver staining, but gives a much higher resolution than other AFLP detection systems.     

应用AFLP技术对不同山羊种群进行遗传多样性检测的方法初探

7种不同山羊品种或种群的基因组DNA经限制性内切酶酶切 ,连接特异性接头 ,用 5条人工设计的与接头序列相识别的AFLP选择性引物 ,进行AFLP -PCR扩增 ,以琼脂糖凝胶电泳检测扩增结果。不同山羊种群基因组DNA的扩增结果具有差异。从而得出结论 :AFLP技术是一种适宜于山羊的遗传检测方法。     

Genomic typing of Escherichia coli O157:H7 by semi-automated fluorescent AFLP analysis

Escherichia coli serotype O157:H7 isolates were analyzed using a relatively new DNA fingerprinting method, amplified fragment length polymorphism (AFLP). Total genomic DNA was digested with two restriction endonucleases (EcoRI and MseI), and compatible oligonucleotide adapters were ligated to the ends of the resulting DNA fragments. Subsets of fragments from the total pool of cleaved DNA were then amplified by the polymerase chain reaction (PCR) using selective primers that extended beyond the adapter and restriction site sequences. One of the primers from each set was labeled with a fluorescent dye, which enabled amplified fragments to be detected and sized automatically on an automated DNA sequencer. Three AFLP primer sets generated a total of thirty-seven unique genotypes among the 48 E. coli O157:H7 isolates tested. Prior fingerprinting analysis of large restriction fragments from these same isolates by pulsed-field gel electrophoresis (PFGE) resulted in only 21 unique DNA profiles. Also, AFLP fingerprinting was successful for one DNA sample that was not typable by PFGE, presumably because of template degradation. AFLP analysis, therefore, provided greater genetic resolution and was less sensitive to DNA quality than PFGE. Consequently, this DNA typing technology should be very useful for genetic subtyping of bacterial pathogens in epidemiologic studies.     

Modified AFLP analysis method for species with small genomes

A modified method for detecting amplified fragment length polymorphism (AFLP) has been developed for species with small genomes. We successfully used a combination of primers with a reduced number of selective bases to differentiate and fingerprint isolates ofColletotrichum acutatum, the causative agent of strawberry black spot, an important strawberry disease.     

Customisation of AFLP analysis for cassava varietal identification.

Amplified fragment length polymorphism (AFLP) markers were used in the characterization of eight cassava varieties. This nonradioactive AFLP system was customized in terms of the choice of restriction enzymes used and the selection of nucleotides added to the 3' end of primers. EcoRI/MseI and HindIII/MseI fragments generally gave monomorphic profiles while ApaI/TaqI fragments produced polymorphic profiles suggesting a genome with high G + C content. It was possible to identify the eight cassava varieties used in this study using CTG as selective bases at the TaqI primer. For cassava, the AFLP system provided a higher number of loci detected per run when compared to RAPD. The reliability accompanying AFLP analysis would thus make it suitable for the characterization of cassava varieties.     

Application of the AFLP method to differentiate Genista tinctoria microsymbionts

The high-resolution amplified fragment length polymorphism technique (AFLP), with single PstI restriction endonuclease and two selective primers (PstI-G and PstI-GC), was used for genomotyping and study of the genomic relationships between Genista tinctoria microsymbionts sampled in England, Poland, and Ukraine. Out of 906 amplification products obtained with both selective primers, 537 markers were polymorphic and could be used to differentiate studied nodule isolates. Cluster analysis, based on AFLP patterns from PCR reaction with PstI-G and PstI-GC primers, separated Genista tinctoria rhizobia into three subgroups according to their geographic origin. The results presented in this paper emphasize the role of AFLP analysis in taxonomic and ecological studies of rhizobia.     

Characterisation of large and small subunit rRNA and mini-exon genes further supports the distinction of six Trypanosoma cruzi lineages

It has been proposed that isolates of Trypanosoma cruzi, the agent of American trypanosomiasis, can be ordered into two primary phylogenetic lineages, first based on multilocus enzyme electrophoresis and random amplified polymorphic DNA, and subsequently based on the 24Salpha rRNA and mini-exon genes. Recent multilocus enzyme electrophoresis and random amplified polymorphic DNA data have additionally shown that the major multilocus enzyme electrophoresis/random amplified polymorphic DNA lineage II is further subdivided into five smaller lineages, designated IIa-IIe. In this study, the precise correspondence between the multilocus enzyme electrophoresis/random amplified polymorphic DNA and rRNA/mini-exon lineages was investigated. Using the 24Salpha rRNA and mini-exon markers in combination, five sets of strains were distinguished, corresponding to the multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I, IIa, IIc, IId and to lineages IIb/IIe together, respectively. The previous categorisation into only two primary lineages based on 24Salpha rRNA and mini-exon characterisation is explained, in part, by the lack of representativeness of the breadth of T. cruzi diversity in earlier study samples. Additionally, a PCR assay based on a length-variable region of the 18S rRNA gene distinguished lineage IIe from lineage IIb. Thus, the six multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages could be readily identified by combining data from the 24Salpha rRNA, mini-exon and 18S rRNA characterisation assays, further supporting the relevance of these genetic units for T. cruzi strain classification and subspecific nomenclature. The recently proposed groups T. cruzi I and T. cruzi II correspond to multilocus enzyme electrophoresis/random amplified polymorphic DNA lineages I and IIb, respectively. Our findings show that T. cruzi lineage characterisation based on a single marker (either mini-exon or 24Salpha rRNA) has insufficient resolution, and leads to important reinterpretations of recent epidemiological and evolutionary studies based on the oversimplified rRNA/mini-exon dichotomic classification of T. cruzi isolates.     

Genomic diversity of Astragalus cicer microsymbionts revealed by AFLP fingerprinting

DNA polymorphism among 36 Astragalus cicer nodule isolates and 9 reference mesorhizobia was evaluated by a simplified PstI based AFLP procedure with three selective primers: Pst-A, Pst-G, and Pst-GC. The DNA profiles were found to be highly specific for nearly each strain, although DNA bands characteristic for most A. cicer microsymbionts were also noted. The overall topologies of dendrograms, generated by AFLP patterns in PCR reaction with three primers, were very similar to one another and to that constructed by phenotyping. Also the strain compositions in the particular clusters on pheno- and genomograms were in good agreement. The obtained results indicate that AFLP technique can be a useful tool for typing of A. cicer rhizobia as well as for studying their diversity.     

Optimization of AFLP fingerprinting of organisms with a large-sized genome: a study on Alstroemeria spp.

 The recently introduced PCR-based DNA fingerprinting technique AFLP (amplified fragment length polymorphism) allows the selective amplification of subsets of genomic restriction fragments. AFLP has been used for multiple purposes such as the construction of linkage maps, marker saturation at specific genomic regions, analysis of genetic diversity and molecular phylogeny and cultivar identification. AFLP can be tailored by varying the number of selective nucleotides added to core primers and can allow accurate amplification, even in complex template mixtures generated from plant species with very large genomes. In this study Alstroemeria, a plant species with a very large genome, was tested for adapting the AFLP protocol. The results indicated that the estimated number of amplification products was close to the observed number when eight selective nucleotides were used but that seven selective nucleotides did not increase the number of amplification products fourfold. However, we found reproducibility in both +7 and +8 fingerprints. Various distributions of selective nucleotides over the various rounds of preamplifications were tested. Preamplification with four selective nucleotides followed by final amplification with eight selective nucleotides produced clear and reproducible AFLP patterns. The effects of GC content of primers and multiple preamplification steps were also discussed. Received: 16 March 1998 / Accepted: 14 July 1998     

Use of Random Amplified Polymorphic DNA (RAPD) Analysis and Genetic Fingerprinting to Differentiate Isolates of Race O, C and T of Bipolaris maydis

Isolates of three races of Bipolaris maydis from China (races O, C and T) were compared using two techniques. Random Amplified Polymorphic DNA (RAPD) analysis using 24 primers indicated that race O and C isolates were more similar to one another than to the race T isolate. Twenty of the primers produced RAPD profiles that were similar for the race O and C isolates but differed for the race T isolate (four primers did not amplify products in any of the isolates). Four primers produced profiles which differed for all three races and two of these (A-09 and B-18) clearly differentiated the race O and race C isolates. Genetic fingerprinting of B. maydis using M13 DNA as a probe differentiated race O and C isolates from the race T with all four restriction enzymes used. Furthermore, when DNA was digested with Hind III, the hybridization profiles of the race O and C isolates differed from one another.     

Trypanosoma congolense: host responses following tsetse transmitted infection of Kilifi isolates in goats

East African x Galla goats, when infected with Trypanosoma congolense isolates from the Kilifi area of Kenya by Glossina morsitans centralis, did not develop the characteristic chancre reaction at the bite sites, whereas bites of tsetse infected with the cloned T. congolense IL.1180 from Serengeti, Tanzania, resulted in chancres in the same goats. Histological changes could not be observed in skin biopsies collected 8 or 9 days after infection with Kilifi isolates. However, all goats became parasitemic about 10 days after challenge. It is concluded that the absence of chancre development is a characteristic feature of T. congolense parasites from Kilifi. The isoenzyme analysis of clones of two T. congolense Kilifi isolates and the T. congolense clone IL.1180 indicated that they belong to different zymodemes. Neutralizing antibodies to homologous metacyclic variable antigen types were detected in six out of seven (86%) of the sera from goats infected with a clone or stock of a T. congolense Kilifi isolate, 20 days after infection. Goats primed by tsetse transmitted infection with a stock or clone of T. congolense from Kilifi and treated with Berenil were, in three out of eight cases (37%), not immune to homologous challenge. It is suggested that the reduced immune response to metacyclic variable antigen types could be a result of the absence of cellular infiltration, i.e., chancre development in the skin at the tsetse bite site. It is concluded that the use of the chancre reaction as a marker for serodeme analysis of recently isolated stocks of T. congolense from Kilifi was not feasible.     

Modified AFLP technique for rapid genetic characterization in plants

The standard amplified fragment-length polymorphism (AFLP) technique was modified to develop a convenient and reliable technique for rapid genetic characterization of plants. Modifications included (i) using one restriction enzyme, one adapter molecule and primer, (ii) incorporating formamide to generate more intense and uniform bands and (iii) using agarose gel electrophoresis. Sea oats (Uniola paniculata L.), pickerel-weed (Pontederia cordata L.), Bermudagrass (Cynodon dactylon L.) and Penstemon heterophyllus Lindl. were used to determine the ability to generate adequate resolution power with both self- and cross-pollinated plant species including cultivars, ecotypes and individuals within populations. Reproducibility of bands was higher in all the AFLP experiments compared to random amplified polymorphic DNA (RAPD). Formamide with or without bovine serum albumin improved band intensities compared to dimethyl sulfoxide and the standard reaction mixture with no organic solvents. Comparison between RAPD and modified AFLP using sea-oats population samples proved that modified AFLP exhibits (i) a low number of faint bands with increased specificity of amplified bands, (ii) a significantly higher number of polymorphic loci per primer, (iii) less primer screening time, (iv) easy scoring associated with fewer faint bands and (v) greatly enhanced reproducibility. The technique described here can be applied with a high degree of accuracy for plant genetic characterization.     

Identification of a genetic marker for isometamidium chloride resistance in Trypanosoma congolense

Isometamidium chloride has remained a very important prophylactic and therapeutic drug against trypanosomosis in cattle since its introduction into the market in the 1950s with, unfortunately, a concomitant development of resistance in trypanosomosis endemic areas. Amplified Fragment Length Polymorphism (AFLP) was used to compare two isogenic clones of Trypanosoma congolense. The parent clone, sensitive to isometamidium, has a CD50 (the curative dose that gives complete cure in 50% of the animals) in the mouse of 0.018 mg/kg and its derivative exposed to increasing doses of isometamidium, has a CD50 that is 94-fold higher. Sixty-four combinations of eight Eco RI and eight Mse I primers were used in comparative AFLP analysis to detect subtle genetic differences between the two clones. Thirty-five polymorphic fragments of DNA that were observed only in the resistant clone were purified and then sequenced. The nucleotide sequences were used in searching the GeneDB T. congolense database to find surrounding sequences upstream of an open reading frame and downstream to a stop codon. The sequences of the open reading frames were subsequently compared to the sequences in the genomic databases. A predicted gene coding for an 854 amino acids protein was thus identified. The protein contains a putative ATP binding site, Walker B and LSGG motifs and eight predicted trans-membrane domains. The gene in the resistant strain of T. congolense has a triplet insertion coding for an extra lysine. Using polymerase chain reaction-restriction fragment length polymorphism, the insertion was sought in the genomes of 35 T. congolense strains isolated from different geographic origins and whose response to isometamidium chloride had been determined through single dose mouse tests. The presence of the insertion, specifying an extra codon was found to always be present in the genomes of T. congolense clones that were resistant to isometamidium chloride.     

Efficient DNA fingerprinting of Clostridium botulinum types A, B, E, and F by amplified fragment length polymorphism analysis

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II Clostridium botulinum strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of C. botulinum. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between C. botulinum groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic C. botulinum strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of C. botulinum type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for C. botulinum group identification.     

AFLP analysis of genetic diversity within and between Arabidopsis thaliana ecotypes

The degree of genetic diversity within and between 21 Arabidopsis thaliana (L.) Heynh ecotypes was estimated by AFLP analysis. Within seven of the 21 ecotypes, a low but significant level of polymorphism was detected, and for five of these ecotypes two or three distinct subgroups could be distinguished. As these ecotypes represent natural populations, this intra-ecotypic diversity reflects natural genetic variation and diversification within the ecotypes. The source of this diversity remains unclear but is intriguing in view of the predominantly self-fertilizing nature of Arabidopsis. Interrelationships between the different ecotypes were estimated after AFLP fingerprinting using two enzyme combinations (EcoRI/MseI and SacI/MseI) and a number of selective primer pairs. SacI recognition sites are less evenly distributed in the genome than EcoRI sites, and occur more frequently in coding sequences. In most cases, AFLP data from only one enzyme combination are used for genetic diversity analysis. Our results show that the use of two enzyme combinations can result in significantly different classifications of the ecotypes both in cluster and ordination analysis. This difference most probably reflects differences in the genomic distribution of the AFLP fragments generated, depending on the enzymes and selective primers used. For closely related varieties, as in the case of Arabidopsis ecotypes, this can preclude reliable classification.     

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.     

Conversion of AFLP markers associated with FHB resistance in wheat into STS markers with an extension-AFLP method.

Amplified fragment length polymorphism (AFLP) has proven a powerful tool for tagging genes or quantitative trait loci (QTLs) of interest in plants. However, conversion of AFLP markers into sequence-tagged site (STS) markers is technically challenging in wheat owing to the complicated nature of its genome. In this study, we developed an "extension-AFLP" method to convert AFLP markers associated with Fusarium head blight (FHB) resistance into STS markers. When an AFLP marker of interest was detected with an EcoRI+3-MseI+4-selective primer combination, the PCR product was used as a template for an additional selective amplification with four primer pairs, in which one additional selective base (either A, C, G, or T) was added to the 3' end of one of the two primers. The extended primer pair that produced the targeted band was further extended by adding each of the four selective nucleotide bases for the next round of selective amplification. Extension selective amplification was performed until the target bands became clear enough for subsequent cloning and sequencing. By using the extension-AFLP method, we successfully converted two AFLP markers located on chromosome 3BS and associated with FHB resistance into STS markers. Our results indicated that the extension-AFLP method is an efficient approach for converting AFLP markers into STS markers in wheat. The developed STS markers might be used for marker-assisted selection (MAS) for FHB resistance in wheat breeding programs.