Amplified Fragment Length Polymorphism Fragments Research Articles

Ecological Factors Generally Not Altitude Related Played Main Roles in Driving Potential Adaptive Evolution at Elevational Range Margin Populations of Taiwan Incense Cedar (Calocedrus formosana).

Population diversification can be shaped by a combination of environmental factors as well as geographic isolation interacting with gene flow. We surveyed genetic variation of 243 samples from 12 populations of Calocedrus formosana using amplified fragment length polymorphism (AFLP) and scored a total of 437 AFLP fragments using 11 selective amplification primer pairs. The AFLP variation was used to assess the role of gene flow on the pattern of genetic diversity and to test environments in driving population adaptive evolution. This study found the relatively lower level of genetic diversity and the higher level of population differentiation in C. formosana compared with those estimated in previous studies of conifers including Cunninghamia konishii, Keteleeria davidiana var. formosana, and Taiwania cryptomerioides occurring in Taiwan. BAYESCAN detected 26 FST outlier loci that were found to be associated strongly with various environmental variables using multiple univariate logistic regression, latent factor mixed model, and Bayesian logistic regression. We found several environmentally dependent adaptive loci with high frequencies in low- or high-elevation populations, suggesting their involvement in local adaptation. Ecological factors, including relative humidity and sunshine hours, that are generally not altitude related could have been the most important selective drivers for population divergent evolution in C. formosana. The present study provides fundamental information in relation to adaptive evolution and can be useful for assisted migration program of C. formosana in the future conservation of this species.

Investigation of Genetic Diversity in Afghan Bread Wheat Genotypes Using SSR and AFLP Markers

Genetic diversity assessment is the principle component for conservation and characterization of germplasm. Genetic diversity study of Afghan bread wheat genotypes is a first step to identify and to select high performance genotypes and distribute to wheat breeding programs. The main objective of this study is to investigate of genetic diversity in 35 Afghan bread wheat genotypes by using Simple Sequence Repeat (SSR) and Amplified Fragment Length Polymorphism (AFLP) markers. DNA extraction according to Cetyl Trimethyl Ammonium Bromide (CTAB) method was conducted and the total genomic DNA was isolated from each variety. Sixty-four SSR primer markers were used and eighteen EcoRI+(N)/MseI+(N) primer combinations with their primer sequences were used for selective polymerase chain reaction (PCR) amplification. Every SSR and AFLP fragment was scored as present (1) or absent (0) within all genotypes under study. Marker/ Value ratio of pairwise genetic distance between genotypes according to the SSRs data was from 0.508 to 0.691 with an average distance of 0.599. Relatively different grouping pattern in comparison to AFLP data observed through cluster analysis. Both types of molecular markers (AFLP and SSR) used in this research proved to be suitable for investigating genetic diversity in the genotypes of Afghan bread wheat, however, AFLP markers gave better view of genetically relationships among genotypes than the SSR markers. The grouping generated by AFLP data showed a special agreement with the origin regions of genotypes (Ariana-07 and Mazar-99 originating from the north of Afghanistan, Lalmi-03 and Kabul-02. Large number of DNA bands identified with AFLP markers might provide a better estimation of genetic similarity than those of SSR markers.

Sequence capture using AFLP-generated baits: A cost-effective method for high-throughput phylogenetic and phylogeographic analysis.

Target sequence capture is an efficient technique to enrich specific genomic regions for high‐throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP‐based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome‐scale data (hundreds or thousands of loci).

AFLP-AFLP in silico-NGS approach reveals polymorphisms in repetitive elements in the malignant genome.

The increasing interest in exploring the human genome and identifying genetic risk factors contributing to the susceptibility to and outcome of diseases has supported the rapid development of genome-wide techniques. However, the large amount of obtained data requires extensive bioinformatics analysis. In this work, we established an approach combining amplified fragment length polymorphism (AFLP), AFLP in silico and next generation sequencing (NGS) methods to map the malignant genome of patients with chronic myeloid leukemia. We compared the unique DNA fingerprints of patients generated by the AFLP technique approach with those of healthy donors to identify AFLP markers associated with the disease and/or the response to treatment with imatinib, a tyrosine kinase inhibitor. Among the statistically significant AFLP markers selected for NGS analysis and virtual fingerprinting, we identified the sequences of three fragments in the region of DNA repeat element OldhAT1, LINE L1M7, LTR MER90, and satellite ALR/Alpha among repetitive elements, which may indicate a role of these non-coding repetitive sequences in hematological malignancy. SNPs leading to the presence/absence of these fragments were confirmed by Sanger sequencing. When evaluating the results of AFLP analysis for some fragments, we faced the frequently discussed size homoplasy, resulting in co-migration of non-identical AFLP fragments that may originate from an insertion/deletion, SNP, somatic mutation anywhere in the genome, or combination thereof. The AFLP–AFLP in silico–NGS procedure represents a smart alternative to microarrays and relatively expensive and bioinformatically challenging whole-genome sequencing to detect the association of variable regions of the human genome with diseases.

English

Among the biological constraints facing maize production in Tanzania is a severe occurrence of maize lethal necrosis disease (MLN) raising an urgent need for application of new approaches. A pool of 22 maize genotypes with promising resistance and susceptibility to MLN infection were evaluated by Amplified fragment length polymorphism (AFLP) DNA fingerprinting analysis to detect genetic variation in the selected lines. Eleven AFLP primer combinations were screened and resulted in the identification of 95 polymorphic AFLP allelic fragments. Genetic similarities among the selected Tanzanian maize landraces and other maize lines were estimated by Unweighted Pair Group of Arithmetic Mean (UPGMA) and genotypes were clustered in three primary groups according to their reaction to MLN disease. Promising resistant and tolerant genotypes were grouped in cluster I and susceptible genotypes in clusters II and III. Landraces were grouped according to agro-ecological locations where they were collected. Unambiguous polymorphic AFLP fragments were eluted, purified and sequenced. Sequencing and nucleotide alignment on Basic Local Alignment Search Tool (BLAST) analysis showed similarities of fragments consistent with transcripts involved in disease resistance and stress responses. Further studies will explore the potential application of the identified AFLP markers and their significant association to MLN disease resistance genes in maize. Key words: Zea mays, AFLP, maize lethal necrosis disease, Tanzania.

Analysis of genetic polymorphisms and tropism in East African Leishmania donovani by Amplified Fragment Length Polymorphism and kDNA minicircle sequencing

Visceral leishmaniasis (VL), the most severe form of leishmaniasis, is caused by Leishmania donovani. In addition to fatal VL, these parasites also cause skin diseases in immune-competent and -suppressed people, post-kala azar dermal leishmaniasis (PKDL) and HIV/VL co-infections, respectively. Genetic polymorphism in 36 Ethiopian and Sudanese L. donovani strains from VL, PKDL and HIV/VL patients was examined using Amplified Fragment Length Polymorphism (AFLP), kDNA minicircle sequencing and Southern blotting. Strains were isolated from different patient tissues: in VL from lymph node, spleen or bone marrow; and in HIV/VL from skin, spleen or bone marrow. When VL and PKDL strains from the same region in Sudan were examined by Southern blotting using a DNA probe to the L. donovani 28S rRNA gene only minor differences were observed. kDNA sequence analysis distributed the strains in no particular order among four clusters (A – D), while AFLP analysis grouped the strains according to geographical origin into two major clades, Southern Ethiopia (SE) and Sudan/Northern Ethiopia (SD/NE). Strains in the latter clade were further divided into subpopulations by zymodeme, geography and year of isolation, but not by clinical symptoms. However, skin isolates showed significantly (p < 0.0001) fewer polymorphic AFLP fragments (average 10 strains = 348.6 ± 8.1) than VL strains (average 26 strains = 383.5 ± 3.8).

Before-after analysis of genetic and epigenetic markers in garlic: A 13-year experiment

Fil: Gimenez, Magali Diana. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnologico Conicet - San Juan. Centro de Investigaciones de la Geosfera y Biosfera. Universidad Nacional de San Juan. Facultad de Ciencias Exactas Fisicas y Naturales. Centro de Investigaciones de la Geosfera y Biosfera; Argentina

Inheritance and gene mapping of the white flower trait in Brassica juncea

Despite being a unique marker trait, white flower inheritance in Brassica juncea remains poorly understood at the gene level. In this study, we investigated a B. juncea landrace with white petal in China. The white petal phenotype possessed defective chromoplasts with less plastoglobuli than the yellow petal phenotype. Genetic analysis confirmed that two independent recessive genes (Bjpc1 and Bjpc2) controlled the white flower trait. We then mapped the BjPC1 gene in a BC4 population comprising 2295 individuals. We identified seven AFLP (amplified fragment length polymorphism) markers closely linked to the white flower gene. BLAST search revealed the sequence of AFLP fragments were highly homologous with the Scaffold000085 and Scaffold000031 sequences on the A02 chromosome in the Brassica rapa genome. Based on this sequence homology, we developed simple sequence repeat (SSR) primer pairs and identified 13 SSRs linked to the BjPC1 gene, including two that were co-segregated (SSR9 and SSR10). The two closest markers (SSR4 and SSR11) were respectively 0.9 and 0.4 cM on either side of BjPC1. BLAST analysis revealed that these marker sequences corresponded highly to A02 in B. juncea. They were mapped within a 33 kb genomic region on B. rapa A02 (corresponds to a 40 kb genomic region on B. juncea A02) that included three genes. Sequence BjuA008406, homologous to AtPES2 in Arabidopsis thaliana and Bra032956 in B. rapa, was the most likely candidate for BjPC1. These results should accelerate BjPC1 cloning and facilitate our understanding of the molecular mechanisms controlling B. juncea petal color.

Levels of Intra-specific AFLP Diversity in Tuber-Bearing Potato Species with Different Breeding Systems and Ploidy Levels.

DNA-based marker analysis of plant genebank material has become a useful tool in the evaluation of levels of genetic diversity and for the informed use and maintenance of germplasm. In this study, we quantify levels of amplified fragment length polymorphism (AFLP) in representative accessions of wild and cultivated potato species of differing geographic origin, ploidy, and breeding system. We generated 449 polymorphic AFLP fragments in 619 plants, representing multiple plants (16–23) from 17 accessions of 14 potato taxa as well as single plants sampled from available accessions (from 3 to 56) of the same 14 taxa. Intra-accession diversities were compared to those of a synthetic ‘taxon-wide’ population comprising a single individual from a variable number of available accessions of each sampled taxon. Results confirm the expected considerably lower levels of polymorphism within accessions of self-compatible as compared to self-incompatible taxa. We observed broadly similar levels of ‘taxon-wide’ polymorphism among self-compatible and self-incompatible species, with self-compatible taxa showing only slightly lower rates of polymorphism. The most diverse accessions were the two cultivated potato accessions examined, the least diverse being the Mexican allohexaploids Solanum demissum and S. iopetalum. Generally allopolyploid self-compatible accessions exhibited lower levels of diversity. Some purported self-incompatible accessions showed relatively low levels of marker diversity, similar to the more diverse self-compatible material surveyed. Our data indicate that for self-compatible species a single plant is highly representative of a genebank accession. The situation for self-incompatible taxa is less clear, and sampling strategies used will depend on the type of investigation. These results have important implications for those seeking novel trait variation (e.g., disease resistance) in gene banks as well as for the selection of individuals for genomics studies. We also show that AFLPs, despite having been largely replaced by other marker types, is highly suitable for the evaluation of within and between accession diversity in genebanks.

Identification and characterization of novel single nucleotide polymorphism markers for fat deposition in muscle tissue of pigs using amplified fragment length polymorphism.

ObjectiveThis study was conducted to identify and evaluate the effective single nucleotide polymorphism (SNP) markers for fat deposition in the longissimus dorsi muscles of pigs using the amplified fragment length polymorphism (AFLP) approach.MethodsSixty-four selective primer combinations were used to identify the AFLP markers in the 20 highest- and 20 lowest-intramuscular fat (IMF) content phenotypes. Five AFLP fragments were converted into simple codominant SNP markers. These SNP markers were tested in terms of their association with IMF content and fatty acid (FA) composition traits in 620 commercially crossbred pigs.ResultsThe SSC7 g.4937240C>G marker showed an association with IMF content (p<0.05). The SSC9 g.5496647_5496662insdel marker showed a significant association with IMF content and arachidonic levels (p<0.05). The SSC10 g.71225134G>A marker revealed an association with palmitoleic and ω9 FA levels (p<0.05), while the SSC17 g.61976696G>T marker showed a significant association with IMF content and FA levels of palmitoleic, eicosenoic, arachidonic, monounsaturated fatty acids, and ω9 FA levels. However, no significant association of SSC8 g.47338181G>A was observed with any IMF and FA levels in this study.ConclusionFour SNP markers (SSC7 g.4937240C>G, SSC9 g.5496647_5496662insdel, SSC10 g.71225134G>A, and SSC17 g.61976696G>T) were found to be associated with IMF and/or FA content traits in commercially crossbred pigs. These findings provide evidence of the novel SNP markers as being potentially useful for selecting pigs with the desirable IMF content and FA composition.

Assessing the genetic diversity and population structure of the endangered Chascolytrum bulbosum (Poaceae, Poeae) using AFLP markers

Chascolytrum bulbosum occurs in shrubby fields with granite outcrops in southern South America. It is considered endangered, due to habitat loss and overgrazing. However, no population genetic studies nor conservation management plan for the species is currently available. Therefore, Amplified Fragment Length Polymorphism (AFLP) markers were used to assess genetic diversity and infer population structure of 13 natural populations of C. bulbosum distributed across southern Brazil. A total of 651 AFLP fragments were produced, of which 98.2% were polymorphic. Genetic diversity was considered moderate (H = 0.150), with genetic differentiation being moderate to high (GST = 0.228; FST = 0.341). Analyses enabled the separation of individuals into two clusters, although AMOVA results suggest only 13.2% variation between them. Estimates of gene flow by migration (Nm) were high in all analyses. Results suggest that no severe bottleneck has occurred recently in populations analyzed in this study. As gene flow occurs broadly through the geographical range of species found in Rio Grande do Sul, we suggest the stimulation of in situ rather than ex situ conservation, and priority populations for conservation of C. bulbosum are indicated.

Genetic variation among Fusarium verticillioides isolates associated with Ethiopian maize kernels as revealed by AFLP analysis

Amplified fragment length polymorphism (AFLP) was used to study the genetic variation among 80 F. verticillioides isolates from kernels of Ethiopian maize, collected from 20 different maize growing areas in four geographic regions. A total of 213 polymorphic fragments were obtained using six EcoRI/MseI primer combinations. Analysis of the data based on all 213 polymorphic AFLP fragments revealed high level of genetic variation in the F. verticillioides entities in Ethiopia. About 58% of the fragments generated were polymorphic. The genetic similarity among F. verticillioides isolates varied from 46% to 94% with a mean Dice similarity of 73%. Unweighted Pair Group Method with Arithmetic Average (UPGMA) analysis revealed two main groups and four subgroups. The principal coordinate analysis (PCO) also displayed two main groups that agreed with the results of UPGMA analysis, and there was no clear pattern of clustering of isolates according to geographic origin. Analysis of molecular variance: (AMOVA) showed that only 1.5% of the total genetic variation was between geographic regions, while 98.5% was among isolates from the same geographic regions of Ethiopia. Eighty distinct haplotypes were recognized among the 80 isolates analyzed. Hence, breeding efforts should concentrate on quantitative resistance that is effective against all genotypes of the pathogen.

Genetic Variability and Proteome Profiling of a Radiation Induced Cellulase Mutant Mushroom Pleurotus florida.

We report the genetic similarity changes between a mutant mushroom (Pleurotus florida, designated as PfCM4) having increased cellulolytic activity developed through radiation mutagenesis and its wild type by amplified fragment length polymorphism (AFLP). On average, 23 AFLP fragments were amplified per primer combination, and a total of 286 polymorphic fragments (78.57% polymorphism) with maximal fragment length of 1365 base pairs (bp) were obtained. The genetic similarity between wild type and PfCM4 was found to be 22.30%. In addition, mycelial and secreted protein profiling by 2D-PAGE showed at least three and five different protein spots in the range of 25 kD to 100 kD, respectively, in PfCM4. It seems that the variation in genetic similarity and different expression of both mycelial and secreted proteins in PfCM4 in comparison to the wild type could likely be correlated with its increased cellulolytic activity effected by the irradiation.

Genetic diversity among synthetic hexaploid wheat accessions (Triticum aestivum) with resistance to several fungal diseases

Synthetic hexaploid wheat (SHW) is known to be an excellent vehicle for transferring large genetic variations especially the many useful traits present in the D genome of Aegilops tauschii Coss. (2n = 2x = 14, DD) for improvement of cultivated wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD). The objectives of the present study were to (1) evaluate genetic diversity among 32 selected SHW accessions with resistance to several fungal diseases using Amplified Fragment Length Polymorphism (AFLP) and Simple Sequence Repeat (SSR) markers and (2) identify diverse SHWs for pyramiding genes conferring resistance to different diseases. These SHWs containing different accessional sources of the D genome were identified from about 1000 SHW accessions developed by the Wheat Wide Crosses program at the International Maize and Wheat Improvement Center, Mexico. Of the 32 SHW accessions eight had resistance to Fusarium head blight (Fusarium graminearum Schw.), seven were resistant to leaf rust (Puccinia triticina Eriks.), eight resistant to Helminthosporium spot blotch [Cochliobolus sativus (Ito et Kurib.) Drechsler ex Dastur (syn.: Bipolaris sorokiniana (Sacc.) Shoem., Helminthosporium sativum Pammel, King et Bakke)], seven resistant to Septoria tritici blotch (Septoria tritici Roberge in Desmaz.), while two were resistant to both Fusarium head blight and leaf rust. Seventeen EcoRI/MseI AFLP primer combinations and 27 highly polymorphic SSR markers including 20 D genome specific markers were screened over all 32 SHW accessions. Among the 703 AFLP fragments scored, 225 were polymorphic across the 32 SHW accessions. Polymorphic information content (PIC) among the SHWs for AFLP ranged from 0.06 to 0.50 with an average PIC of 0.24. Major allelic frequency from SSR analysis ranged from 0.23 to 0.81 with an average of 0.45. Number of alleles per locus for the SSR markers ranged from 3 to 15 with an average allele number of 7.4. Average gene diversity and PIC for the SSR markers was 0.69 and 0.66, respectively, with the highest values being for the D genome specific markers. Cluster analysis showed distinct groups among the SHW accessions studied. Mantel statistics between the distance matrices from AFLP and SSR analyses showed a moderate but significant correlation (r = 0.52**). Our results indicate that the SHW accessions studied possess substantial genetic diversity and are valuable user-friendly pre-breeding materials for breeding improvement of wheat with resistance to Fusarium head blight, leaf rust, Helminthosporium spot blotch, and Septoria tritici blotch. The most diverse SHW accessions can be used for pyramiding resistance genes for different diseases.

Lack of Hybridization between Naturally Sympatric Populations of Red and Blacktail Shiner (Cyprinella lutrensisandC. venusta) in Texas, but Evidence of Introgression among Three Lineages of theC. lutrensisSpecies Group

Hybridization is more common in actinopterygian fishes than any other group of vertebrates. This is especially true for members of the family Cyprinidae; for example, Hubbs found 68 different combinations of inter-generic and intra-generic hybridization among cyprinids collected east of the continental divide. Hybridization between two cyprinid species, the red shiner and the blacktail shiner (Cyprinella lutrensis and C. venusta), has been described in detail in Georgia where C. lutrensis is an introduced species. However, hybridization has not been thoroughly assessed where the two species are naturally sympatric. Our specific objectives were to determine the extent of ongoing hybridization between the two species using nuclear markers and morphometrics and to determine the extent of historical introgression using mitochondrial DNA (mtDNA). We collected 100 individuals from four different locations along the Bosque River and an additional 100 individuals from four locations along the Paluxy River. We used amplified fragment length polymorphism (AFLP) to verify species identification and determine hybrid status of each individual. A total of 56 AFLP fragments were scored, with 82.14% (47 fragments) being polymorphic (95% criterion). Based on these nuclear markers, we only identified two hybrids out of the 200 specimens analyzed; one from the Bosque River, Texas and one from the Paluxy River, Texas. There were no instances of introgression of mitochondrial DNA (mtDNA) from one species into the nuclear background of the other species. We did, however, discover the sympatric occurrence of three mtDNA lineages of C. lutrensis within a single nuclear gene pool; there was only one mtDNA lineage of C. venusta. Overall body shape was assessed using a truss network and was found to be statistically different with red shiner having the shorter and deeper body. The minimal amount of hybridization inferred from AFLP data, in combination with the absence of mtDNA introgression and limited morphological overlap, indicates that pre- or postzygotic isolating mechanisms effectively minimize genetic exchange between naturally sympatric populations of C. lutrensis and C. venusta within these two river systems.

Genetic and phenotypic diversity for drought tolerance in perennial ryegrass (Lolium perenne L.)

Perennial ryegrass (Lolium perenne L.) is a widely grown species in temperate regions as forage grass as well as for recreational and bioenergy production purposes. Information on perennial ryegrass genetic background facilitates breeding programs by providing an assessment of genetic diversity in exotic material. Genetic diversity of 104 genotypes of perennial ryegrass was evaluated using phenotypic drought traits and amplified fragment length polymorphism (AFLP) data. A high variation was observed for the drought tolerance traits. Chlorophyll fluorescence (F v /F m ) explained 88.8% of the whole variation observed in the collection, while re-growth accounted for 9.1% of all variation. A principal component analysis on the basis of phenotypic drought tolerance traits classified perennial ryegrass collection into three clusters. Three AFLP primer pairs produced a total of 210 fragments, 202 of which were polymorphic among all accessions. The genetic diversity of the collection was high with an average similarity coefficient of 0.46 and the average polymorphic information content of 0.29. The principal component analysis based on AFLP data did not cluster genotypes into any major group. A total of six AFLP fragments, identified as being prevalent in drought tolerant genotypes, together with the high degree of genetic homogeneity found, could provide a choice of selecting genotypes from this perennial ryegrass collection for a drought tolerance breeding program.

Genome-wide macrosynteny among Fusarium species in the Gibberella fujikuroi complex revealed by amplified fragment length polymorphisms.

The Gibberella fujikuroi complex includes many Fusarium species that cause significant losses in yield and quality of agricultural and forestry crops. Due to their economic importance, whole-genome sequence information has rapidly become available for species including Fusarium circinatum, Fusarium fujikuroi and Fusarium verticillioides, each of which represent one of the three main clades known in this complex. However, no previous studies have explored the genomic commonalities and differences among these fungi. In this study, a previously completed genetic linkage map for an interspecific cross between Fusarium temperatum and F. circinatum, together with genomic sequence data, was utilized to consider the level of synteny between the three Fusarium genomes. Regions that are homologous amongst the Fusarium genomes examined were identified using in silico and pyrosequenced amplified fragment length polymorphism (AFLP) fragment analyses. Homology was determined using BLAST analysis of the sequences, with 777 homologous regions aligned to F. fujikuroi and F. verticillioides. This also made it possible to assign the linkage groups from the interspecific cross to their corresponding chromosomes in F. verticillioides and F. fujikuroi, as well as to assign two previously unmapped supercontigs of F. verticillioides to probable chromosomal locations. We further found evidence of a reciprocal translocation between the distal ends of chromosome 8 and 11, which apparently originated before the divergence of F. circinatum and F. temperatum. Overall, a remarkable level of macrosynteny was observed among the three Fusarium genomes, when comparing AFLP fragments. This study not only demonstrates how in silico AFLPs can aid in the integration of a genetic linkage map to the physical genome, but it also highlights the benefits of using this tool to study genomic synteny and architecture.

Comparison of two detection systems to reveal AFLP markers in plants

Since their development, AFLP (amplified fragment length polymorphism) markers have been used for a wide variety of analyses and, up to this day, are considered highly informative, robust, and reproducible molecular markers. Originally, the visualization of the amplified fragments was done in polyacrylamide gels, followed by silver staining or by developing in an X-ray plate, when radioactivity is used. In the last 14 years, capillary electrophoresis of fluorescently labeled fragments has been gradually replacing gel-based systems. However, the latter continue to be better for isolating and cloning AFLP fragments. In this report, we compare the results obtained by capillary electrophoresis with those from silver staining. We found that if fluorescence-labeled amplification products are loaded in a polyacrylamide gel, duplicated bands (doublets) are seen. This phenomenon is probably due to a delay in the migration of the strand that carries the fluorophore. Therefore, we recommend a minimum separation of 4 bp from the nearest fragment to the target fragment for its unambiguous identification and isolation. If this requirement is not fulfilled, an alternative is to make new amplifications using the same primer combination, but with unlabeled primers.

Genetic Diversity and Population Structure of Sardinian Myrtle (Myrtus communis L.) Selections as Obtained by AFLP Markers

Myrtle is an aromatic plant typical of the Mediterranean maquis. It is widely exploited in pharmaceutical, cosmetic, and food industries, whereas in Italy, it is mainly used for the production of the typical liqueur. Amplified fragment length polymorphism (AFLP) markers were used to evaluate the genetic variability of some Sardinian Myrtus communis L. candidate cultivars. The AFLP selective amplification produced 138 reproducible AFLP fragments, 96% of which were polymorphic. STRUCTURE analysis divided the myrtle accessions into two main genetic groups (K = 2). The two clusters showed different numbers of individuals. Most of the individuals belonged to Cluster B, whereas only eight genotypes were attributed to Cluster A. Unweighted pair group method with Arithmetic Mean (UPGMA) dendrogram segregated all the myrtle cultivars into five main groups, displaying a partial congruence with the division observed by STRUCTURE analysis. The analysis of the genetic diversity distribution in a candidate cultivar selection displayed a geographical gradient of myrtle from north to south, which reflects the Sardinian shape, and from west to east, which reflects the Sardinian mountain distribution.

Genetic variation among pathotypes of Verticillium dahliae Kleb. from cotton in western Turkey revealed by AFLP

Cotton (Gossypium hirsutum L.) is crucial for the textile industry worldwide. Among the diseases attacking cotton, Verticillium wilt caused by Verticillium dahliae Kleb. is the most significant. Isolates of V. dahliae can be classified into defoliating and non-defoliating pathotypes. Thirty-two isolates of the non-defoliating pathotype and one isolate of a virulent, defoliating pathotype were analysed by the amplified fragment length polymorphism (AFLP) method. Three hundred and forty AFLP fragments were obtained with nine primer combinations. The number of total bands per primer pair ranged from 16 to 81, with an average of 37.7. The average polymorphism information content (PIC) value for the AFLP products was 0.50. Using the genotypic data, genetic distance analysis was performed. The maximum variation was found between isolates (Vd11) Nazilli and (Vd16) Soke, at a value of 0.79 and the minimum variation was found between isolates (Vd20) Aydin and (Vd14) Soke, at 0.24. The unweighted paired group method with arithmetic averages cluster analysis (UPGMA) was used to discriminate the V. dahliae isolates into five subgroups. Defoliating pathotypes (Vd33) from Soke province formed a single subgroup. As a result, it was found that there was significant variation among Verticillium isolates. AFLP analysis is an efficient and effective marker technology for determining genetic relationships among Verticillium isolates.