Amplified Fragment Length Polymorphism Markers Research Articles

Genetic diversity and structure of Tunisian and Indian date palm (Phoenix dactylifera and sylvestris) cultivars and genotypes revealed by AFLP markers

The date palm breeding programs need to discover valid genetic fingerprints to characterize cultivars and assess their genetic diversity. This study assessed the genetic diversity among thirty-nine date palm cultivars from Tunisia (Phoenix dactylifera) and India (Phoenix sylvestris) by using six AFLP (Amplified Fragment Length Polymorphism) markers. 360 loci were amplified, with 127 loci polymorphic (34.35 %). The Jaccard's similarity coefficient ranged from 0.161 to 0.931, with the mean genetic distances of 0.568. AFLP's average marker index value was 7.28, with a resolving power of 10.91. The analysis of population structure showed two main clusters with a clear separation between Tunisian and Indian cultivars.Furthermore, the heatmap analysis allowed the identification of 10 bands specific to the Indian accessions, which were not detected in Tunisian genotypes. These loci could be linked to genes involved in adapting the species in Indian lands, which allowed the study of the genetic diversity of date palm resources of different origins, confirming the existence of at least two origins of domestication. Additionally, identifying AFLP loci specific to P. dactylifera and P. sylvestris will significantly contribute to breeding programs by exploiting species-specific polymorphisms.

Serpentine environment prevails over geographic distribution in shaping the genetic diversity of Medicago lupulina L.

Edaphic conditions of serpentine soils, naturally rich in heavy metals, act as a strong selection pressure that shapes specific metal-tolerant ecotypes. Medicago lupulina L. (black medick) is not only a widespread plant species that prefers calcareous and dry soil types but also grows at the borders of serpentine formations. It can also be found in waste and disturbed habitats. This is a species with reported phytoremediation potential, however, there is no published data regarding the impact of the environment on the genetic distribution of this species. The aim of our research was to explore how selection pressure of serpentine soils affects genetic diversity of M. lupulina and to test heavy-metal accumulation capacity of this species. Specimens of 11M. lupulina populations were collected from serpentine outcrops located in Central and Eastern Bosnia as well as from non-serpentine sites. Soil and plant samples were analyzed for the total contents of heavy metals using air-acetylene flame atomic absorption spectroscopy. Genetic diversity was analyzed using AFLP (Amplified Fragment Length Polymorphism) markers. Serpentine soils showed high nickel, cobalt, chromium and iron concentrations. Nickel and manganese concentrations in soil samples and plant material showed statistically significant correlation. Although plants in two populations show the ability to extract Ni, M. lupulina does not show hyperaccumulating properties. Despite severe selective pressure, genetic diversity in serpentine populations is not reduced. Analyses of intrapopulation and interpopulation genetic diversity showed significant genetic differentiation among populations which is not related to their geographic distance. Population from non-metalliferous soil showed clear separation from all other populations. Diversity data suggest that serpentine populations maintain genetic diversity by undetected mechanisms and that edaphic factors rather than geography influence genetic structure analyzed M. lupulina populations.

Diversity of Genetic and Vegetative Compatibility Group of Colletotrichum coccodes Isolates from Chile Using Amplified Fragment Length Polymorphism Markers.

Colletotrichum coccodes (Wallr.) Hughes is an asexual fungus with five vegetative compatibility groups. It was postulated that C. coccodes was isolated at the center of origin of potato at one time, and due to the movement of potato around the globe, the fungus was established on each continent but became bottlenecked and genetically unable to form stable heterokaryons via vegetative compatibility grouping (VCG) studies. The objectives of this study were (i) to determine if the VCGs around the world are related to the VCGs in Chile, (ii) to determine the diversity of C. coccodes populations in Chile, and (iii) to find any evidence for a cryptic sexual life cycle for this fungus. Worldwide C. coccodes populations have been found to be genetically correlated and belong to one or more C. coccodes-identified VCGs. The most distributed VCG in Chile was VCG2, which is the most common VCG in North America. We hypothesize that one or more VCGs had spread from Chile to the rest of the world. Precautions and further studies should be investigated by using other molecular markers and gene sequencing.

Novel amplified fragment length polymorphism (AFLP) markers for typing medically relevant Fusarium and allied fusarioid genera

Fusariosis is an emerging mycosis caused by diverse Fusarium and allied fusarioid genera that are characterized by spindle-shaped macroconidia. These fungi possess a broad ecological distribution, causing infections in a wide diversity of hosts, spanning the animal and plant kingdoms. The spectrum of human fusariosis encompasses superficial lesions like keratitis and onychomycosis to invasive fungal diseases. Notable genera within the medically relevant fusarioid group include Neocosmospora, Fusarium s. str., and Bisifusarium. While Neocosmospora species (formerly F. solani species complex) are primary causative agents of human fusariosis, instances involving Fusarium s. str. and Bisifusarium (formerly F. dimerum species complex) have been reported. There is an urgent need for DNA-based markers to explore the epidemiology of these emerging fusarioid pathogens using molecular methods. We took advantage of fusarioid genomes available in NCBI (n = 20) to optimize the development of novel amplified fragment length polymorphism (AFLP) markers by conducting in-depth in silico analyses to refine their applicability for studies on these pathogens’ genetic epidemiology. In-silico screening highlighted eight primer pair combinations (C1–C8) to be tested in vitro. The AFLP protocol was used for genotyping 40 medically relevant fusarioid fungi. Based on the overall scored AFLP markers (77–93 fragments), the values of polymorphism information content (PIC = 0.3474–0.3725), marker index (MI = 0.0038–0.0056), effective multiplex ratio (E = 26.3750–40.4750), resolving power (Rp = 40.1500–54.6000), discriminating power (D = 0.7978–0.8857), expected heterozygosity (H = 0.4476–0.4949), and mean heterozygosity (Havp = 0.0001) demonstrated the utility of these primer combinations for discriminating Neocosmospora, Fusarium s. str., and Bisifusarium species. Of relevance, some AFLP panels were better than others at studying genetic trends in Neocosmospora (#2 EcoRI-AT/MseI-TA, #3 EcoRI-AA/MseI-TT, and #5 EcoRI-AT/MseI-AG) or Fusarium s. str. (mainly #2 EcoRI-AT/MseI-TA and #6 EcoRI-GA/MseI-TT) and Bisifusarium (#1 EcoRI-GA/MseI-AG and #6 EcoRI-GA/MseI-TT), and these combinations will better resolve disease transmission routes. Our DNA fingerprint assay has proven effective by exhibiting rapidity, reproducibility, and high discriminatory capabilities, which represents a valuable asset in the ongoing efforts to combat fusariosis and enhance our scientific understanding of medically relevant Fusarium and allied fusarioid genera.

STUDY OF NON-CODING AREAS OF THE DNA GENOME OF DIFFERENT ANIMAL SPECIES

STUDY OF NON-CODING AREAS OF THE DNA GENOME OF DIFFERENT ANIMAL SPECIES

Genetic and Morphological Variation of Belgian Cyperus esculentus L. Clonal Populations and Their Significance for Integrated Management

Cyperus esculentus is an invasive troublesome neophyte in many arable crops across the globe. Analysis of the genetic and morphological profile of local C. esculentus clonal populations may be useful in explaining differential herbicide sensitivity found among distinct clonal populations and spatial distribution patterns. In this study, 35 Belgian C. esculentus clonal populations, evenly spread across the entire infestation area (30,689 km2) and covering a great diversity of farm and soil types, and hydrological and environmental conditions, were genetically characterized using amplified fragment length polymorphism (AFLP) profiling. These clonal populations were also grouped into different morphological clusters using data from shoot, tuber, and inflorescence number, fresh tuber biomass, individual fresh tuber weight, and number of germinable seeds collected in three consecutive years. Of the 271 AFLP markers, 207 were polymorphic. The gene diversity among clonal populations was 0.331 and three genetically distinct clusters were identified. Depending on observation year, clonal populations were grouped in four to five morphologically distinct clusters that closely aligned with the genetic clusters. The genetically distinct clusters differed in their geographical distribution pattern and range as well as in their morphological characteristics. Clonal populations belonging to clusters with broad distribution ranges produced numerous viable seeds. Clusters with clonal populations that produced large tubers were less widespread than the cluster with clonal populations producing many small tubers. The results suggest that tuber size, tuber number, and fecundity may all play an important role in the spread of C. esculentus. Morphotyping may be very useful in designing effective preventive and curative C. esculentus management strategies.

Molecular Characterization of Olive (Olea europaea L.) Cultivars in Duhok Governorate

The olive tree (Olea europaea L.) was considered one of the most cultivated crops in the Mediterranean region, and it had an economically important value due to it began consumed as a table olive and oil production. In this study, AFLP and SRAP techniques were used to identify 15 cultivars of olive plants that were collected from 12 different locations in the Duhok governorate, eleven primer combinations of sequence-related amplified polymorphism (SRAP) and ten primer combinations of amplified fragment length polymorphism (AFLP) were used to fingerprinting these cultivars. The results show that the SRAP marker had the least genetic distance 0.241between (Shtla falay olive and Khodeiri olive) and the highest value 0.946 was found between (Zawita olive and Sorani olive). While AFLP marker show the lowest value of 0.107 between (Nzarky olive and Dger olive) and the highest value of 0.916 between (Amedi olive and Shtla falay olive). Cluster analysis based on AFLP and SRAP combined data clearly differentiated the genotype and it showed it was a powerful tool to evaluate the genetic distance and fingerprinting of these tested cultivars. Establishing the genetic relationships between cultivars and species, and the construction of genetic maps, had the main aims for the researchers in plant breeding programs and to understand the plant evolution lingues, and as a conclusion, this study demonstrates that AFLP and SRAP markers both are powerful tools in plants tree genetics.

Mendelian segregation for parthenogenetic embryo development at the diploid level in the flowering plant Erigeron.

Parthenogenesis is the capacity of organisms to develop embryos from unfertilized eggs. When parthenogenesis is coupled with unreduced gamete formation (apomeiosis), genetically maternal progeny result. Genetic elucidation of this form of reproduction in plants, apomixis, has important agronomic implications. However, genetic characterization of apomeiosis and parthenogenesis has been problematic in part because the traits usually co-occur and are restricted to polyploids. In this work, the inheritance of parthenogenetic embryo development, by itself, was studied at the diploid level. Progeny resulting from a cross between a diploid (2n = 18), heterozygous, parthenogenetic pollen donor, and a diploid, wildtype, sexual seed parent were evaluated. Paternity was tested with conserved orthologous sequence (COS) markers, reproductive development of F1s was evaluated with microscopy of cleared ovules, and an amplified fragment length polymorphism (AFLP) marker (Eagc × Macg.615) co-segregating with parthenogenesis was characterized at the sequence level. Of 102 diploid biparental progeny, 47 exhibited parthenogenetic embryo and endosperm development, and 55 lacked development of the egg and central cell. This result is consistent with Mendelian inheritance for a single locus (P = 0.43). Isolation and sequencing of the AFLP marker indicates that it is likely a portion of a Ty-Gypsy retrotransposon. Attempts to develop a sequence-characterized amplified region marker from the AFLP were unsuccessful. This work shows that parthenogenesis can be transmitted simply at the diploid level. This advance is key in the development of a tractable system in Erigeron aimed at the identification of the parthenogenesis locus using genetic mapping strategies.

Assessment of the genetic variations of sago palm Metroxylon sagu in three regions of Sarawak, Malaysia using amplified fragment length polymorphism (AFLP) marker

BackgroundSago palm (Metroxylon sagu) is an important staple crop in the rural area of Sarawak, Malaysia. The palms grow well in the rough, swampy peat, from low flooded areas to uplands and acidic to neutral soils. Among the starch-producing crops, this palm is the most productive and promising, where it can store a significant amount of carbohydrate in the trunk. Due to the lack of molecular study of sago palm, the work described here aimed to develop molecular markers for identifying M. sagu and accessing the genetic variations of sago palm in different locations of Sarawak.ResultsAmplified fragment length polymorphism was employed to determine the genetic variations of sago palm between fifteen populations collected from three different locations: Mukah, Pusa and Samarahan. Based on 35,016 polymorphic fragments generated from 32 primer combinations, polymorphic information content (PIC), marker index (MI) and resolving power (RP) averaged 0.269, 0.005157 and 51.52039 per primer combination, respectively, were obtained, signifying the effectiveness and accuracy of the AFLP marker. Mukah showed higher diversity (I = 0.428, H = 0.232), while Pusa showed the lowest (I = 0.367, H = 0.240). The average value of the Shannon information index was 0.404. AMOVA showed 99% of the variation was found within the population. The Mantel test was performed with 9999 permutations. A significantly positive correlation (r = 0.220, p = 0.010) was observed between the genetic divergence of the population (Nei genetic distance) with the geographical distance.ConclusionsOur study considered AFLP is as an effective and reliable method to assess the genetic variations and the identification of M. sagu.Graphical

Evaluation of growth and yield of indigo ecotypes in different sowing dates and genetic diversity analysis using AFLP markers

AbstractIndigo (Indigoferatinctoria L.), as a forgotten crop, has immense industrial and medicinal value. In this research, the effect of sowing date on the leaf, seed, and dye yield of indigo was examined at Jiroft, Iran, during the 2013 and 2014 growing seasons. The experiment was arranged as a split‐plot based on a randomized complete block design with three replications. Planting dates (5 May, 20 May, and 5 June) and indigo ecotypes as landraces of Jiroft, Roudbar, Rigan, and Ghale‐ganj were considered as main and subplots, respectively, aiming to evaluate their effects on pod number/plant, 1,000‐seed weight, seed number/pod, leaf dry weight, seed yield, as well as indigo dye percentage and yield. The results indicated that the indigo content was greater at the second sowing date (0.68%) than the first (0.66%) and third (0.65%) dates. Further, the indigo percentage was greater in Jiroft and Roudbar ecotypes compared with Rigan and Ghale‐ganj. Seed yield at the second and third sowing dates significantly increased (560.3 kg ha–1) and reduced (188.4 kg ha–1), respectively, compared with the first sowing date (261.4 kg ha–1). Furthermore, the highest value of leaf dry weight, indigo yield, and seed yield were obtained from the Jiroft ecotype at the second sowing date; in contrast, the lowest of these parameters were recorded in the Ghale‐ganj ecotype at the latest sowing date. Moreover, the genetic diversity of ecotypes was studied using the amplified fragment length polymorphism (AFLP) marker. Based on the dendrogram, the four ecotypes of indigo were divided into three groups; thus, Jiroft and Rudbar, geographically close to each other and almost the same, were in one group, and Reagan and Ghale‐Ganj were in separate groups. Overall, based on this research results, Jiroft and 20 May could be suggested as the best ecotype and sowing date of indigo for the examined region. Obtained results can also be useful for expanding and facilitating the cultivation of neglected plants.

Genetic characterization of oleaginous bottle gourd (Lagenaria siceraria) germplasm from Côte d'Ivoire using agromorphological and molecular markers

AbstractBeing difficult to regenerate and maintain the seeds, the oleaginous bottle gourd was investigated using nine agromorphological traits and 31 amplified fragment length polymorphism (AFLP) markers. Specifically, the study was conducted to determine the intra-specific variability of a total of 173 accessions, which were identified from five agro-ecological regions from Côte d'Ivoire (Centre, East, North and South). Then, the genetic diversity and relationships within accessions were studied using AFLP markers. This characterization using both morphological and AFLP markers was realized in order to ultimately build a reliable core collection. The discriminant analysis, using nine quantitative traits, reveals plant length and seeds number per fruit as discriminating characteristics. From the accessions used for the agromorphological study, 148 were able to be differentiated by the AFLP markers. A range of 52 to 113 bands were amplified per primer combination. As revealed by the analysis of molecular variance (AMOVA), 28% of the total variation resides among accessions and 72% occurs within populations. The AMOVA computed in order to differentiate cultivars, displayed the same trends when no prior grouping of accessions was considered. The differentiation within cultivar (97%) was more than that, among cultivars (3%). Tree topologies inferred by neighbour-joining analysis reflected no clear cut off grouping.To group accessions, we used a Bayesian clustering analysis which exhibited two clusters. Using the informativeness of the primer combinations analysed in the present study, an orientation was given for the choice of the accessions which would be used to build a core collection.

AFLP-based assessment of genetic variation in certain Indian elite cultivars of Cymbopogon species

Cymbopogon, one of the major aromatic genera of Poaceae family, is mainly cultivated for its essential oils that hold promising medicinal and commercial value. In India, several improved cultivars of Cymbopogon species are developed for commercial cultivation and conceded by means of agronomic and chemotypic traits. Information on genetic variability in these commercial cultivars is still limited which poses a problem in further improvement of oil grasses. The present study aimed to investigate the genetic variation among certain improved Indian cultivars of three commercially important Cymbopogon sp. i.e., C. flexuosus, C. winterianus and C. martinii using amplified fragment length polymorphism (AFLP) markers. A total of 94 robust loci were amplified using 8 AFLP primer combinations. Among the total amplified fragments, 72 were polymorphic which showed 76.59% polymorphism across all the germplasm. Polymorphic information content (PIC) and marker index (MI) values showed significant positive correlation (r2 =0.936, p < 0.005) in determining the discriminatory power of the AFLP primer combinations. A higher effective multiplex ratio (EMR) and MI values were recorded for the primer combinations that generated higher polymorphism across the genotypes. To obtain the genetic information at the intra- and inter species level, genetic diversity indices were calculated. Genetic heterogeneity was noticed in the order C. martinii>C. flexuosus>C. winterianus. UPGMA-phenogram and principal coordinate analysis (PCA) separated all the cultivars as per the chemotaxonomic series with a few exceptions. Molecular diversity explained and diagnostic fragments developed through AFLP markers in this study could provide a detailed cataloguing of Cymbopogon cultivars for genetic conservation purpose and also useful for exploration and improvement of diverse set of germplasm.

RELATIONSHIP BETWEEN GENETIC AND ENVIRONMENTAL CHARACTERISTICS OF LITHUANIAN POPULATIONS OF PURPLE LOOSESTRIFE (LYTHRUM SALICARIA)

The present study evaluated genetic diversity of Lithuanian populations of Lythrum salicaria in relation to parameters of riparian environment. Growing along Nemunas, Seaside and Lielupė river basins, 15 populations were examined using amplified fragment length polymorphism markers. Molecular data were related to the river basins, type of land use and cover, natural vice versa regulated fragments of the rivers. Population mean genetic diversity parameters were as follows: percentage of polymorphic loci (57.2), expected heterozygosity (0.183), polymorphismc information content (0.218). Mantel test revealed correlation (R2 = 0.0986, p = 0.01) between genetic and geographic distance of populations. Greater genetic diversity within, rather than among populations (ΦPT = 0.213) was observed. According to the Bayesian clustering, studied populations are admixtures of two gene pools. Analysis of molecular variance revealed significant differentiation between populations belonging to distinct river basins, between populations from natural vs. regulated fragments of the rivers.

Strain Degeneration in White Button Mushroom (Agaricus bisporus) Using Amplified Fragment Length Polymorphism

Background: The growing population and limited agricultural land have pushed the demand for higher food production per land area. Edible mushrooms are a rich source of protein. Agaricus bisporus is the most common edible mushroom with high economic value. One of the most important steps in the production process of white button mushroom is the production of spawn. It is highly important to make pure high-quality mycelium during the production of spawn. Studies have shown that at this stage of production, a high level of morphological changes occurs in some mushrooms, which causes the formation of fluffy-shaped mycelium. This phenomenon is a kind of strain degeneration in edible mushrooms and is one of the biggest concerns of the producers of white button mushroom. Objectives: The purpose of this study was to investigate the nature of stroma (strain degeneration) in white button mushroom, and to identify the causes of genetic changes. Methods: In this experiment, ten successive subcultures were produced from the mother mycelium on potato dextrose agar medium (pH: 6.2), and the genetic changes of mycelium were examined after ten successive subcultures. DNA extraction was performed after sampling of the mother, normal, and fluffy mycelia. Six pairs of designed primers and propagated parts were loaded into six polyacrylamide gels. DNA fragments were visualized using silver staining. Genetic comparisons were made between the mother, normal, and fluffy mycelia by the amplified fragment length polymorphism (AFLP) method, and the results were evaluated using POPGENE software. Results: The AFLP marker produced 137 fragments for the H2 strain, of which 57 fragments had polymorphism between the mother, normal, and fluffy mycelia. For the 737 strain, 149 fragments were produced, of which 73 fragments had polymorphism between the mother, normal, and fluffy mycelia. On average, the mother, normal, and fluffy mycelia had about 42 and 49% polymorphism for the H2 and 737 strains, respectively. The degree of polymorphism among the samples confirmed genetic differences between the mother, normal, and fluffy mycelia. Conclusions: Based on the results of the present study, we concluded that strain degeneration, which manifests as the fluffy mycelium of white button mushroom, has a genetic origin. These genetic differences can be attributed to diverse factors, such as the phenomenon of heterokaryosis, asexual recombination, and the formation of mitosis crossing over. These factors and their effects require further evaluations.

Constructing the wolfberry (Lycium spp.) genetic linkage map using AFLP and SSR markers

Genetic linkage maps are important for quantitative trait locus (QTL) and marker-assisted selection breeding. The wolfberry (Lycium spp.) is an important food and traditional medicine in China. However, few construction genetic linkage maps have been reported because of the lack of genomic and genetic resources. In this study, a population of 89 F1 seedings was derived from a cross between two heterozygous parents, L. chinense var. potaninii ‘BF-01’ (female) and L. barbarum var. auranticarpum ‘NH-01’ (male), in order to construct a genetic linkage map using simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers based on the double pseudo-test cross mapping strategy. The resulting genetic map consisted of 165 markers (74 AFLPs and 91 SSRs) distributed across 12 linkage groups and spanned a total length of 557.6 cM with an average distance of 3.38 cM between adjacent markers. The 12 linkage groups contained 3 to 21 markers and ranged in length from 8.6 to 58.3 cM. Twenty-nine segregated markers distributed in the map were mainly located on LG4 and LG9 linkage groups at P<0.05. This is the first linkage map of Lycium species using SSR and AFLP markers, which can serve as basis for improving genes and selective breeding of the genome assembly.

Amplified Fragment Length Polymorphism (AFLP) analysis in Cyamopsis spp. for Genetic Diversity Study

The aim of present work is to study genetic diversity and phylogenetic relationship among 13 guar varieties from different germplasm conservation centers of India. A total of 64 primer combinations were tested and 24 primer combinations producing best DNA fingerprints were selected for final analysis. The AFLP marker gives keen amplification with 1,671 polymorphic bands and 62 unique bands ranging from 55 bp to 350 bp. Maximum numbers of unique bands were produced with E-ACT/M-CAG primer combination. This analysis allows determining association with an undefined feature and helps in identification of these genotypes. The phylogenetic relationship was explained by a dendrogram generated with UPGMA method. These guar genotypes may be used to develop new varieties with significant improvement in quality and quantity of gum.

Study of Plant Genetic Variation through Molecular Markers: An Overview

This article refers to viewing the role of molecular markers during analyzing the genome of plants and their importance in plant biotechnology. In recent years, we observed the role of molecular techniques in programs for improving plant breeding and preserving genetic resources has been observed, and molecular and biochemical indicators which represent basic material through determining the diversity between genotypes for indicators it is never affected by external surrounding conditions as always in the phenotype features. Molecular markers of DNA have been widely applied to answer a range of questions related to taxonomy, molecular evolution, population genetics, and genetic diversity, as well as monitoring trade in plants and food products , in addition to its having a role in studying gene expression , genetic mapping, and studies of species evolution providing fast and accurate results. In this work, the advantages and limitations of the molecular techniques applied in plant sciences such as: RAPD (Random Amplification Polymorphic DNA Marker); ISSR (Inter Simple Sequence Repeat Marker); SSR (Simple Sequence Repeat Marker); AFLP (Amplified Fragment Length Polymorphic Marker); RFLP (Restriction Fragment Length Polymorphism Marker); SNP (Single Nucleotide Polymorphism) and Real Time PCR.

Evidence for introgressive hybridization of wild black‐necked pheasant with the exotic ring‐necked pheasant during the past 50 years in the Hyrcanian zone, an integrative molecular and morphological approach

Hybridization with non-native-related taxa, especially with introgression, is a major conservation problem, as it may disturb local adaptations, resulting in population decline and biodiversity loss. It is important to identify hybrid individuals, which can be difficult to break up morphologically. Using morphometric and genetic approaches [multivariate analysis, microsatellites, and amplified fragment length polymorphism (AFLP)], this research attempts to classify the interaction and the prevalence of introgressive hybridization in four native pheasant subspecies of Phasianus colchicus with non-native P. c. torquatus. According to principal component analysis and canonical discriminant function results, hybrid individuals are placed between these two major groups of native and exotic birds. Also, we assessed the population structure, genetic variation, and gene flow between native subspecies and exotic pheasants using 16 microsatellite loci in 129 samples. Our findings revealed that hybridization occurred between two native subspecies, P. c. talischensis and P. c. persicus, and exotic subspecies (P. c. torquatus). Observation of hybrids in P. c. persicus were almost three times higher than P. c. talischensis. The levels of the genetic variation within and among the natural populations were investigated using the AFLP markers method to identify hybrids. A total of 202 distinct bands were amplified using four primer combinations, with 179 of them being polymorphic. AMOVA analysis revealed a low degree of genetic differentiation among the populations. The hybrid samples were closely related to the P. c. talischensis and P. c. persicus populations, according to the UPGMA clusters and discriminant analysis of principal components. It appears that disruption of gene flow between pheasant populations in the Hyrcanian zone is a serious issue. The data from molecular markers prompted us to raise a concern about the genetic integrity in some native subspecies in the Iranian plateau. As a result, a clear cut-level can now be used to differentiate the hybrids, which is also supported by morphological evidence. A direct relationship was found between farmed pheasants and hybrid frequency. Farmed individuals should be closely monitored, and non-native taxa should not be released into the wild.

Exploring genetic diversity, population structure, and phylogeography in Paracoccidioides species using AFLP markers.

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection acquired after inhalation of Paracoccidioides propagules from the environment. The main agents include members of the P. brasiliensis complex (phylogenetically-defined species S1, PS2, PS3, and PS4) and P. lutzii. DNA-sequencing of protein-coding loci (e.g., GP43, ARF, and TUB1) is the reference method for recognizing Paracoccidioides species due to a lack of robust phenotypic markers. Thus, developing new molecular markers that are informative and cost-effective is key to providing quality information to explore genetic diversity within Paracoccidioides. We report using new amplified fragment length polymorphism (AFLP) markers and mating-type analysis for genotyping Paracoccidioides species. The bioinformatic analysis generated 144 in silico AFLP profiles, highlighting two discriminatory primer pairs combinations (#1 EcoRI-AC/MseI-CT and #2 EcoRI-AT/MseI-CT). The combinations #1 and #2 were used in vitro to genotype 165 Paracoccidioides isolates recovered from across a vast area of South America. Considering the overall scored AFLP markers in vitro (67–87 fragments), the values of polymorphism information content (PIC = 0.3345–0.3456), marker index (MI = 0.0018), effective multiplex ratio (E = 44.6788–60.3818), resolving power (Rp = 22.3152–34.3152), discriminating power (D = 0.5183–0.5553), expected heterozygosity (H = 0.4247–0.4443), and mean heterozygosity (Havp = 0.00002–0.00004), demonstrated the utility of AFLP markers to speciate Paracoccidioides and to dissect both deep and fine-scale genetic structures. Analysis of molecular variance (AMOVA) revealed that the total genetic variance (65-66 %) was due to variability among P. brasiliensis complex and P. lutzii (PhiPT = 0.651–0.658, P < 0.0001), supporting a highly structured population. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for P. brasiliensis s. str. (χ2 = 1.025; P = 0.3113), P. venezuelensis (χ2 = 0.692; P = 0.4054), and P. lutzii (χ2 = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found for P. americana (χ2 = 8.909; P = 0.0028) and P. restrepiensis (χ2 = 4.571; P = 0.0325) with a preponderance of MAT1-1. Geographical distributions confirmed that P. americana, P. restrepiensis, and P. lutzii are more widespread than previously thought. P. brasiliensis s. str. is by far the most widely occurring lineage in Latin America countries, occurring in all regions of Brazil. Our new DNA fingerprint assay proved to be rapid, reproducible, and highly discriminatory, to give insights into the taxonomy, ecology, and epidemiology of Paracoccidioides species, guiding disease-control strategies to mitigate PCM.

Genetic Analyses of Saprolegnia Strains Isolated from Salmonid Fish of Different Geographic Origin Document the Connection between Pathogenicity and Molecular Diversity.

Saprolegnia parasitica is recognized as one of the most important oomycetes pests of salmon and trout species. The amplified fragment length polymorphism (AFLP) and method sequence data of the internal transcribed spacer (ITS) were used to study the genetic diversity and relationships of Saprolegnia spp. collected from Canada, Chile, Japan, Norway and Scotland. AFLP analysis of 37 Saprolegnia spp. isolates using six primer combinations gave a total of 163 clear polymorphic bands. Bayesian cluster analysis using genetic similarity divided the isolates into three main groups, suggesting that there are genetic relationships among the isolates. The unweighted pair group method with arithmetic mean (UPGMA) and principal coordinate analysis (PCO) confirmed the pattern of the cluster analyses. ITS analyses of 48 Saprolegnia sequences resulted in five well-defined clades. Analysis of molecular variance (AMOVA) revealed greater variation within countries (91.01%) than among countries (8.99%). We were able to distinguish the Saprolegnia isolates according to their species, ability to produce oogonia with and without long spines on the cysts and their ability to or not to cause mortality in salmonids. AFLP markers and ITS sequencing data obtained in the study, were found to be an efficient tool to characterize the genetic diversity and relationships of Saprolegnia spp. The comparison of AFLP analysis and ITS sequence data using the Mantel test showed a very high and significant correlation (r2 = 0.8317).