AFLP Analysis Research Articles

AFLP Analysis of Genetic Diversity Evaluation in Pear (Pyrus communis L.) in Kurdistan Region/Iraq

AFLP Analysis of Genetic Diversity Evaluation in Pear (Pyrus communis L.) in Kurdistan Region/Iraq

The Emergence of Cat-Transmitted Sporotrichosis Driven by Sporothrix brasiliensis in Piauí, Brazil.

In Brazil, the emergence of feline sporotrichosis, a neglected subcutaneous mycosis primarily transmitted by cats and caused by the fungus Sporothrix brasiliensis, has been monitored via molecular tools. This approach underscores the geographic expansion of this disease and highlights the critical role of molecular surveillance in understanding its epidemiology and guiding public health interventions. We investigated three domestic cats (Felis catus) with multiple skin lesions in Picos, Piauí, Brazil. The cats were examined, and samples were collected for laboratory diagnosis, including cytological evaluation, fungal culture, and molecular characterisation. The molecular analysis involved a one-tube multiplex probe-based qPCR assay for the diagnosis of Sporothrix species, mating-type determination, and amplified fragment length polymorphism (AFLP, EcoRI-GA/MseI-AG) to assess genetic similarity and potential origin. All three cats were diagnosed with sporotrichosis caused by the hypervirulent S. brasiliensis. A probable case of zoonotic transmission has been reported in a 12-year-old girl who developed Parinaud oculoglandular syndrome after contact with one of the cats with sporotrichosis. The molecular analysis revealed that the cat isolates were MAT 1-2 and formed a single cluster according to the AFLP analysis, suggesting direct transmission (cat-cat) and a potential founder effect. The isolates were also closely related to strains from Pernambuco and Southeast Brazil, indicating a possible introduction from these regions. Identifying S. brasiliensis in Piauí emphasises the need for increased awareness and control measures to prevent further spread. The predominance of the MAT1-2 idiomorphs and the genetic similarity among outbreak isolates suggest clonal expansion, which could have significant implications for public health and veterinary practices. Considering its zoonotic potential and environmental adaptability, a One Health approach is crucial for managing and controlling the spread of cat-transmitted sporotrichosis.

Genomic DNA polymorphism in <i>Myrica gale</i> (Myricaceae) in the Lebyazhiy state Nature Reserve (the southern coast of the gulf of Finland)

The analysis of genomic DNA polymorphism is one of widely used approaches for studying the genetic structure of natural populations. It has been successfully applied to various plants. However, many species have not yet been studied, which is primarily due to methodological difficulties in isolating well-purified and non-degraded genomic DNA. These difficulties are due to the fact that plants possess numerous bioorganic compounds (polysaccharides, polyphenols, lipids, etc.) that contaminate DNA and significantly reduce its quality. Such species include marsh waxweed (Myrica gale L.), a perennial sub-Atlantic shrub with presumably vegetative propagation (in nature, waxweed seedlings are quite rare). We developed a simple protocol for isolation of high-quality genomic DNA from waxweed leaves and performed AFLP analysis of 42 plants of this species from three subpopulations in the Lebyazhiy Nature Reserve. Using three primer pairs, we isolated 22 amplification fragments, 8 of which were monomorphic. For the remaining 14 fragments, the average level of their polymorphism was low: depending on the subpopulation studied, it varied from 0.079 to 0.129. As shown by our analysis, all three studied subpopulations are polymorphic with a predominance of two common AFLP-genotypes. The corresponding plants are apparently the vegetative descendants of the founders. The rare AFLP-genotypes (represented by just one or two plants; a total of 12 such genotypes were identified) are likely the result of mutational and recombination processes. Our data give evidence that in the life cycle and dispersal of waxweed, the role of sexual reproduction is also noticeable.

Allium goumenissanum (Allium sect. Codonoprasum), a new species for Bulgaria and new localities in Greece: testing an integrated approach to species identification

Allium sect. Codonoprasum represents an evolutionarily young and rapidly radiating group of bulbous geophytes, with a significant proportion of polyploids and minor morphological differences between species. In the last 20 years, dozens of new species of this section have been described from the Mediterranean. Still, very little new information has been obtained on most of them since their description, especially concerning cytogenetic, molecular, and distribution data. Allium goumenissanum is a recently described species of this section, known only from three nearby localities in northern Greece. During the last 10 years, the senior authors' team collected population samples of an unknown species of this section from the southern Balkans, which appeared to be representative of A. goumenissanum. Using an integrated approach employing a range of methods (cytogenic, molecular, morpho- and micromorphological methods), the populations of an unknown species were compared with morphologically similar species A. goumenissanum and A. stamatiadae. AFLP, ITS sequencing and genome size analyses revealed that all the populations of the unknown species represent A. goumenissanum, and that this species is differentiated genetically and in genome size from A. stamatiadae. ITS sequencing indicates an allotriploid origin of A. goumenissanum from unknown ancestors. The characterization of the morphology and reproduction of A. goumenissanum is completed and micromorphological and anatomical characteristics of the leaves of both species are newly published. The distribution of A. goumenissanum, based on new data, now includes a more extensive area of north central Greece and the species has been found as a new one for Bulgaria.

In vitro virulotyping, antifungal susceptibility testing and DNA fingerprinting of Microsporum canis strains of canine and feline origin

Microsporum canis is considered the common dermatophyte agent associated with ringworm in felines and canines. In the present study, we sampled n = 548 felines and canines for the probable isolation of M. canis. The rate of isolation from the cats and dogs was 70.27 % (52/74) and 1.68 % (8/474), respectively and Persian cats were found to be highly susceptible to M. canis infection. The strains were evaluated for their production of phospholipase, lipase, catalase, and hemolysis and their ability to grow at 35 ℃. All the strains were identified as low producers of catalase and n = 17 strains exhibited high thermotolerance ability. Terbinafine was found to be the most effective antifungal drug and fluconazole was the least effective, in vitro. AFLP analysis revealed three genotypes of M. canis with 15 sub-clusters showing ≥ 90 % similarity and 7 sub-clusters exhibiting 100 % similarity. However, the phenotypic characters cannot be attributed based on the AFLP profiles.

Modern technologies for studying the genome of mycobacteria

Molecular technologies play a leading role in the laboratory diagnosis of tuberculosis and mycobacteriosis. The successes in studying the genome of Mycobacterium have contributed to significant progress in understanding the evolution, variability, and genetic diversity of pathogens, as well as the development of diagnostic technologies, including research into resistance to anti-tuberculosis drugs.
 The aim of this research is to conduct a comparative study of the spectrum of modern technologies for studying the genomes of mycobacteria and their impact on the efficiency of the laboratory diagnosis of tuberculosis.
 Materials and methods: a search for sources of information was carried out in the PubMed, Medline, Web of Science, and Google Scholar databases. Materials related to the technology of molecular diagnosis of tuberculosis and mycobacteriosis and for determining the susceptibility of pathogens to anti-tuberculosis drugs were selected.
 Results: it was determined that the modern methods for studying the genome of mycobacteria include amplification technologies (PCR analysis), hybridization, restriction, spoligotyping, sequencing, and their various combinations. The main methods are standard and modified protocols of PCR (RAPD-PCR, AP-PCR, rep-PCR, Real-time PCR, Inverse PCR, TB-LAMP, HIP, LM-PCR). Genomic Restriction Analysis can be used in studies of MTBC and NTM strains (RFLP, AFLP analysis, MIRU-VNTR genotyping). The most effective method for genome analysis is WGS. Complex methods that utilize a combination of molecular technologies allow for the direct detection of mycobacteria in clinical samples.
 Conclusions: the widespread application of genomic technologies in the study of mycobacteria will contribute to the effective implementation of the global WHO strategy for the prevention, treatment, and control of tuberculosis and mycobacteriosis

Nutritional, metabolic and genetic profiling of ‘Cerato’ and ‘Curniciello’ bean landraces from Caserta, Southern Italy

Italy represents a territory rich in common bean landraces, most of which have not yet been characterized. Therefore, the proximal composition and metabolic profiles (fatty acid composition, total and free amino acids) as well as total polyphenols and antioxidant capacities of both ‘Cerato’ and ‘Curniciello’ dry beans, cultivated in Caserta’s rural areas (Southern Italy) were evaluated, in comparison with other local known dry beans. ‘Cerato’ dry beans have a lower content of crude proteins (21.18 vs 23.41 g/100 g), lipids (1.27 vs 2.08 g/100 g) and total amino acids (16.01 vs. 17.89 g/100 g) with respect to ‘Curniciello’ dry beans, considering the average values of two different harvest years (2020–2021), although slight statistical differences were found when the two harvest years were analysed separately. Two essential fatty acids (n-6 linoleic and n-3 alpha-linolenic) and oleic acid were the most abundant fatty acids in both dry beans, (∼90% of the total). Subsequently, the trypsin and chymotrypsin inhibitory activities in raw and boiled (2 h) dry beans, as well as the α-amylase and α-glucosidase inhibitory activities were investigated, considering their capability to reduce crude protein and carbohydrates intake and assimilation. In addition, AFLP analysis of two landraces shows different polymorphic patterns useful for their authentication and traceability. Overall, our data provide a starting point for promoting the cultivation and consumption of ‘Cerato’ and ‘Curniciello’ dry beans, thus contributing to the preservation of local culinary traditions and Italian biodiversity.

Phylogeographic pattern of the high-alpine plant species Eritrichium nanum (Boraginaceae) within the Carpathians

The Carpathians represent not only a European hotspot of plant diversity for both species richness and endemism, but also an important stepping-stone area in historical migrations between the flora of the Asian and European mountain systems and a starting point of postglacial recolonizations for many species. Yet, until recent years, phylogeographical studies for alpine or arctic-alpine plants were focused on the Alps, whereas peripheral mountain ranges, including the Carpathians, were either neglected or insufficiently sampled. In this study, we aimed to complement the Alpine phylogeographic structure of an emblematic high-alpine European endemic taxon, Eritrichium nanum, by focusing on the Carpathian range of the species. We sampled nine populations from the South-Eastern Carpathians and performed ITS1 sequencing and AFLP fingerprinting. In case of ITS1 region, all the populations, no matter of their geographic origin, presented the same ribotype. The AFLP analysis indicated that, within the Carpathians, the extant populations of E. nanum comprised two major allopatric lineages. One important result of the research was the discovery that the species’ sole important genetic break was located in the Southern Carpathians, separating populations of the Retezat Mountains from all the others in the Carpathians.

An Interactive, Online Web Map Resource of Global Fusarium oxysporum ff. spp. Diversity and Distribution.

An Interactive, Online Web Map Resource of Global Fusarium oxysporum ff. spp. Diversity and Distribution.

Genome variability of domestic tomato varieties: data from AFLP analysis.

Tomato Solanum lycopersicum L. is one of the main vegetable crops, accessions and cultivars of which are characterized by a low level of genomic polymorphism. Introgressive tomato breeding uses related wild Solanum species to improve cultivars for stress tolerance and fruit quality traits. The aim of this work was to evaluate the genome variability of 59 cultivars and perspective breeding lines of S. lycopersicum and 11 wild tomato species using the AFLP method. According to the AFLP analysis, four combinations of primers E32/M59, E32/M57, E38/M57, and E41/M59, which had the highest PIC (polymorphism information content) values, were selected. In the process of genotyping a collection of 59 cultivars/lines of S. lycopersicum and 11 wild tomato accessions, the selected primers revealed 391 fragments ranging in size from 80 to 450 bp, of which 114 fragments turned out to be polymorphic and 25 were unique. Analysis of the amplif ication spectra placed wild tomato accessions into separate clades. Sister clades included cultivars of FSCV breeding resistant to drought and/or cold and, in part, to late blight, Alternaria, Septoria, tobacco mosaic virus and blossom end rot, as well as tomato accessions not characterized according to these traits, which suggests that they have resistance to stress factors. In accessions of distant clades, there was clustering on the basis of resistance to Verticillium, cladosporiosis, Fusarium, tobacco mosaic virus, gray rot, and blossom end rot. The combination of ac cessions according to their origin from the originating organization was shown. The primer combinations E32/M59, E32/M57, E38/M57 and E41/M59 were shown to be perspective for genotyping tomato cultivars to select donors of resistance to various stress factors. The clade-specif ic fragments identif ied in this work can become the basis for the development of AFLP markers for traits of resistance to stress factors.

Taxonomic analysis of certain taxa of Epipactis in Slovenia

The predominantly allogamous Broad-leaved Helleborines (Epipactis helleborine) and the predominantly autogamous Narrow-lipped Helleborines (Epipactis leptochila) represent two taxonomically complex groups with great morphological diversity and close genetic relationships at the subspecies level. They are influenced by environment, clinal variability, hybridization between allogamous taxa, and in particularly by speciation through transitions from allogamy to autogamy. We analysed floral traits, genome size and performed molecular analyses (Internal transcribed spacer region, ITS; Amplified Fragment Length Polymorphism, AFLP) of several individuals of E. helleborine and E. leptochila aggregate from different geographical regions in Slovenia, focusing on intermediate traits of E. helleborine subsp. moratoria, E. helleborine subsp. leutei and E. leptochila subsp. neglecta. In order to successfully distinguish studied taxa, the main attention should be paid to the shape of the epichil and the ovary, as well as to the size ratio of sepals and petals. Among the microfloral characters, the shape and size of the lower margin of the stigma and staminodes are found to be supportive. E. neglecta clearly shows intermediate floral characteristics between E. leptochila and E. leutei, suggesting that the transition to autogamy evolved E. neglecta as an intermediate stage in the speciation process, coinciding with the colonisation of forest sites with lower light intensity. Analysis of genome size revealed significant differences, with E. neglecta having the greatest value. Using molecular markers, we were able to determine two main ribotype clusters within the E. helleborine group, which were also determined by AFLP analyses. E. leutei and E. neglecta formed two clearly defined subclusters within the E. leptochila lineage. The results of the presented study suggest that combining molecular markers and genome size with information on how variation in floral morphology is distributed, using multivariate analyses, is the most reliable way to gain improved insight into the relationships among the taxa analysed.

Genetic Diversity among Single, Double, And Triple Cross Hybrids in Beit Alpha Cucumber (Cucumis sativus)

Genetic variation was investigated in fifteen single, eighteen three-way, and nine double hybrid crosses and their parents along with nine elite commercial cucumber hybrids. Six lines (NS5, NS9, NS17, NS29, NS31, and NS33) were used to develop these cross hybrids. Based on AFLP and SSR analysis, the studied cucumber genotypes have a narrow genetic base. The results also showed genetic differentiation compared to commercial varieties. Out of 1352 bands produced from 12 AFLP primer pairs, only 47 were polymorphic. Similarly, out of 26 SSR primers, only one primer was polymorphic. The highest heterozygosity index (0.6044), polymorphic information content value (0.5392), and marker Index (0.6044) were observed with M-CTT_E-AAG AFLP primer combination, while the highest effective multiplex ratio (2.8246) and resolving power (4.8421) were observed with M-CTC_E-AGC AFLP primer combination. Genetic distance estimation revealed clear distinctiveness of the studied genotypes with the control varieties. The cluster analysis by UPGMA showed two main clusters covering 84% of the studied genotypes grouped. This research shows that double and three-way hybrid crosses can be used in cucumber hybrid breeding.

Paphiopedilum charlesworthii var. lannaense, a new slipper orchid from Northern Thailand identified by morphological and AFLP analyses

A new endemic slipper orchid in the family Orchidaceae from Northern Thailand was described according to morphological and molecular analyses. Morphological information specifies the new slipper orchid Paphiopedilum charlesworthii var. lannaense to be similar to Paphiopedilum charlesworthii (Rolfe) Pfitzer 1895, with the exception of its staminode being obovate-obcordate with yellow color, glittery and rough surface. Molecular analysis by AFLP technique indicates that Paphiopedilum charlesworthii var. lannaense can be included as a member of section Paphiopedilum and is closely related to Paphiopedilum coccineum Perner, H. & Herrmann, R. (2000) (syn. Paphiopedilum barbigerum var. coccineum), from which it differs morphologically by dorsal sepal and petal characters. The morphological and molecular evidence supported that Paphiopedilum charlesworthii var. lannaense is a new slipper orchid in the genus Paphiopedilum (Cypripedioideae, Orchidaceae).

Genetic diversity and population structure of the rare species Artemisia hololeuca detected by AFLP analysis

ISHS VIII International Scientific and Practical Conference on Biotechnology as an Instrument for Plant Biodiversity Conservation (Physiological, Biochemical, Embryological, Genetic and Legal Aspects) Genetic diversity and population structure of the rare species Artemisia hololeuca detected by AFLP analysis

Epigenomic stability assessment during cryopreservation and physiology among various strains of Chromochloris zofingiensis (Chlorophyceae) and their genetic variability revealed by AFLP and MS-AFLP

Chromochloris zofingiensis (Dönz) Fucíková & L.A.Lewis, due to its production of highly valuable carotenoids such as astaxanthin, is a model organism in biotechnology. Since the recognition of this physiological property, many biotechnological applications have only used a single strain (SAG 211-14 = CCAP 211/14 = UTEX 32 = ATCC 30412) to produce biomass and carotenoids. However, multiple acquisitions of strains putatively belonging to the same species raised the question of the conspecificity of those strains and their properties. In this study, the conspecificity of the available strains, which are deposited axenically in SAG, was tested using SSU and ITS rDNA sequencing and AFLP (EcoRI/PstI) analyses. The comparison of SSU and ITS rDNA sequences as well as the AFLP patterns revealed that the investigated strains formed two very similar groups, (1) SAG 211-14, SAG 4.80, SAG 31.80, and SAG 34.80 and (2) SAG 221-2. All strains belonged to one species, C. zofingiensis, and represented one monophyletic lineage within the so-called DO-group of the Chlorophyceae. The robustness to cryopreservation and the subsequent epigenetic variability was detected using the methylation-sensitive AFLP (EcoRI/MspI and EcoRI/HpaII) among the five Chromochloris strains. All strains showed a high rate of survival (54.4–98.1%) during cryopreservation. The methylation patterns varied between precryo and postcryo in all strains detected among three time points (before, shortly after, and 8 weeks after cryopreservation), showing that the MS-AFLP technique has the potential to detect epigenetic effects occurring in response to cryopreservation and other stresses. Finally, the potential of these five strains for usage in biotechnological applications was proven by growing them in aerated cultures with and without additional carbon dioxide supply. The comparison showed that all strains produced high amounts of biomass and carotenoids under aeration with additional CO2 and were therefore suitable in biotechnology.

Morpho-agronomic, biochemical and molecular analysis of genetic diversity in the Mesoamerican common bean panel.

The common bean (Phaseolus vulgaris L.) is of great importance to the food and nutritional security of many populations, and exploitation of the crop's genetic diversity is essential for the success of breeding programs. Thus, the aim of the present study was to evaluate the genetic diversity of 215 common bean accessions, which included cultivars, obsolete cultivars, improved lines, and landraces using morpho-agronomic and biochemical traits, and amplified fragment length polymorphism markers (AFLP). Genetic parameters, box plots, Pearson's correlation analysis, and Ward's hierarchical clustering were used to analyze the data. The Jaccard similarity coefficient and neighbour-joining clustering method were used for molecular analysis. A wide variability among the accessions was observed for morpho-agronomic and biochemical traits. Selective accuracy (Ac) and broad-sense heritability (h2) values were high to intermediate for all traits, except seed yield. Ward's hierarchical clustering analysis generated six groups. AFLP analysis also revealed significant differences among the accessions. There was no correlation between the differences based on genetic markers and those based on morpho-agronomic and biochemical data, which indicates that both datasets are important for elucidating the differences among accessions. The results of the present study indicate great genetic diversity among the evaluated accessions.

Isolation and HPLC assisted quantification of two iridoid glycoside compounds and molecular DNA fingerprinting in critically endangered medicinal Picrorhiza kurroa Royle ex Benth: implications for conservation.

The online version contains supplementary material available at 10.1007/s12298-021-00972-w.

Identification and genetic analysis of two maize CMS-T mutants obtained from out-space-flighted seeds

Male sterility is widely utilized for hybrid seed production. In this study, two new found male sterile mutants SauS4 and SauS5 were obtained from space flighted seeds of maize inbred line RP125. Then, genetic analysis, molecular markers identification, and cytological observation were conducted to confirm their male sterile types. For genetic analysis, the above two male sterile mutants were continuously backcrossed with two maize inbred line 18Hong and RP125, and four stable male sterile lines SauS4(18Hong), SauS5(18Hong), SauS4(RP125), SauS5(RP125) were generated by six-generation backcross. Restoring and maintaining relationship analysis showed that both Hui313 and Zifeng1 didn’t rescue the male sterility SauS4(18Hong) and SauS5(18Hong). Using CMS mitochondria-specific primers for PCR detection suggested that only a 440 bp band unique to CMS-T type was amplified in SauS4(18Hong), SauS5(18Hong), SauS4(RP125), and SauS5(RP125). Sequencing results showed that these bands sequences were identical in DNA level which compared with T-urf13. Cytological observations showed that the main abortion stages of SauS4 and SauS5 were at the middle stage of uninucleate microspores under the two nuclear backgrounds of 18Hong and RP125, exhibiting the characteristics of sporophyte sterility. All the above results pointed out the two male sterile mutants SauS4 and SauS5 belonged to the CMS-T type. Interestingly, some mitochondrial genome difference between SauS4(RP125) and SauS5(RP125) were revealed by AFLP analysis.

Meristic characters of the Sebastes inermis species complex in northern Kyushu based on species identification by AFLP analysis

Meristic characters of the <i>Sebastes inermis</i> species complex in northern Kyushu based on species identification by AFLP analysis

Pathogenic and genetic variability of Ralstonia solanacearum strains from the Philippines

AbstractIn the Philippines, bacterial wilt caused by Ralstonia solanacearum is one of the most important diseases affecting vegetables and banana. In this study, 89 strains of R. solanacearum isolated from various hosts were screened for their biovar, phylotype, pathogenicity, and genetic diversity. Foreign strains were included for comparison with these Philippine strains. Results of the biochemical and multiplex‐PCR tests divided the Philippine strains into five biovars (1, 2, 3, 4, and N2) and three phylotypes (I, II, and IV). Three potato strains belonged to biovar N2/phylotype IV. Pathogenicity tests divided the strains into five pathogenicity types based on their virulence in tomato, potato, eggplant, sweet pepper, and tobacco. Strains classified as biovar N2 were weakly pathogenic to potato (pathogenicity type III) and almost all strains isolated from banana were not pathogenic to the test plants except potato (pathogenicity type V). The results of AFLP analysis divided the strains into four clusters. Cluster 1 was composed of strains isolated from solanaceous crops, ginger (Zingiber officinale), and Morus sp. from the Philippines and other Asian countries. Cluster 2 grouped the potato strains (biovar N2) from the Philippines and Japan and blood disease bacterium strains from Indonesia. Cluster 3 contained the local and foreign strains isolated from potato (biovar 2) and banana (biovar 1). Cluster 4 consisted only of the tomato strain from the USA.