Amplified Fragment Length Polymorphism Genotyping Research Articles

Genotyping and antifungal susceptibility testing of Sporothrix brasiliensis isolates from Southern Brazil

Sporotrichosis is an implantation mycosis caused by the dimorphic fungus Sporothrix and mostly involves cutaneous and subcutaneous tissues and the lymphatic vessels. Among more than 50 different species, only Sporothrix schenckii, Sporothrix globosa and Sporothrix brasiliensis are frequently reported to cause infections in humans. Sporothrix brasiliensis is remarkably virulent and has been spreading rapidly in Brazil and other Latin American countries. In this study, we aimed to determine the genetic relatedness and antifungal susceptibility of Sporothrix strains by analysing 89 isolates from humans and cats in Curitiba, Southern Brazil. Calmodulin sequencing identified 81 S. brasiliensis and seven S. schenckii isolates. Amplified fragment length polymorphism genotyping analysis showed feline and human isolates clustering together. In vitro susceptibility testing with seven antifungals demonstrated a broad activity against all tested S. brasiliensis isolates, with no significant differences in minimal inhibitory concentration (MIC) values between feline and human isolates. Resistance was solely observed in one human isolate against itraconazole and posaconazole, with MICs of ≥16 μg/mL against both antifungals. Whole genome sequencing (WGS) analysis on this isolate and two related susceptible isolates did not reveal any unique substitutions in resistance-associated genes, including cyp51, hmg and erg6, when compared to two related susceptible isolates. The novel antifungal olorofim exhibited excellent activity against this large isolate collection, with all isolates considered as susceptible. Altogether, we indicate zoonotic transmission based on genotyping and revealed a broad activity of seven common antifungals, including olorofim, against a large S. brasiliensis isolate collection.

Genetic diversity and structure of Crupina vulgaris (common crupina): a noxious rangeland weed of the western United States

Common crupina (Crupina vulgaris) is a federal noxious weed in the western USA that is currently the target of a classical biological control programme using the fungus Ramularia crupinae. We first identified and determined the location of populations of the two varieties of common crupina in the western United States and assessed the pattern of genetic diversity and structure of these populations. We found seven AFLP (Amplified Fragment Length Polymorphism) genotypes for 326 plants in 17 populations. AFLP genotypes correlated with two taxa, either C. vulgaris var. vulgaris or C. vulgaris var. brachypappa. This annual species is outcrossing, but relies on selfing when pollination does not occur, which may explain why less than 1% of the genetic variation is within populations. We found strong population genetic structuring and can typically predict genotype or variety for a given location. Researchers and managers will be able to predict and survey for differential efficacy of R. crupinae on the different genotypes and varieties during initial biological control field releases, thereby increasing the likelihood of successful biocontrol establishment and impact.

P257 Amplified fragment length polymorphism fingerprinting supports the absence of correlation of genotype with clinical phenotype or source of the isolate in Aspergillus flavus infections

Poster session 2, September 22, 2022, 12:30 PM - 1:30 PMObjectives Aspergillus flavus accounts for ∼ 10% of bronchopulmonary aspergillosis and is the second leading etiological agent of invasive aspergillosis worldwide. It is the commonest cause of fungal rhinosinusitis and ocular mycoses in tropical countries including India. We report amplified fragment length polymorphism genotyping of a set of clinical and environmental isolates to unravel the genetic diversity of A. flavus in India and to further determine correlation between isolate genotype and the source/clinical phenotype of the Aspergillus infection.MethodsTwo sets of morphologically identified isolates of A. flavus from clinical (n = 71) and environmental sources (n = 22) were included in the study using a stratified random sampling method. Clinical strains were isolated from lower respiratory tract specimens (n = 22), sinus and sino-nasal biopsies (n = 25), corneal samples (n = 12), and others (n = 12). Environmental strains were isolated from different niches like air, soil, and infected crop samples. Aspergillus fumigatus F145 was used as an out-group. DNA was extracted from the fungal broth culture following the method of Lee et al. AFLP was done as per an earlier described method using HpyCH41V and Mse1 (New England BioLabs) for restriction digestion followed by selective amplification of restriction-digested products with 1 mm HpyCH4 IV primer with one selective residue (5-Flu-GTA-GACTGCGTACCCGTC-3’), 1 mm MseI primer with four selective residues (5-GATGAGTCCTGACTAATGAA). Fingerprint data was analyzed in BioNumerics software version 7.6 (Applied Maths, Belgium). The phylogenetic tree was constructed using Pearson's Correlation coefficient. An AFLP genotype was assigned to a cluster using an arbitrary cut-off value of ≥90% fingerprint similarity. The distribution of genotypes among different categories was analyzed by Fisher's exact test or Pearson's χ 2 goodness of fit test with Yates correction factor as appropriate and two-tailed P-values of <.05 were considered statistically significant.ResultsThe analyses revealed a total of 16 AFLP genotypes with 5 major clusters (≥ 5 isolates) reflecting the extent of genetic diversity in A. flavus. Genotype VIII encompassed predominantly clinical isolates (P <.01) and genotype XI with majority of isolates from environmental sources (P <.0001). The strains which were isolated from invasive and non-invasive forms or from different sites (pulmonary, sinus, and ocular) did not diverge into separate or unique clusters. Although the genotypes had an asymmetric distribution in different clinical presentations as revealed by the χ2 goodness of fit test, none of the genotypes was exclusively responsible for causing a particular infection. Isolates from the north zone of India shared genotypes with those from the southern region of the country. Three isolates formed a separate genotype XVI and diverged from the A. flavus cluster by 42% fingerprint similarity. Partial β-tubulin and calmodulin gene sequencing-based phylogeny reconstruction placed those three isolates in A. tamarii clade of the A. flavus species complex.Conclusions: This study suggests that every genotype of A. flavus has the potential of causing an allergic, non-invasive or invasive infection. Further, A. flavus sensu stricto was predominantly (97%) isolated from clinical specimens revealing the majority of infections/colonization are caused by this species compared to other members in the Flavi complex.

High genetic diversity and differentiation of an extremely narrowly distributed and critically endangered decaploid rose (Rosa praelucens): implications for its conservation

Rosa praelucens is a critically endangered decaploid alpine rose with an extremely narrow geographic distribution in Northwestern Yunnan, China. We sampled almost all the extant individuals (527 individuals in 31 natural locations and 56 individuals preserved in three local living collections) to assess the genetic variation and to probe the genetic connectivity among the individuals and populations based on three cpDNA intergenic spacers and six fluorescent amplified fragment length polymorphism (AFLP) markers. The morphological traits from seven populations were also measured. R. praelucens exhibited high levels of morphological variation, genetic diversity, and differentiation. The extant individuals were clustered into eight groups in neighbor-net networks, and subsequent Bayesian analysis assigned them into three larger gene pools, both in accordance with their morphological traits, especially flower color. The living collections embraced two private cpDNA haplotypes and included three out of the species’ total eight AFLP genotypes. Rhizome clonal growth, decaploid, and mixed breeding system may largely contribute to high genetic diversity and differentiation in R. praelucens. We concluded that the endangered status of R. praelucens may mainly be due to habitat fragmentation and loss and inherent reproductive difficulties, rather than low genetic diversity. The populations contributing higher cpDNA genetic diversity, representing more AFLP genotypes, and encompassing private cpDNA haplotypes should be given conservation priority by creating plant-micro reserves. The living collections should also be targeted for further ex situ conservation, population recovery, and reintroduction of R. praelucens plants.

VARIATION IN AGGRESSIVENESS AND AFLP AMONG Alternaria solani ISOLATES FROM INDONESIA

&lt;p&gt;&lt;em&gt;Alternaria solani &lt;/em&gt;is a necrotroph fungus that causes three-phased diseases in tomato. Management of the pathogen by using resistant cultivars requires knowledge on the aggressiveness and genetic diversity of the fungus. The aims of this study were to isolate &lt;em&gt;A. solani &lt;/em&gt;from major tomato and potato producing areas in Indonesia and to study their aggressiveness and genetic variability. Twenty two&lt;strong&gt; &lt;/strong&gt;&lt;em&gt;A. solani&lt;/em&gt;&lt;strong&gt; &lt;/strong&gt;isolates were recovered from early blighted tomato and potato in Central and West Java.&lt;strong&gt; &lt;/strong&gt;&lt;em&gt;A. alternata&lt;/em&gt;&lt;strong&gt; &lt;/strong&gt;was also isolated from tomato leaves in West Java and North Sumatra, indicating that early blight in Indonesia may be caused by more than one &lt;em&gt;Alternaria &lt;/em&gt;species. Resistance tests of four tomato genotypes to selected&lt;strong&gt; &lt;/strong&gt;&lt;em&gt;A. solani&lt;/em&gt;&lt;strong&gt; &lt;/strong&gt;isolates revealed that local isolates were more aggressive in inciting early blight and stem lesion than an imported isolate from USA. This implies that introduced breeding materials must be tested to local isolates to obtain effective resistance genes. Cluster analysis based on amplified fragment length polymorphism (AFLP) obtained from &lt;em&gt;Eco&lt;/em&gt;RI+AG and&lt;strong&gt; &lt;/strong&gt;&lt;em&gt;Mse&lt;/em&gt;I+C primer amplification separated 28 local and Taiwan isolates from the US isolate, which was coincided with aggressiveness separation between the local isolates and the US isolate. Three clusters of AFLP genotypes which did not associate with geographic origin were observed among tropical isolates. The low genetic diversity among the Indonesian isolates suggests clonal population structure with wide distribution. Successful local tomato breeding requires the availability of local&lt;strong&gt; &lt;/strong&gt;&lt;em&gt;A. solani &lt;/em&gt;collection with well-characterized aggressiveness level and molecular diversity to obtain effective resistance genes.&lt;/p&gt;

Low genetic variation of invasive Fallopia spp. in their northernmost European distribution range.

Knowledge about the reproduction strategies of invasive species is fundamental for effective control. The invasive Fallopia taxa (Japanese knotweed s.l.) reproduce mainly clonally in Europe, and preventing spread of vegetative fragments is the most important control measure. However, high levels of genetic variation within the hybrid F. × bohemica indicate that hybridization and seed dispersal could be important. In Norway in northern Europe, it is assumed that these taxa do not reproduce sexually due to low temperatures in the autumn when the plants are flowering. The main objective of this study was to examine the genetic variation of invasive Fallopia taxa in selected areas in Norway in order to evaluate whether the taxa may reproduce by seeds in their most northerly distribution range in Europe. Fallopia stands from different localities in Norway were analyzed with respect to prevalence of taxa, and genetic variation within and between taxa was studied using amplified fragment length polymorphism (AFLP). Taxonomic identification based on morphology corresponded with identification based on simple sequence repeats (SSR) and DNA ploidy levels (8× F. japonica, 6× F. × bohemica and 4× F. sachalinensis). No genetic variation within F. japonica was detected. All F. × bohemica samples belonged to a single AFLP genotype, but one sample had a different SSR genotype. Two SSR genotypes of F. sachalinensis were also detected. Extremely low genetic variation within the invasive Fallopia taxa indicates that these taxa do not reproduce sexually in the region, suggesting that control efforts can be focused on preventing clonal spread. Climate warming may increase sexual reproduction of invasive Fallopia taxa in northern regions. The hermaphrodite F. × bohemica is a potential pollen source for the male‐sterile parental species. Targeted eradication of the hybrid can therefore reduce the risk of increased sexual reproduction under future warmer climate.

Molecular Characterization and Antifungal Susceptibility Testing of Sequentially Obtained Clinical Cryptococcus deneoformans and Cryptococcus neoformans Isolates from Ljubljana, Slovenia.

To retrospectively investigate the epidemiology of cryptococcosis in Ljubljana, Slovenia. Forty-six sequentially obtained isolates from 19 patients were subjected to amplified fragment length polymorphism (AFLP) genotyping, microsatellite typing, mating- and serotype PCRs and antifungal susceptibility testing. Majority of the isolates were Cryptococcus deneoformans (n=29/46; 63%) followed by Cryptococcus neoformans (n=16/46; 34.8%) and their interspecies hybrid (n=1/46; 2.2%). Mating-type α was predominant, two mating-type a C. deneoformans isolates and one mating-type a/α isolate were observed. Several mixed infections were found by microsatellite typing; one patient had a persisting C. deneoformans infection for>2.5years. For C. deneoformans, the in vitro antifungal MIC90 and susceptibility ranges were for amphotericin B 0.25µg/ml (0.031-0.25µg/ml), 5-fluorocytosine 0.25µg/ml (0.063-4µg/ml), fluconazole 8µg/ml (0.5-16µg/ml), voriconazole 0.063µg/ml (0.008-0.125µg/ml), posaconazole 0.063µg/ml (0.008-0.063µg/ml) and itraconazole 0.063µg/ml (0.031-0.125µg/ml). For C. neoformans, these values were for amphotericin B 0.25µg/ml (0.063-0.5µg/ml), 5-fluorocytosine 1µg/ml (0.063-1µg/ml), fluconazole 16µg/ml (0.5-64µg/ml), voriconazole 0.125µg/ml (0.008-0.25µg/ml), posaconazole 0.063µg/ml (0.008-0.063µg/ml) and itraconazole 0.063µg/ml (0.031-0.125µg/ml). Majority of the cases were caused by C. deneoformans; mating-type α was predominant. Several mixed infections were identified by AFLP genotyping and microsatellite typing. Despite antifungal therapy, a cryptococcal isolate could persist for years. Voriconazole, itraconazole and posaconazole were the most potent antifungal drugs.

Invasive Russian Knapweed (Acroptilon repens) Creates Large Patches Almost Entirely by Rhizomic Growth

Russian knapweed is an outcrossing perennial invasive weed in North America that can spread by both seed and horizontal rhizomic growth leading to new shoots. The predominant mode of spread at the local scale and dispersal at the long-distance scale informs control but has not been quantitatively researched. We used amplified fragment-length polymorphisms (AFLPs) of DNA collected from 174 shoots in two discrete patches of Russian knapweed at each of three locations in Montana. Out of the 174 shoots collected, we found nine AFLP genotypes. Three out of the six patches were monotypic; the other three patches each had one rare genotype. No genotypes were shared between patches. The maximum diameter of a genet (a genetic individual) was 56.5 m. These results indicate that patch expansion at the local scale is almost entirely by rhizomes that spread and develop new shoots. At the long-distance scale, dispersal is by seed. Controlling seed development through biological control and herbicide use may be effective at stopping long-distance dispersal but may not affect expansion of existing patches.

Genetic variation of Sporothrix globosa isolates from diverse geographic and clinical origins in China

Sporothrix globosa is the main causative agent of sporotrichosis, a common mycosis that usually affects the skin, in China. Despite increasing efforts in the molecular identification of this fungal pathogen, its modes of transmission and epidemiology remain poorly understood. The goals of this study were to assess the genetic diversity of S. globosa using amplified fragment length polymorphism (AFLP) analysis and to assess the correlation of AFLP profiles with the geographic origins, growth rates, clinical forms, and antifungal susceptibilities of S. globosa isolates. AFLP analysis of 225 clinical S. globosa isolates from eight provinces or municipalities in China identified eight distinct clustering groups (I–VIII), with groups I, II and IV being the most common. The AFLP genotypes showed distinct distribution patterns among different regions within Jilin Province and between northern and southern China, but there was no obvious association between the AFLP genotypes and the growth rates, clinical forms or antifungal susceptibilities of the S. globosa isolates. These results expand our understanding of the genetic variation of S. globosa and suggest that AFLP analysis is a potentially useful tool for studying the epidemiology of this fungal pathogen.Emerging Microbes & Infections (2017) 6, e88; doi:10.1038/emi.2017.75; published online 11 October 2017

Cryptococcus neoformans population diversity and clinical outcomes of HIV-associated cryptococcal meningitis patients in Zimbabwe.

HIV and cryptococcal meningitis co-infection is a major public health problem in most developing countries. Cryptococcus neoformans sensu stricto is responsible for the majority of HIV-associated cryptococcosis cases in sub-Saharan Africa. Despite the available information, little is known about cryptococcal population diversity and its association with clinical outcomes in patients with HIV-associated cryptococcal meningitis in sub-Saharan Africa. In a prospective cohort, we investigated the prevalence and clinical outcome of Cryptococcusneoformans sensu stricto meningitis among HIV-infected patients in Harare, Zimbabwe, and compared the genotypic diversity of the isolates with those collected from other parts of Africa. Molecular typing was done using amplified fragment length polymorphism genotyping and microsatellite typing. The majority of patients with HIV-associated Cryptococcusneoformans sensu stricto meningitis in this cohort were males (n=33/55; 60.0 %). The predominant Cryptococcus neoformans sensu stricto genotype among the Zimbabwean isolates was genotype AFLP1/VNI (n=40; 72.7 %), followed by AFLP1A/VNB/VNII (n=8; 14.6 %), and AFLP1B/VNII was the least isolated (n=7; 12.7 %). Most of the isolates were mating-type α (n=51; 92.7 %), and only four (7.3 %) were mating-type a. Overall in-hospital mortality was 55.6 % (n=30), and no difference between infecting genotype and clinical outcome of patient (P=0.73) or CD4+ counts (P=0.79) was observed. Zimbabwean Cryptococcusneoformans sensu stricto genotypes demonstrated a high level of genetic diversity by microsatellite typing, and 51 genotypes within the main molecular types AFLP1/VNI, AFLP1A/VNB/VNII and AFLP1B/VNII were identified. This study demonstrates that Cryptococcusneoformans sensu stricto in Zimbabwe has a high level of genetic diversity when compared to other regional isolates.

Diversity of Oxalis tuberosa Molina: a comparison between AFLP and microsatellite markers

Traditional crops contribute to food security and agroecological sustainability, but their diversity is increasingly threatened by complex interplays of local and global sociocultural and economic change. Molecular markers are powerful tools to measure and characterize this diversity, and comparisons among different molecular marker systems are necessary to assess their appropriateness in different research contexts. Using a common sample set, we compare amplified fragment length polymorphism (AFLP) and microsatellite (simple sequence repeats; SSRs) techniques to assess their utility in research on the Andean tuber crop oca (Oxalis tuberosa Molina, Oxalidaceae). We find that 26 of 27 individuals have distinct AFLP genotypes, and all 27 individuals have distinct SSR genotypes. Both markers systems cluster samples in agreement with morphotype groups and separate clusters with similar strength, but more variation occurs within AFLP-based clusters than within SSR-based clusters. In addition, correlation between marker systems of pairwise distances is positive and significant (R = 0.831, p = 0.001). Ultimately, we discuss each system’s advantages and disadvantages for future oca diversity research.

Genetic and Morphological Variation of Two Rare Species of Lepidium (Brassicaceae) on Mangere Island, Chatham Islands, and Implications for Their Conservation Management

The genus Lepidium in New Zealand comprises a number of threatened and endemic species. Two recently described species, Lepidium panniforme and L. oblitum, have a restricted distribution on the remote and difficult to access Mangere Island, Chatham Islands. We examined the genetic and morphological variation of L. panniforme and L. oblitum on Mangere Island to assess the best conservation management options. Amplified fragment length polymorphism (AFLP) data and morphological data were used to detect finer scale variation in L. panniforme and L. oblitum. Bayesian analysis of AFLP genotypes detected up to four genetic clusters on Mangere Island. The morphological data were also variable, although better reflected the taxonomic treatment of the Mangere Island plants. The conservation implications of such high genetic and morphological variation over such a small spatial scale are discussed, to propose the best method for ensuring the ongoing survival of these island endemics.

Propagule pressure, genetic structure, and geographic origins of Chondrilla juncea (Asteraceae): An apomictic invader on three continents

Assessing propagule pressure and geographic origins of invasive species provides insight into the invasion process. Rush skeletonweed (Chondrilla juncea; Asteraceae) is an apomictic, perennial plant that is invasive in Australia, South America (Argentina), and North America (Canada and the United States). This study comprehensively compares propagule pressure and geographic structure of genotypes to improve our understanding of a clonal invasion and enhance management strategies. • We analyzed 1056 native range plants from Eurasia and 1156 plants from three invaded continents using amplified fragment length polymorphism (AFLP) techniques. We used measures of diversity (Simpson's D) and evenness (E), analysis of molecular variance, and Mantel tests to compare invasions, and genotype similarity to determine origins of invasive genotypes. • We found 682 unique genotypes in the native range, but only 13 in the invaded regions. Each invaded region contained distinct AFLP genotypes, suggesting independent introduction events, probably with different geographic origins. Relatively low propagule pressure was associated with each introduction around the globe, but levels of among-population variation differed. We found exact AFLP genotype matches between the native and invaded ranges for five of the 13 invasive genotypes. • Invasion dynamics can vary across invaded ranges within a species. Intensive sampling for molecular analyses can provide insight for understanding intraspecific invasion dynamics, which can hold significance for the management of plant species, especially by finding origins and distributions of invasive genotypes for classical biological control efforts.

Molecular genotyping of Acinetobacter spp. isolated in Arizona, USA, using multilocus PCR and mass spectrometry

Acinetobacter spp. are a diverse group of Gram-negative bacteria frequently implicated in nosocomial infections. Genotypic methods have been instrumental in studying Acinetobacter, but few offer high resolution, rapid turnaround time, technical ease and high inter-laboratory reproducibility, which has hampered understanding of disease incidence, transmission patterns and diversity within this genus. Here, we further evaluated multilocus PCR electrospray ionization/mass spectrometry (PCR/ESI-MS), a method that is simple and robust, and provides both species characterization and strain-level resolution of Acinetobacter spp. on a single platform. We examined 125 Acinetobacter isolates from 21 hospitals, laboratories and medical centres spanning four counties in Arizona, USA, using PCR/ESI-MS. We compared PCR/ESI-MS with an in-house amplified fragment length polymorphism (AFLP) genotyping scheme. PCR/ESI-MS demonstrated that Acinetobacter spp. from Arizonan hospitals had similar species and strain distributions to other US civilian hospitals. Furthermore, we showed that the PCR/ESI-MS and AFLP genotypes were highly congruent, with the former having the advantages of robust inter-laboratory reproducibility, rapid turnaround time and simple experimental set-up and data analysis. PCR/ESI-MS is an effective and high-throughput platform for strain typing of Acinetobacter baumannii and for identification of other Acinetobacter spp., including the emerging nosocomial pathogens Acinetobacter pittii and Acinetobacter nosocomialis.

Evolution of the polyploid north-west Iberian Leucanthemum pluriflorum clan (Compositae, Anthemideae) based on plastid DNA sequence variation and AFLP fingerprinting

The genus Leucanthemum is a species-rich polyploid complex from southern and central Europe, comprising 41 species with ploidy ranging from 2x to 22x. The present contribution aims at reconstructing the evolutionary history of a geographically isolated species group (the L. pluriflorum clan) from the north-west Iberian Peninsula comprising the diploid L. pluriflorum, the tetraploids L. ircutianum subsp. pseudosylvaticum and L. × corunnense (a putative hybrid taxon based on crossing between L. pluriflorum and L. merinoi), and the hexaploids L. sylvaticum and L. merinoi. Chromosome number variation (determined flow cytometrically) and sequence variation were analysed for two intergenic spacer regions on the plastid genome (psbA-trnH and trnC-petN) for individuals from 54 populations in combination with amplified fragment length polymorphism (AFLP) fingerprinting of 246 representative individuals from these populations. Plastid sequence data revealed that all surveyed members of the L. pluriflorum clan possess plastid haplotypes that are closely related to each other and distinctly separated from other Leucanthemum species. AFLP fingerprinting resulted in allopolyploid fragment patterns for most of the polyploid populations, except for the tetraploid L. × corunnense and a further tetraploid population in northern Galicia, which cluster with the diploids rather than with the other polyploids. In silico modelling of (auto)tetraploid AFLP genotypes further corroborates the allopolyploid nature of L. ircutianum subsp. pseudosylvaticum, L. sylvaticum and L. merinoi. The present study provides evidence for recognizing one diploid (L. pluriflorum), one autotetraploid (L. corunnense), one allotetraploid (L. pseudosylvaticum) and one allohexaploid (L. sylvaticum with the two geographically and ecologically differentiated subspecies subsp. sylvaticum and subsp. merinoi) in the L. pluriflorum clan. It also has implications for the understanding of biogeographical patterns in the Iberian Peninsula.

Patterns of genetic diversity in natural populations of Paspalum agamic complexes

Paspalum has many multiploid species displaying a wide range of ploidy levels and reproductive systems including apomixis. However, not much is known about the genetic structure of natural populations of the apomictic species of Paspalum. The aim of this work was to evaluate the genetic diversity of several natural populations belonging to five species of Paspalum. A total of 13 populations were analyzed using amplified fragment length polymorphism (AFLP). The AFLP data revealed maximal genotypic diversity and significant levels of genetic diversity in diploid and mixed diploid–tetraploid populations of P. denticulatum and P. rufum, where all individuals represent different genotypes. This may be mainly due to the reproductive system of diploid members and the gene flow from diploids to polyploids. The pure populations of tetraploids consist of either multiple genotypes (P. nicorae) or of one dominant genotype with a few deviated genotypes (P. denticulatum and P. lividum). Here, the main source of variability may be the residual sexuality, which continues generating new genotypic combinations. The hexaploid populations of P. buckleyanum consist of a single AFLP genotype and each population represents a particular genotype suggesting that populations arose from independent polyploidization events. This study represents one of the first reports of genetic diversity in natural populations of several Paspalum agamic complexes. Apomixis in these five species may be acting as a successful method for the dispersion of better adapted genotypes.

Population Genetic Structures of Puccinia striiformis in Ningxia and Gansu Provinces of China.

Wheat stripe rust, caused by the fungal pathogen Puccinia striiformis f. sp. tritici, is one of the most destructive plant diseases in China. Gansu and Ningxia Provinces are considered to be the key areas for over-summering of this pathogen in China. In this study, 283 P. striiformis f. sp. tritici isolates were collected in these two provinces. Amplified fragment length polymorphism (AFLP) was used to analyze the population genetics and to infer the chance of population exchanges between different geographic locations and seasons. The genotypic diversity of the Gansu population (0.514) was slightly higher than that of the Ningxia population (0.489). The occurrence of frequent population exchanges between these two regions was observed, showing that 40 AFLP genotypes were shared by the populations of the two provinces. Gene flow between these two regions in autumn and spring subpopulations was also detected. The genotype distribution in three populations of Ningxia from opposite sides of the Liupan Mountains revealed possible significant effects of the mountains on limiting gene flow and population exchange. Phylogenetic analysis confirmed the possibility of recombination in some of the studied subpopulations in both provinces.

Cytological and Molecular Characterization of Genetic Diversity in Stenotaphrum

St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is a warm‐season turfgrass broadly distributed across the southern United States. Here, we investigated genetic diversity and ploidy levels in publicly available plant introductions and cultivars of St. Augustinegrass as an aid to more effective use of these materials in breeding programs. Ploidy assignment of genotypes was problematic in some cases because of a lack of agreement between flow cytometry–inferred ploidy level and chromosome counts indicating that DNA content of higher ploidy genotypes was not a simple multiple of the diploid genome. Cytological investigations indicated five different ploidy levels (diploid, triploid, aneuploid, tetraploid, and hexaploid) with chromosome numbers ranging from 2n = 2x = 18 to 2n = 6x = 54. Principal coordinate and cluster analyses separated genotypes into distinct groups that were mostly congruent with ploidy levels. Moreover, analysis of molecular variance results based on amplified fragment length polymorphism genotyping indicated that 46% of the total variation could be explained by differences between ploidy levels. A clear positive correlation was observed between ploidy level and number of scored bands, with polyploids showing an increased number of bands. Variation in chromosome number is an important source of genetic variation in S. secundatum, and knowledge of the genetic relationships among accessions of this species can be an important consideration for the proper utilization of this germplasm in applied cultivar development.

Yersinia enterocoliticaof Porcine Origin: Carriage of Virulence Genes and Genotypic Diversity

Yersinia enterocolitica is an important foodborne pathogen, and pigs are recognized as a major reservoir and potential source of pathogenic strains to humans. A total of 172 Y. enterocolitica recovered from conventional and antimicrobial-free pig production systems from different geographic regions (North Carolina, Ohio, Michigan, Wisconsin, and Iowa) were investigated to determine their pathogenic significance to humans. Phenotypic and genotypic diversity of the isolates was assessed using antibiogram, serogrouping, and amplified fragment length polymorphism (AFLP). Carriage of chromosomal and plasmid-borne virulence genes were investigated using polymerase chain reaction. A total of 12 antimicrobial resistance patterns were identified. More than two-thirds (67.4%) of Y. enterocolitica were pan-susceptible, and 27.9% were resistant against β-lactams. The most predominant serogroup was O:3 (43%), followed by O:5 (25.6%) and O:9 (4.1%). Twenty-two of 172 (12.8%) isolates were found to carry Yersinia adhesion A (yadA), a virulence gene encoded on the Yersinia virulence plasmid. Sixty-nine (40.1%) isolates were found to carry ail gene. The ystA and ystB genes were detected in 77% and 26.2% of the strains, respectively. AFLP genotyping of isolates showed wide genotypic diversity and were grouped into nine clades with an overall genotypic similarity of 66.8-99.3%. AFLP analysis revealed that isolates from the same production system showed clonal relatedness, while more than one genotype of Y. enterocolitica circulates within a farm.

Molecular evidence for Neotyphodium fungal endophyte variation and specificity within host grass species

Host specificity of Neotyphodium species symbiotic with three grass species, Festuca arundinacea, Festuca pratensis and Lolium perenne, was studied based on comparisons of amplified fragment length polymorphisms (AFLP) between hosts and their corresponding endophytes. Endophytic fungi were isolated from 24 accessions of host plants. Neotyphodium identity was determined based on morphological characteristics observed in cultures and polymerase chain reaction analysis using specific primers. The results of AFLP data analysis revealed high genetic variation in plant and fungal endophyte species. Plant AFLP genotypes from different species clustered in three distinctive groups, congruent with species. A cluster analysis of AFLP data grouped endophytic isolates according to their host species and secondarily according to their host geographic distribution. The result of the AMOVA on AFLP data accounted for a large and significant proportion of genetic variation due to differences among plant and endophyte species. Phylogenetic groups of isolates corresponded to their respective host genotypes based on maximum parsimony phylograms. Comparisons of the two phylograms illustrated a significant congruence between nodes and branches of host and endophyte clades. These results strongly suggest host specificity of Neotyphodium fungal endophytes with their geographically distant host grasses within each species.