Amplified Fragment Length Polymorphism Analysis Research Articles

Development and validation of novel SNP markers for the rapid identification of natural hybrids of the 11 closely related pufferfish species (Takifugu spp.) distributed in Japan

Accurate identification of interspecific hybrids is vital for breeding programs in hybridizing taxa, as well as for the use of hybrids in aquaculture and fisheries. The use of species-specific single nucleotide polymorphisms (SNPs) has become the standard for the molecular identification of hybrids. However, it remains challenging to use these SNPs to identify hybrids involving multiple candidate parental species. Here, we developed and validated SNP markers for the identification of hybrids of the 11 closely related pufferfish species (genus Takifugu) distributed in Japan; these species have different aquacultural characteristics, such as growth rate, physiology, behavior, and toxicity. In total, 1232 species-specific SNPs were identified through double-digest restriction-site-associated DNA sequencing. The species had 11 (T. flavipterus) to 295 (T. chrysops) species-specific SNPs. We validated 33 genome-wide species-specific SNPs using TaqMan® genotyping assays for the 11 species and their hybrids, which were identified using amplified fragment length polymorphism analyses. No false negative or positive assignments were observed in 77 and 30 specimens of pure species and F1 hybrids, respectively. This marker combination allows rapid and reliable identification of natural hybrids, which will be useful for pufferfish aquaculture and will enhance our understanding of the natural hybridization between various Takifugu species.

Rapid Genetic Assessment of Carrot Varieties Based on AFLP Analysis

It is necessary to regard the biodiversity of carrot as a genetic source of useful and indispensable components for the human diet. Amplified fragment length polymorphism (AFLP) markers were used to discriminate eight carrot genotypes with different root colors. With the combination of enzymes Tru9I, PstI, and three sets of primer pairs corresponding to adapters joint to the restricted sites, 92 loci were produced, including 60 polymorphic ones. Each of the three primer sets showed high efficiency, according to estimations of PIC (0.34, 0.34, and 0.41), D (0.36, 0.67, and 0.67), Rp (5.5, 11.3, and 15), and HE (0.32, 0.49, and 0.49). The genetic distances were calculated using values of Nei’s coefficient. The most genetically similar were Chantenay Coeur Rouge and Colmar a Coeur Rouge at a distance of 0.12, whereas the most distant were Saint Valery and Purple Dragon at the highest distance of 0.34. Confirming its genetic identity, Purple Dragon and Gelber Goliath with purple and yellow roots were the most detached varieties from others at distances of 0.23–0.34 and 0.23–0.28, respectively. Male sterile Berlicum breeding accessions were well distinct from other orange-colored varieties at the highest distance of 0.30 from Deep Purple F1. Slight modifications including the facilitation of gel staining enables the wide use of the AFLP method for genetic diversity assessment in carrot breeding accessions.

Varietal Descriptors for the Distinction of Underutilized Varieties of Sechium edule (Jacq) Swartz.

Sechium edule (Jacq.) Sw. (Cucurbitaceae) is a species native to Mexico and Central America. The collection, characterization, and evaluation of accessions maintained in genebanks is essential for the conservation of this species. However, there are no specific varietal descriptors that differ from those used in a phenetic approach and are adapted to international registration guidelines to help distinguish, improve, cluster, and protect intraspecific variants of common use and those obtained by breeding. Therefore, 65 morphological descriptors (qualitative and quantitative) were evaluated in 133 accessions obtained from Mexico, Guatemala, and Costa Rica located in the National Germplasm Bank of S. edule in Mexico. These characteristics were observed to be phenetically stable for five generations under the same agroclimatic conditions. In addition, an analysis of amplified fragment length polymorphism (AFLP) was applied to 133 samples from a set of 245 accessions. According to the multivariate analysis, 26 of the 65 descriptors evaluated (qualitative and quantitative) enabled differentiation of varieties of S. edule. The AFLP analysis showed a high level of polymorphism and genetic distance between cultivated accessions and their corresponding wild ancestor. The variations in S. edule suggest that the morphological characteristics have differentiated from an essentially derived initial edible variety (ancestral original variety), but unlike other cucurbits, there is no evidence of the ancestral edible for Sechium since the seed is unorthodox and there are no relicts.

Assessment of Genetic Diversity among Wild Ruta chalepensis L. from the North of Jordan

Ruta chalepensis, known as Fringed Rue, is a small shrub of the Rutaceae family. To date, there is no record of its natural distribution across Jordan, a country located in the eastern part of the Mediterranean basin, and there are no previous studies on its genetic diversity in the region. Therefore, this study was conducted to assess the genetic diversity of R. chalepensis in the northern parts of Jordan using morphological trait and amplified fragment length polymorphism (AFLP) analyses. For the morphological traits, the analysis of variance indicated that there were significant differences between the identified populations. The Shannon diversity indices showed relatively high values, indicating the existence of a high variability among the identified populations. The principal component analysis explained 82% of the variation between the collected plants, and a clear separation of the collected individuals from the Jarash-A, Jarash-B and Ajloun-B populations from the rest of the populations was observed. The heatmap clustering was in general agreement with the results of the principal component analysis, with the plant height, rachis length and plant width considered as the discriminative traits. The AFLP analysis using eight different primer combinations generated 59 polymorphic bands, with an average polymorphism information content value of 0.32. The phylogenetic analysis identified three main clusters, with the first cluster including 65% of the individuals collected from the Jarash and Ajloun provinces, with a clear separation of the Jarash-B population. The AMOVA revealed that the genetic variation between the populations contributed 30% of the total genetic variation, while the variation within the populations explained 70%. In conclusion, morphological traits and molecular markers were used successfully to assess the genetic diversity among wild R. chalepensis from the north of Jordan, and such data can be used for future conservation plans and utilization purposes.

Short-length Homologous Region exhaustive Search algorithm (SHRS): A primer design algorithm for differentiating bacteria at the species, subspecies, or strain level based on a whole genome sequence

In fields such as the food industry, it is very important to identify target bacteria at the species level or lower for optimal product quality control. Bacteria identification at the subspecies or lower level requires time-consuming and high-cost analyses such as multi-locus sequence typing and amplified fragment length polymorphism analyses. Herein, we developed a primer design algorithm for precisely identifying bacteria based on a whole genome DNA sequence that is easy to apply. The algorithm designs primer sets that produce fragments from all input sequences and maximizes the differences in the amplicon size or amplicon sequence among input sequences. We demonstrate that the primer sets designed by the algorithm clearly classified six subspecies of Lactobacillus delbrueckii, and we observed that the resolution of the method is equal to that of a multi-locus sequence analysis. The algorithm allows the easy but precise identification of bacteria within a short time. (SHRS is available freely from PyPI under the MIT license.)

Asexually propagated Agave tequilana var. azul exhibits variation in genetic markers and defence responses to Fusarium solani.

Agave (Agave tequilana var. azul) is considered a crop with low genetic diversity because it has been propagated vegetatively for centuries for commercial purposes, and consequently, it could be equally susceptible to pests and diseases. However, the present study employs plant material derived from field-grown plants exhibiting phenotypic variability in susceptibility to agave wilt. The offshoots from rhizomes of these plants were reproduced in vitro and classified as potentially resistant or susceptible. Amplified fragment length polymorphism analysis confirmed wide genetic differences among individuals, but these differences were not correlated with the observed phenotypic variability in resistance. Propagated plantlets were inoculated with Fusarium solani in two time-lapse confrontations for 72 h and 30 days. The early biochemical response showed statistically superior levels in the accumulation of shikimic acid, phenolic compounds, and chitinase activity in potentially resistant plantlets. There was an inverse correlation of these early biochemical responses and salicylic acid and the incidence of diseased root cells in isogenic plantlets in the 30-day confrontation with F. solani, suggesting that these activities and accumulation of molecules were primordial in the defence against this pathogen.

Ploidy, genetic diversity and speciation of the genus Aronia

Speciation in Aronia is complex based on Amplified Fragment Length Polymorphism (AFLP) and ploidy analysis of 132, mostly wild, accessions. There are four species of Aronia and seven taxonomic groups comprised of species by ploidy combinations. A. melanocarpa can be diploid or tetraploid, with diploid forms occurring in New England and tetraploid forms occurring primarily outside of New England. A. arbutifolia was only found as a tetraploid and did not appear to occur throughout parts of New England as is generally accepted. It is likely that numerous misidentifications of A. arbutifolia have occurred historically. Almost all wild A. prunifolia were tetraploid, with the exception of a single triploid plant. Evidence from AFLP analysis and several accessions expressing a continuum of morphological characteristics between those of A. arbutifolia and A. melanocarpa suggest that A. prunifolia is of interspecific hybrid origin. The occurrence of a natural triploid A. prunifolia accession and our ability to easily create triploid A. prunifolia progeny from diploid A. melanocarpa by tetraploid A. arbutifolia crosses supports the hybrid formation of the A. prunifolia species. Hybrid A. prunifolia origin is also supported by the prevalence of A. prunifolia accessions in geographic areas where A. arbutifolia and diploid A. melanocarpa interface. Most likely, tetraploid A. prunifolia found in the wild result from repeated formation of triploid F1 interspecific hybrids and the triploid bridge mechanism. AFLP analysis, along with morphology and phenology, suggest that a new species of black-fruited Aronia (melanocarpaS) exists in the southern part of the A. melanocarpa geographic range. A. melanocarpaS plants are placed on a separate branch of the AFLP dendrogram that is separate from other Aronia species. Several accessions with large, wide leaves, large fruits and non-rhizomatous growth were found to be A. mitschurinii, an intergeneric hybrid between Aronia and Sorbus. All A. mitschurinii accessions were tetraploid.

Obtain inter-section hybrids between Primula forbesii (Sect. Monocarpicae) and P. saxatilis (Sect. Cortusoides)

To breed excellent cultivars with prosperous fragrant flowers, long flowering period, and high resistance to hardiness, three Primula forbesii (Sect. Monocarpicae) cultivars (2n=2x=18) and P. saxatilis (Sect. Cortusoides) (2n=2x=24) were used as parents for hybridization. Fourteen progenies were obtained from three cross combinations of P. forbesii as maternal parent by embryo rescue. Eventually, 12 progenies were proved to be true hybrids (2n=2x=21) by flow cytometry, chromosome counting and amplified fragment length polymorphism analysis. All the hybrids had long lasting flowering period just like P. forbesii, and their leaf back and scapes were not covered by white powder like P. saxatilis. The hybrid progenies were more similar to their male parent P. saxatilis by phenotypes analysis. Pollen of hybrids was abnormal and the germination rates were very low (3.18%–0.23%). Phenomena such as chromosome outside the equatorial plate, lagging chromosomes, chromosome bridges, micronucleus and polyads happened during the process of pollen mother cell meiosis, resulting pollen abortion finally. The results of backcrossing with their parents as pollen donators indicated that the pollen was able to germinate on the stigma surface and pollen tubes could extend into the hybrids’ ovules. It was showed that the inter-section hybrids could be used as female parent, but could not be used as pollen donator in continuing hybridization.

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).

AFLP analysis of Genetic diversity and Phylogenetic relationships of Vigna radiata (L) Wilczek

Among the important pulse-yielding plants Vigna radiata is important. In the Indian subcontinent, it is an essential source of dietary protein particularly in the vegetarian population. It contains abundant nutrients with biological activities. Soaked seeds are eaten as a good source of protein and mature seeds are cooked or can be sprouted for human consumption. Mung beans and its sprouts contain chemical constituents like flavonoids, phenolic acids, organic acids, amino acids, carbohydrates, and lipids. It is related to different biological activities like antioxidant, antimicrobial, antitumor, anti-inflammatory, anti-diabetic, and antihypertensive effects, etc. The seeds are a traditional source of cures for paralysis, rheumatism, coughs, fevers, and liver ailments. As a green manure or cover crop, the plant can be grown, enriching the soil with the nitrogen formed on its roots. The powerful Amplified Fragment Length Polymorphism (AFLP) is a molecular marker used for a variety of applications like assessing genetic diversity within species or among closely related species, inferring population-level phylogenies, generating genetic maps, and determining the relatedness among cultivars. AFLP has become extremely beneficial in the study of taxa including bacteria, fungi, and plants, where much is still unknown about the genomic makeup of various organisms. A study was made to assess the genetic diversity and phylogenetic relationships of a set of five varieties of Vigna radiata (L) Wilczek using the AFLP technique. Five different varieties of Vigna radiata viz. B1, TARM 2, PDM 84, TM 99, and TM 98 were subjected to AFLP analysis. A total of 471 fragments were scored across all the 12 AFLP primer sets used and the results were used to plot a dendrogram. It was observed that these five varieties formed three distinct groups among which the aromatic B1 variety formed a distinct group.

MALDI-TOF MS characterisation, genetic diversity and antifungal susceptibility of Trichosporon species from Iranian clinical samples.

Trichosporonosis is an emerging fungal infection caused by Trichosporon species, a genus of yeast-like fungi, which are frequently encountered in human infections ranging from mild cutaneous lesions to fungemia in immunocompromised patients. The incidence of trichosporonosis has increased in recent years, owing to higher numbers of individuals at risk for this infection. Although amphotericin B, posaconazole and isavuconazole are generally effective against Trichosporon species, some isolates may have variable susceptibility to these antifungals. Herein, we evaluated the species distribution, genetic diversity and antifungal susceptibility profiles of Trichosporon isolates in Iran. The yeasts were identified by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS). Phylogenetic analysis was performed based on amplified fragment length polymorphism (AFLP). The in vitro susceptibilities of eight antifungal agents were analysed using the Clinical and Laboratory Standards Institute broth microdilution methods. The isolates belonged to the species Tasahii (n=20), Tjaponicum (n=4) and Tfaecale (n=3). A dendrogram of the AFLP analysis demonstrated that Tasahii and non-asahii Trichosporon strains (Tjaponicum and Tfaecale) are phylogenetically distinct. While voriconazole was the most active agent (GM MIC=0.075μg/ml), high fluconazole MICs (8μg/ml) were observed for a quarter of Trichosporon isolates. The GM MIC value of amphotericin B for Tasahii and non-asahii Trichosporon species was 0.9μg/ml. The distribution and antifungal susceptibility patterns of the identified Trichosporon species could inform therapeutic choices for treating these emerging life-threatening fungi.

Evaluation of genetic stability in olive callus-induced and meristem-induced shoots using flow cytometry and amplified fragment length polymorphism techniques

BackgroundIn vitro culture of olive, as an economically valuable tree, has fundamentally a genotype-dependant low micropropagation rate which needs to be improved in already established and newly released cultivars. Various plant tissue culture media, planting systems and growth factors were evaluated in two promissing Iranian olive cultivars ˈAminˈ and ˈMeshkatˈ and the commercial Spanish cultivar ˈArbequinaˈ.ResultsThe results showed that cultivars have their specific optimal media, i.e. ˈAminˈ in the MS with 4 mg/L zeatin, ˈArbequinaˈ in the OM with 1 mg/L zeatin, and ˈMeshkatˈ in the OM and MS with 2 mg/L zeatin, which produced significantly a higher number of axillary shoots than other media. The results also indicated a significant improvement in the growth indices of ˈAminˈ (number of axillary shoots) when cultured using periodical mini bioreactor (PMB) in the VS medium. In comparison with VS, OM did not reveal any significant differences when both culturing systems (PMB and semi-solid media (SSM)) were used.Regarding the effect of carbon source and light intensity, mannitol and 2000 cd sr m−2 greatly enhanced ˈArbequinaˈ growth indices (main shoot length and growth quality). The results of genetic stability of callus induced shoots (CIS) and meristem induced shoots (MIS) revealed that 2C DNA value assessed by partec flow cytometery (FCM) had 0.01, 0.03 and 0.08 pg discrepencies in ˈAminˈ, ˈArbequinaˈ and ˈMeshkatˈ, repectively. The Amplified Fragment Length Polymorphism (AFLP) results also indicated that the cultivars were classified regardless of the micropropagation origin (CIS or MIS), except for ˈArbequinaˈ. The AFLP findings showed that ˈArbequinaˈ had the highest dispersal (7–38%) in CIS and MIS, while the Iranian cultivar of ˈMeshkatˈ (5–9%) had the highest stability.ConclusionsThis study indicated the importance of in vitro growth parameters for improving the micropropagation indices of olive cultivars. It showed that optimized protocols (OM, PMB, zeatin, mannitol and 2000 cd sr m−2) co-produced larger calli resulting in indirect organogenesis. Based on FCM and AFLP analysis, it can be concluded that true-to-typeness of micropropagated olive was cultivar-dependent.

Copper Ions Induce DNA Sequence Variation in Zygotic Embryo Culture-Derived Barley Regenerants.

In vitro tissue culture could be exploited to study cellular mechanisms that induce sequence variation. Altering the metal ion composition of tissue culture medium affects biochemical pathways involved in tissue culture-induced variation. Copper ions are involved in the mitochondrial respiratory chain and Yang cycle. Copper ions may participate in oxidative mutations, which may contribute to DNA sequence variation. Silver ions compete with copper ions to bind to the complex IV subunit of the respiratory chain, thus affecting the Yang cycle and DNA methylation. The mechanisms underlying somaclonal variation are unknown. In this study, we evaluated embryo-derived barley regenerants obtained from a single double-haploid plant via embryo culture under varying copper and silver ion concentrations and different durations of in vitro culture. Morphological variation among regenerants and the donor plant was not evaluated. Methylation-sensitive Amplified Fragment Length Polymorphism analysis of DNA samples showed DNA methylation pattern variation in CG and CHG (H = A, C, or T) sequence contexts. Furthermore, modification of in vitro culture conditions explained DNA sequence variation, demethylation, and de novo methylation in the CHG context, as indicated by analysis of variance. Linear regression indicated that DNA sequence variation was related to de novo DNA methylation in the CHG context. Mediation analysis showed the role of copper ions as a mediator of sequence variation in the CHG context. No other contexts showed a significant sequence variation in mediation analysis. Silver ions did not act as a mediator between any methylation contexts and sequence variation. Thus, incorporating copper ions in the induction medium should be treated with caution.

Genetic and Epigenetic Changes Are Rapid Responses of the Genome to the Newly Synthesized Autotetraploid Carassius auratus.

Whole genome duplication events have occurred frequently during the course of vertebrate evolution. To better understand the influence of polyploidization on the fish genome, we herein used the autotetraploid Carassius auratus (4n = 200, RRRR) (4nRR) resulting from the whole genome duplication of Carassius auratus (2n = 100, RR) (RCC) to explore the genomic and epigenetic alterations after polyploidization. We subsequently performed analyses of full-length transcriptome dataset, amplified fragment length polymorphism (AFLP) and methylation sensitive amplification polymorphism (MSAP) on 4nRR and RCC. By matching the results of 4nRR and RCC isoforms with reference genome in full-length transcriptome dataset, 649 and 1,971 novel genes were found in the RCC and 4nRR full-length geneset, respectively. Compared to Carassius auratus and Megalobrama amblycephala, 4nRR presented 3,661 unexpressed genes and 2,743 expressed genes. Furthermore, GO enrichment analysis of expressed genes in 4nRR revealed that they were enriched in meiosis I, whereas KEGG enrichment analysis displayed that they were mainly enriched in proteasome. Using AFLP analysis, we noted that 32.61% of RCC fragments had disappeared, while 32.79% of new bands were uncovered in 4nRR. Concerning DNA methylation, 4nRR exhibited a lower level of global DNA methylation than RCC. Additionally, 60.31% of methylation patterns in 4nRR were altered compared to RCC. These observations indicated that transcriptome alterations, genomic changes and regulation of DNA methylation levels and patterns had occurred in the newly established autotetraploid genomes, suggesting that genetic and epigenetic alterations were influenced by autotetraploidization. In summary, this study provides valuable novel insights into vertebrate genome evolution and generates relevant information for fish breeding.

A pseudo-outbreak of Rhinocladiella similis in a bronchoscopy unit of a tertiary care teaching hospital in London, United Kingdom.

Outbreaks of fungal infections due to emerging and rare species are increasingly reported in healthcare settings. We investigated a pseudo-outbreak of Rhinocladiella similis in a bronchoscopy unit of a tertiary care teaching hospital in London, UK. We aimed to determine route of healthcare-associated transmission and prevent additional infections. From July 2018 through February 2019, we detected a pseudo-outbreak of R.similis isolated from bronchoalveolar lavage (BAL) fluid samples collected from nine patients who had undergone bronchoscopy in a multispecialty teaching hospital, during a period of 8months. Isolates were identified by MALDI-TOF mass spectrometry. Antifungal susceptibility testing was performed by EUCAST broth microdilution. To determine genetic relatedness among R.similis isolates, we undertook amplified fragment length polymorphism analysis. To determine the potential source of contamination, an epidemiological investigation was carried out. We reviewed patient records retrospectively and audited steps taken during bronchoscopy as well as the subsequent cleaning and decontamination procedures. Fungal cultures were performed on samples collected from bronchoscopes and automated endoscope washer-disinfector systems. No patient was found to have an infection due to R.similis either before or after bronchoscopy. One bronchoscope was identified to be used among all affected patients with positive fungal cultures. Physical damage was found in the index bronchoscope; however, no fungus was recovered after sampling of the affected scope or the rinse water of automated endoscope washer-disinfectors. Use of the scope was halted, and, during the following 12-month period, Rhinocladiella species were not isolated from any BAL specimen. All pseudo-outbreak isolates were identified as R.similis with high genetic relatedness (>90% similarity) on ALFP analysis. The study emphasises the emergence of a rare and uncommon black yeast R.similis, with reduced susceptibility to echinocandins, in a bronchoscope-related pseudo-outbreak with a potential water-related reservoir. Our findings highlight the importance of prolonged fungal culture and species-level identification of melanised yeasts isolated from bronchoscopy samples. Possibility of healthcare-associated transmission should be considered when R.similis is involved in clinical microbiology samples.

Ex vitro Rooting, Acclimatization and Genetic Stability of Lonicera caerulea var. kamtschatica

Abstract Ex vitro rooting and acclimatization of two cultivars ‘Wojtek’ and ‘Zojka’ of blue honeysuckle (Lonicera caerulea var. kamtschatica Sevast.) were studied. To the ex vitro conditions were transferred rooted and unrooted shoots. The post-effect of auxin type and concentration as well as microcutting and soil substrate types were tested. The genetic stability of the plantlets in relation to the mother plants by using amplified fragment length polymorphism (AFLP) and inter simple sequence repeat (ISSR) markers has been also determined. It has been found that in vitro rooted cuttings of both cultivars showed a higher survival rate (max. 88%) and better growth and development when they were rooted on a medium containing a low auxin level (1.0 mg·dm−3). The results of the second experiment showed successful ex vitro rooting of blue honeysuckle shoots without auxin treatment. Higher ex vitro rooting and survival rate in the greenhouse have been observed for ‘Wojtek’ (max. 96%) than ‘Zojka’ (max. 88%). Better growth and development of shoots and roots were observed on peat alone or a mixture of peat and perlite as compared to a mixture of peat and sand. The micropropagated plantlets appeared similar to mother plants. Molecular analysis confirmed a high level of genetic stability of blue honeysuckle after 2 years of in vitro propagation. However, among the cultivars studied, ‘Wojtek’ showed slightly higher genetic stability than ‘Zojka’ (99.5% and 97.7%, respectively). For ‘Zojka’ plants, the degree of variation was comparable for AFLP and ISSR markers. For ‘Wojtek’, no polymorphism was detected using the ISSR analysis in contrast to the AFLP analysis.

Virulence and Molecular Diversity in the Kabatiella zeae Population Causing Maize Eyespot in China.

Maize eyespot, caused by Kabatiella zeae, has become a major yield-limiting factor in maize planting areas in northeast China. Limited information is available on pathotypes, virulence, and the genetic diversity of the K. zeae population. We analyzed virulence and genetic diversity of 103 K. zeae isolates collected from six provinces in China with differential hosts and the amplified fragment length polymorphism (AFLP) technique, respectively. To evaluate the virulence, 103 isolates were inoculated on nine differential hosts (maize inbred lines)-E28, Shen137, Qi319, B73, Danhuang34, Zi330, Mo17, Huangzaosi, and CN165-and grouped into 23 pathotypes and three virulence groups according to the coded triplet nomenclature system on differential hosts. AFLP analysis resolved the set of isolates into four genetic diversity clusters (DICE similarity values of 76%). Genetic variation of K. zeae among and between pathotypes revealed that the pathogen population had a high genotypic diversity. The correlation between pathotypes, virulence, and genetic diversity grouping was low. A correlation between AFLP groups and geographic locations was detected.

Habitat matters - Strong genetic and epigenetic differentiation in Linum catharticum from dry and wet grasslands.

Plant species differ in their ecological amplitude, with some species occurring in very different habitats under strongly differentiated environmental conditions. We were interested in to what extent the occurrence of Linum catharticum in dry calcareous grasslands (Bromion) and wet litter meadows (Molinion), two habitats on opposing ends concerning, for example, moisture level, is reflected on the genetic and epigenetic level. Using AFLP (amplified fragment length polymorphisms) and MSAP (methylation sensitive amplification polymorphisms) analyses, we studied the genetic and epigenetic variation of L. catharticum from calcareous grasslands and litter meadows. From each habitat, we included five study sites with 16 individuals per sampling location. We observed lower genetic than epigenetic diversity, but considerable differentiation among habitats, which was stronger on the genetic than the epigenetic level. Additionally, we observed a strong correlation of genetic and epigenetic distance, irrespective of geographic distance. The dataset included a large portion of fragments exclusively found in individuals from one or the other habitat. Some epigenetic fragments even occurred in different methylation states depending on the habitat. We conclude that environmental effects act on both the genetic and epigenetic level, producing the clear differentiation among plant individuals from calcareous grasslands and litter meadows. These results may also point into the direction of ecotype formation in this species.

Genetic variation of Anoectochilus formosanus revealed by ISSR and AFLP analysis

Anoectochilus formosanus is an expensive medicinal folk herb in Taiwan. The plant has been used to treat hypertension, diabetes, and hepatitis by native people for a long time. In this study, 4 typical lines of A. formosanus, red-stem, green-stem, narrow-leaf, and round-leaf, were collected and screened to identify the line-specific SSR (single sequence repeat) markers. In addition, we found the narrow-leaf line exhibited the highest polysaccharide content (3.39 ± 0.74%, polysaccharide wt/plant dried wt) which is more than twofold of the lowest content of the red-stem line (1.27 + 0.41%). In the culture program construction, the extent of the somaclonal variation among 20 regenerated shoots from the same parental individual of a narrow-leaf line was revealed using the amplified fragment length polymorphism (AFLP) technique. The aim of this study is to preliminarily investigate the genetic variation of A. formosanus through polymorphic marker screening, which will benefit to the cultivation of A. formosanus with high polysaccharide content in future work. The results showed that 6 out of 81 primers screened, generating a total of 26 markers, were able to differentiate the 4 lines in the inter single sequence repeat (ISSR) analysis. On the other hand, 17 sets of AFLP primers were chosen to detect the somatic variation among the excised shoots derived from the same individual. The genetic variation among the regenerated shoots ranged from 0.00% to 5.43%. The primer pair B6 exerts the most powerful selection ability in the AFLP analysis among 20 somaclonal samples used, whereas the E4 primer pair the lowest. The AFLP primer pairs become useful tools for selecting the lowest somaclonal variation from the regenerated shoots of A. formosanus under the tissue culture program.

Molecular Subtyping of Salmonella enterica serovar Enteritidis isolated from Taiwan during 1992-1998 by amplified fragment length polymorphism and pulsed-field gel electrophoresis

Over the past 10 years, Salmonella enterica serovar Enteritidis has emerged as a new serovar and a frequent cause of food-borne diseases in Taiwan. We used two molecular subtyping methods to investigate the development of the infections by S. enterica Enteritidis in Taiwan. Twenty-eight strains of S. enterica Enteritidis isolated from Taiwan during the period of 1992 to 1998 were collected. The primer pairs, MseI+C/EcoRI+0 and MseI+G/PstI+C, were used in amplified fragment length polymorphism (AFLP) analyses, and the restriction enzyme, AvrII, was used in pulsed-field gel electrophoresis (PFGE). Twenty-two AFLP profiles with 53-59 fragments in each profile were found by using the primer pair, MseI+G/PstI+C. In the PFGE analysis, 7 PFGE types were identified. Some of the collected strains, especially the imported strains, had closer relationships with the SE 02 strain isolated from Taipei County. As our results indicate, AFLP analysis was time-saving, easy to perform, and highly discriminative. In this study, AFLP and PFGE were used to analyze the S. enterica Enteritidis isolated from patients in Taiwan. These results provide the epidemiological distribution of these isolated strains and additional evidence to illustrate the sources of the food-borne pathogens.