AFLP Profiles Research Articles

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.

Genetic Variability in Balkan Paleoendemic Resurrection Plants Ramonda serbica and R. nathaliae Across Their Range and in the Zone of Sympatry.

The genus Ramonda includes three Paleoendemic and Tertiary relict species that survived in refugial habitats of the Balkan Peninsula (R. nathaliae and R. serbica) and the Iberian Peninsula (R. myconi). They are all “resurrection plants,” a rare phenomenon among flowering plants in Europe. Ramonda myconi and R. nathaliae are diploids (2n = 2x = 48), while R. serbica is a hexaploid (2n = 6x = 144). The two Balkan species occur in sympatry in only two localities in eastern Serbia, where tetraploid potential hybrids (2n = 4x = 96) were found. This observation raised questions about the existence of gene flow between the two species and, more generally, about the evolutionary processes shaping their genetic diversity. To address this question, genetic markers (AFLP) and an estimate of genome size variation were used in a much larger sample and at a larger geographic scale than previously. The combination of AFLP markers and genome size results suggested ongoing processes of interspecific and interploidy hybridization in the two sites of sympatry. The data also showed that interspecific gene flow was strictly confined to sympatry. Elsewhere, both Ramonda species were characterized by low genetic diversity within populations and high population differentiation. This is consistent with the fact that the two species are highly fragmented into small and isolated populations, likely a consequence of their postglacial history. Within sympatry, enormous variability in cytotypes was observed, exceeding most reported cases of mixed ploidy in complex plant species (from 2x to >8x). The AFLP profiles of non-canonical ploidy levels indicated a diversity of origin pathways and that backcrosses probably occur between tetraploid interspecific hybrids and parental species. The question arises whether this diversity of cytotypes corresponds to a transient situation. If not, the question arises as to the genetic and ecological mechanisms that allow this diversity to be maintained over time.

Improving plant-based genotoxicity bioassay through AFLP technique for trace metal-contaminated water: insights from Myriophyllum aquaticum (Vell.) Verdc. and Cd

In this work, we evaluated whether the species Myriophyllum aquaticum (Vell.) Verdc. can be a promising material for devising reliable eco-toxicological tests for Cd-contaminated waters. Plants of M. aquaticum were exposed to Cd, using different concentrations (1 mg L−1, 2.5 mg L−1, 5 mg L−1, and 10 mg L−1; experiment 1) and exposure times (2.5 mg L−1 for 3 days, 7 days, 14 days, and 21 days; experiment 2). Plant growth and Cd accumulation were monitored during the treatment period, and Cd genotoxicity was assessed by analyzing Cd-induced changes in the AFLP fingerprinting profiles using famEcoRI(TAC)/MseI(ATG) and hexEcoRI(ACG)/MseI(ATG) pairs of primers. Root and shoot growth was reduced already at the lowest Cd concentration used (about 20% reduction for roots and 60% for shoots at 1 mg L−1; experiment 1) and after 7 days (about 50% reduction for roots and 70% for shoots; experiment 2). The primer combinations produced 154 and 191 polymorphic loci for experiments 1 and 2, respectively. Mean genetic diversity (He) reduction among the treatment groups was observed starting from 2.5 mg L−1 (He 0.211 treated vs 0.236 control; experiment 1) and after 3 days (He 0.169 treated vs 0.261 control; experiment 2), indicating that results obtained from AFLP profiles did not match with plant growth measurements. Therefore, our results showed that M. aquaticum proved to be a suitable model system for the investigation of Cd genotoxicity through AFLP fingerprinting profile, whereas the more classic eco-toxicological tests based only on biometric parameters could not correctly estimate the risk associated with undetected Cd genotoxicity.

Mentha longifolia uçucu yağlarının sitolojik, fizyolojik, genetik ve morfolojik etkilerine genel bir bakış

The present study was aimed to impress the cytological, physiological, genetic, and morphological effects of the essential oil obtained by hydro-distillation from Mentha longifolia (L.) HUDSON (L.) HUDSON against maize seeds. These naturally occurred compounds can be used instead of synthetic chemicals. To determine their biological activity, the oil was applied to Zea mays L. seeds at different doses. It was observed that the germination rate and mitotic division of the root tips were decreased with the increasing of the applied doses. Genomic stability was established by RAPD and the genetic differences between doses with AFLP technique. The alterations of RAPD and AFLP profiles did not support germination rate and mitotic index results while supporting each other. To determine the effects of the volatile on gene expression, the soluble protein level, and hormone levels were measured and the changes in the protein profile were determined by SDS-PAGE analyses. The independent changes were observed at hormones, soluble protein level, and protein profiles. It was concluded that the different content of oils showed a complicated effect on maize seeds. The maize seeds showed different reactions for protection.

<i>Xanthomonas albilineans</i> (Ashby) Dowson: Study of Its Genetic Variability in Sugarcane Samples in Cuba

Four isolates of the phytopathogenic bacterium <i>Xanthomonas albilineans (Xa)</i> from samples taken in commercial fields of different Cuban provinces and two from the cultivar L55-5, artificially infected in the Camagüey and Matanzas Experimental Stations, were used for the extraction of total nucleic acids according to the CTAB protocol. They were amplified by PCR using four primer combinations: A: P1-M1, B: P1-M2, C: P2-M1 and D: P2-M2. Direct sequencing of the PCR products was performed on all samples and the nucleotide sequences were used to generate partial consensus of 16-23S rRNA, which were analyzed with the BLASTn program. In all the isolates evaluated, the presence of a common band at the height of 620 bp was visualized and the AFLP profile showed that there is homology when comparing the sequences corresponding to the 16-23S rDNA of <i>X. albilineans</i> of the different isolates; the phylogenetic analysis reflected a grouping of the isolates of the species, regardless of its geographical origin, different from others of the genus <i>Xanthomonas</i>. The comparison of the Cuban isolates with eight strains published in GenBank, showed marked similarity with them, with 100% nucleotide identity and percentage of coverage with a genome fragment of the GPE PC73 strain from Guadalupe, KO905 from the United States and a strain from Brazil. The predominance of the generalized circulation in Cuba of a single serovar is presumed.

Sidik Jari DNA Material Kultur Jaringan Menggunakan SSR dan AFLP

Kultur jaringan dimanfaatkan sebagai alat dalam program pemuliaan dan perbanyakan material komersial kelapa sawit. Untuk mengontrol proses kultur jaringan di laboratorium, analisis DNA dapat dilakukan dalam usaha menjamin kebenaran informasi identitas serta untuk mengetahui kestabilan genetik material pada tiap tahap proses kultur jaringan. Penelitian ini dilakukan untuk mengkaji ekstraksi DNA material kultur jaringan kelapa sawit serta sidik jari DNA pada material kultur pada tiap tahapan proses kultur jaringan, menggunakan 11 marka SSR dan 6 kombinasi primer selektif AFLP. Hasil menunjukkan bahwa protokol ekstraksi DNA yang dilakukan dapat digunakan untuk memperoleh DNA dengan kuantitas dan kualitas yang cukup untuk PCR-SSR dan PCR-AFLP. Profil SSR yang sama ditunjukkan pada semua cuplikan material yang dianalisis pada tiap tahap proses kultur jaringan. Terdapat variasi hasil sidik jari DNA menggunakan AFLP, dimana terdapat profil AFLP yang berbeda pada material yang sama pada tahap kalus dan eksplan, serta embrio dan ramet. Perbedaan profil DNA pada material yang sama pada tahap kultur yang berbeda tersebut menunjukkan adanya perubahan genetik material kultur yang mungkin disebabkan oleh pengaruh proses kultur jaringan. SSR dapat digunakan untuk identifikasi atau verifikasi identitas material kultur, sedangkan marka DNA yang menunjukkan ketidakstabilan genetik material kultur dapat digunakan untuk kajian lebih lanjut mengenai perubahan genetik material kultur dalam kaitannya dengan abnormalitas klon.

Genetic evidence of multiple introductions and mixed reproductive strategy in the peregrine earthworm Pontoscolex corethrurus

Pontoscolex corethrurus is a well-known invasive earthworm in tropical zone which is believed to have originated from the Guayana Shield in South America and was described as parthenogenetic. A recent phylogenetic study revealed four cryptic species in the P. corethrurus complex (L1, L2, L3 and L4), among them L1 was particularly widespread and was proposed as P. corethrurus sensu stricto. Here, our aims were to investigate the genetic variation of P. corethrurus L1 in its presumed native and introduced ranges and to examine its reproductive strategy. An extensive dataset of 478 cytochrome oxidase I gene (COI) sequences, obtained in specimens sampled all around the world, revealed a weak COI haplotype diversity with one major haplotype (H1) present in 76% of the specimens. Analyses of the genetic variation of 12 L1 populations were done using both nuclear (226 AFLP profiles) and mitochondrial (269 COI sequences) genetic information. The high AFLP genotype diversity at the worldwide scale and the fact that no genotype was shared among populations, allowed to reject the ‘super-clone’ invasion hypothesis. Moreover, a similar level of mean genetic diversity indices were observed between the introduced and native ranges, a pattern explained by a history of multiple introductions of specimens from different parts of the world. At last, occurrence of identical AFLPs genotypes (i.e. clones) in several population confirmed asexual reproduction, but recombination was also revealed by gametic equilibrium analysis in some populations suggesting that P. corethrurus L1 may have a mixed reproductive strategy.

Phylogenetic Assignment of Ralstonia pseudosolanacearum (Ralstonia solanacearum Phylotype I) Isolated from Rosa spp.

During the last two years, greenhouse cultivation of rose (Rosa spp.) in the Netherlands has been challenged by an uncommon bacterial disease. Affected plants suffered from chlorosis, stunting, wilting, and necrosis. The bacterial isolates obtained from the different Rosa spp. cultivars were all identified as phylotype I, sequevar 33 from the 'Ralstonia solanacearum species complex' (RSSC), actually reclassified as Ralstonia pseudosolanacearum. The work in this paper considers the genetic diversity and the phylogenetic position of 129 R. pseudosolanacearum isolates from the outbreak. This was assessed by AFLP based on four different primer combinations and MLP using partial sequences of the egl, mutS, and fliC genes. The AFLP revealed identical profiles for all the isolates, irrespective of their association with Rosa sp. propagating material, Rosa spp. plants for cut flowers, or water used in the different greenhouse cultivations. These AFLP profiles were unique and diverged from profiles of all other reference isolates in the RSSC included. Furthermore, MLP on egl, fliC, and mutS gene sequences clearly demonstrated that all R. pseudosolanacearum isolates clustered in phylotype I, as a distinct monophyletic group. Interestingly, this monophyletic group also included phylotype I strain Rs-09-161 from eggplant (Solanum melongena), isolated in 2009 in India. AFLP and MLP were both efficient in revealing the genetic divergence from the RSSC isolates included. The phylogenetic tree constructed from the AFLP profiles was, in general, in agreement with the one obtained from MLP. Both phylogenetic trees displayed a similar clustering, supported by high posterior probabilities. Both methodologies clearly demonstrated that the R. pseudosolanacearum isolates from Rosa spp. grouped in a monophyletic group inside phylotype I, with a particular correspondence to a strain present in India, as revealed in MLP.

AFLP Approach Reveals Variability in Phragmites australis: Implications for Its Die-Back and Evidence for Genotoxic Effects.

Phragmites australis is a subcosmopolitan species typical of wetlands being studied in Europe for its disappearance from natural stands, a phenomenon called reed die-back syndrome (RDBS). Although it is conjectured that low genetic variability contributes to RDBS, this aspect remains neglected to this day. Using a molecular fingerprinting approach and a sequence analysis of the trnT-trnL/rbcL-psaI regions of cpDNA, this study aimed to compare the genetic structure of stable vs. RDBS-affected P. australis stands from five wetlands of central Italy. Beforehand, in order to characterize the health condition of reed populations, the occurrence of the main macromorphological descriptors for RDBS was considered on 40 reed stands. Soil samples were also collected to examine the total content of heavy metals. The current study analyzed cpDNA in 19 samples and AFLP profiles in 381 samples to investigate the genetic structure of Phragmites populations. Based on the multinomial-Dirichlet model, an analysis of candidate loci under selective pressure was also performed. The relationships among AFLP data, RDBS descriptors and chemicals were evaluated with the use of Linear Mixed Models. The analysis of the cpDNA shows the occurrence of the haplotypes M (the most widespread), and K here recorded for the first time in Italy. Three new haplotypes were also described. The DNA fingerprinting analysis has produced a total of 322 loci (98% polymorphic) and shows the medium-to-high amount of genetic diversity. The significant genetic differentiation among wetlands (Fst = 0.337) suggests either low gene flow or small effective population size. Moreover, the low amount of outlier loci (only 5; l.5% of the total), seems to indicate the scarce occurrence of selective pressure upon the reed’s genome. Genetic diversity increased in relationship to the decrease in diameter and of flowering buds of the reed, two of the trends associated with the die-back. The current study rejects the hypothesis that genetic diversity massively contributed to RDBS. Moreover, significant relationships between genetic diversity and the total concentration of some heavy metals (Cr, Cu, and Zn) were highlighted, indicating possible genotoxic effects on P. australis. The current study represents a fact-finding background useful for the conservation of common reed.

Authentication of commercially important tuna species landed in Tuticorin coast of Tamil Nadu, India by SE-AFLP method

Seafood serves as a valuable protein source for human population. Among the seafood, tuna is considered as one of the commercially important species worldwide. Five tuna species namely Euthynnus affinis, Auxis thazard, Katsuwonus pelamis, Thunnus albacares and Thunnus obesus are commonly landed in the Tuticorin coast of Tamil Nadu. Among the five species, the exportable quality and grade of the meat is as follows: T. albacares&gt; T. obesus&gt; K. pelamis&gt; E. affinis&gt; A. thazard. In recent years, mislabeling is done by replacing high value tuna meat with low value tuna meat or with other low value fish meat to earn illegally. Usually, the species identification is done based on the morphological features, but this cannot be applied for the processed fish. PCR based identification methods have gained importance in the identification of fish species. PCR-AFLP is one of the molecular based methods, which can differentiate even closely related species. In this study, SE-AFLP method was employed to differentiate the above five tuna species viz., E. affinis, A. thazard, K. pelamis, T. albacares and T. obesus. Species specific AFLP marker was obtained in the primer combination of EcoR1 for fresh tuna. Band Sharing Index (BSI) analysis was also performed to find the similarities and variation among the five tuna species. AFLP profile of unknown tuna products was compared with the standard AFLP profile and the tuna species authentication was done by analysing BSI score.

Systematics of Simplicia Kirk (Poaceae, Agrostidinae) - an endemic, threatened New Zealand grass genus.

A new species of the New Zealand endemic grass Simplicia, Simplicia felix is described. The new species is segregated from and compared with Simplicia buchananii and Simplicia laxa. Simplicia felix occurs mostly in lightly shaded areas of seasonally dry alluvial forest. A distribution map and an assessment of the conservation status of the new species are presented. Genetic variation in the genus was examined, building on previously published work but including additional sampling. Analysis of nrDNA ITS and ETS and plastid trnL intron and trnL–F intergenic spacer sequences show Simplicia felix to be more closely related to Simplicia laxa than to Simplicia buchananii. NeighborNet analyses of AFLP profiles for the three species of Simplicia show each to consist of distinct clusters of genotypes well separated from each other.

Assessment of diversity and genetic relationships of Neonectria ditissima: the causal agent of fruit tree canker.

Background Neonectria ditissima is one of the most important fungal pathogens of apple trees, where it causes fruit tree canker. Information about the amount and partitioning of genetic variation of this fungus could be helpful for improving orchard management strategies and for breeding apple cultivars with high levels of genetically determined resistance. In this study single-spore Neonectria isolates originating from both the same and from different perithecia, apple cultivars and apple orchards in Sweden and Belgium, were evaluated for AFLP- and SSR-based genetic similarity and for mating system.ResultsSeven SSR loci produced a total of 31 alleles with an average of 4 alleles per locus, while 11 AFLP primer combinations produced an average of 35 fragments per primer combination and 71 % polymorphic fragments. An AFLP-based analysis of molecular variance (AMOVA) revealed that 89 % of the variation was found within orchards and 11 % between orchards. Genetic similarity among the studied isolates was illustrated with a principal coordinate analyseis (PCoA) and a dendrogram. AFLP-based Jaccard’s similarity coefficients were the highest when single-ascospore isolates obtained from the same perithecium were compared, medium-high for isolates from different perithecia on the same tree, and lowest when isolates from different trees were compared.ConclusionsBased on the results of PCoA and AMOVA analysis, isolates from the same or geographically close orchards did not group together. Since AFLP profiles differed also when single-ascospore isolates from the same perithecium were compared, the mating system of N. ditissima is most likely heterothallic.

Insight into the genomic diversity and relationship of Astragalus glycyphyllos symbionts by RAPD, ERIC-PCR, and AFLP fingerprinting.

We assessed the genomic diversity and genomic relationship of 28 Astragalus glycyphyllos symbionts by three methodologies based on PCR reaction, i.e., RAPD, ERIC-PCR, and AFLP. The AFLP method with one PstI restriction enzyme and selective PstI-GC primer pair had a comparable discriminatory power as ERIC-PCR oneand these fingerprinting techniques distinguished among the studied 28 A. glycyphyllos symbionts 18 and 17 genomotypes, respectively. RAPD method was less discriminatory in the genomotyping of rhizobia analyzed and it efficiently resolved nine genomotypes. The cluster analysis of RAPD, ERIC-PCR, and AFLP profiles resulted in a generally similar grouping of the test strains on generated dendrograms supporting a great potential of these DNA fingerprinting techniques for study of genomic polymorphism and evolutionary relationship of A. glycyphyllos nodulators. The RAPD, ERIC-PCR, and AFLP pattern similarity coefficients between A. glycyphyllos symbionts studied was in the ranges 8–100, 18–100, and 23-100 %, respectively.

Assessment of genetic and epigenetic changes during cell culture ageing and relations with somaclonal variation in Coffea arabica

Long-term cell cultures were used in coffee to study the cytological, genetic and epigenetic changes occurring during cell culture ageing. The objective was to identify the mechanisms associated with somaclonal variation (SV). Three embryogenic cell lines were established in Coffea arabica (2n = 4x = 44) and somatic seedlings were regenerated after 4, 11 and 27 months. Phenotyping and AFLP, MSAP, SSAP molecular markers were performed on 199 and 124 plants, respectively. SV were only observed from the 11 and 27-month-old cultures, affecting 30 and 94 % of regenerated plants, respectively. Chromosome counts performed on 15 plants showed that normal plants systematically displayed normal chromosome numbers and that, conversely, aneuploidy (monosomy) was systematically found in variants. The allopolyploid structure of C. arabica allowed aneuploid cells to survive and regenerate viable plants. No polymorphic fragments were observed between the AFLP and SSAP electrophoretic profiles of mother plants and those of the in vitro progeny. Methylation polymorphism was low and ranged between 0.087 and 0.149 % irrespective of the culture age. The number of methylation changes per plant—normal or variant—was limited and ranged from 0 (55–80 % of the plants) to 4. The three cell lines showed similar SV rate increases during cell culture ageing and produced plants with similar molecular patterns indicating a non random process. The results showed that cell culture ageing is highly mutagenic in coffee and chromosomal rearrangements are directly linked to SV. Conversely, the analysis of methylation and transposable elements changes did not reveal any relation between the epigenetic patterns and SV.

Genetic Diversity of Dry Bean (Phaseolus vulgaris L.) Accessions of Kenya Using SSR Markers

AFLP technique was applied to evaluate the genetic diversity among 35 common beans varieties of market class drawn from four populations. Two AFLP primers produced 324 polymorphic loci with an estimated variance of 53.45 and a within population variance of 83%. The pair wise matching indices (SM) based on the AFLP profiles were subjected to UPGMA cluster analysis. The pair wise matching coefficients among P. vulgaris generated a dendogram with the similarity coefficient values ranging from 0.66 to 0.89. Neis’ genetic Distance (Ds) among the four populations from which the bean samples were drawn varied from 0.01 to 0.09. The genetic distance observed between the dry bean from the highlands and the dry lands was lower (0.0186), compared to those from western province and market centres (0.0326). From the analysis, moderate to high genetic diversity was observed. These findings can be used to guide future bean breeding studies and germplasm management in Kenya.

Determination of biodiversity of Coprinus comatus using genotyping and metabolic profiling tools.

Coprinus comatus strains (CCMs) originating from Poland were identified using ITS region sequencing. Based on the sequences obtained, the genetic relationship between the CCM strains was determined and a clear separation of all strains into two main clusters was obtained. The Coprinus strains were also genetically characterized for the first time by the AFLP technique. The analysis showed that the CCMs separated into four main clusters and a high complication of a UPGMA-based dendrogram was achieved. C. comatus strains included in the analysis displayed an AFLP profile similarity level in the range from 44 to 66%. The highest similarity coefficient, 0.490, was found between CCM12 and CCM13, and the lowest (0.202) between the CCM2 and CCM5 isolates. Biolog FF MicroPlates were applied to obtain data on utilization of 95 carbon sources and mycelial growth. The analysis allowed comparison of the functional diversity of the CCM strains and revealed a broad variability within the analyzed Coprinus species based on substrate utilization profiles. Significant differences (2-48) have been shown in the substrate richness values. The Biolog experiments proved to be a good profiling technology for studying the diversity in shaggy manes due to metabolic differences and demonstrated that all the strains might be considered individually. It is evident that the strain metabolic grouping does not correlate with the grouping based on the ITS sequences and AFLP profiles, however, some similarities may be observed.

Genetic and metabolic intraspecific biodiversity of Ganoderma lucidum.

Fourteen Ganoderma lucidum strains from different geographic regions were identified using ITS region sequencing. Based on the sequences obtained, the genomic relationship between the analyzed strains was determined. All G. lucidum strains were also genetically characterized using the AFLP technique. G. lucidum strains included in the analysis displayed an AFLP profile similarity level in the range from 9.6 to 33.9%. Biolog FF MicroPlates were applied to obtain data on utilization of 95 carbon sources and mitochondrial activity. The analysis allowed comparison of functional diversity of the fungal strains. The substrate utilization profiles for the isolates tested revealed a broad variability within the analyzed G. lucidum species and proved to be a good profiling technology for studying the diversity in fungi. Significant differences have been demonstrated in substrate richness values. Interestingly, the analysis of growth and biomass production also differentiated the strains based on the growth rate on the agar and sawdust substrate. In general, the mycelial growth on the sawdust substrate was more balanced and the fastest fungal growth was observed for GRE3 and FCL192.

Molecular, Physiological and Phenotypic Characterization of Paracoccus denitrificans ATCC 19367 Mutant Strain P-87 Producing Improved Coenzyme Q10.

Coenzyme Q10 (CoQ10) is a blockbuster nutraceutical molecule which is often used as an oral supplement in the supportive therapy for cardiovascular diseases, cancer and neurodegenerative diseases. It is commercially produced by fermentation process, hence constructing the high yielding CoQ10 producing strains is a pre-requisite for cost effective production. Paracoccus denitrificans ATCC 19367, a biochemically versatile organism was selected to carry out the studies on CoQ10 yield improvement. The wild type strain was subjected to iterative rounds of mutagenesis using gamma rays and NTG, followed by selection on various inhibitors like CoQ10 structural analogues and antibiotics. The screening of mutants were carried out using cane molasses based optimized medium with feeding strategies at shake flask level. In the course of study, the mutant P-87 having marked resistance to gentamicin showed 1.25-fold improvements in specific CoQ10 content which was highest among all tested mutant strains. P-87 was phenotypically differentiated from the wild type strain on the basis of carbohydrate assimilation and FAME profile. Molecular differentiation technique based on AFLP profile showed intra specific polymorphism between wild type strain and P-87. This study demonstrated the beneficial outcome of induced mutations leading to gentamicin resistance for improvement of CoQ10 production in P. denitrificans mutant strain P-87. To investigate the cause of gentamicin resistance, rpIF gene from P-87 and wild type was sequenced. No mutations were detected on the rpIF partial sequence of P-87; hence gentamicin resistance in P-87 could not be conferred with rpIF gene. However, detecting the mutations responsible for gentamicin resistance in P-87 and correlating its role in CoQ10 overproduction is essential. Although only 1.25-fold improvement in specific CoQ10 content was achieved through mutant P-87, this mutant showed very interesting characteristic, differentiating it from its wild type parent strain P. denitrificans ATCC 19367, which are presented in this paper.

Brazil nuts are subject to infection with B and G aflatoxin-producing fungus, Aspergillus pseudonomius

The exploitation of the Brazil nut is one of the most important activities of the extractive communities of the Amazon rainforest. However, its commercialization can be affected by the presence of aflatoxins produced by fungi, namely Aspergillus section Flavi. In the present study, we investigated a collection of Aspergillus nomius strains isolated from Brazil nuts using different approaches, including morphological characters, RAPD and AFLP profiles, partial β-tubulin and calmodulin nucleotide sequences, aflatoxin patterns, as well as tolerance to low water activity in cultured media. Results showed that most of the isolates do belong to A. nomius species, but a few were re-identified as Aspergillus pseudonomius, a very recently described species. The results of the analyses of molecular variance, as well as the high pairwise FST values between A. nomius and A. pseudonomius suggested the isolation between these two species and the inexistence of gene flow. Fixed interspecific nucleotide polymorphisms at β-tubulin and calmodulin loci are presented. All A. pseudonomius strains analyzed produced aflatoxins AFB1, AFB2, AFG1 and AFG2. This study contains the first-ever report on the occurrence in Brazil nuts of A. pseudonomius. The G-type aflatoxins and the mycotoxin tenuazonic acid are reported here for the first time in A. pseudonomius.

Rapid genomic and transcriptomic alterations induced by wide hybridization: Chrysanthemum nankingense × Tanacetum vulgare and C. crassum × Crossostephium chinense (Asteraceae)

BackgroundHybridization is a major driver of evolution in plants. In a number of plant species, the process of hybridization has been revealed to be accompanied by wide-ranging genetic and epigenetic alterations, some of which have consequences on gene transcripts. The Asteraceae family includes a number of polyploid species, and wide crossing is seen as a viable means of genetically improving ornamental species such as Chrysanthemum spp. However, the consequences of hybridization in this taxon have yet to be characterized.ResultsAmplified fragment length polymorphism (AFLP), methylation sensitive amplification polymorphism (MSAP) and cDNA-AFLP profiling of the two intergeneric hybrids C. nankingense × Tanacetum vulgare and C. crassum × Crossostephium chinense were employed to characterize, respectively, the genomic, epigenomic and transcriptomic changes induced by the hybridization event. The hybrids’ AFLP profiles included both the loss of specific parental fragments and the gain of fragments not present in either parent’s profile. About 10% of the paternal fragments were not inherited by the hybrid, while the corresponding rate for the maternal parent fragments was around 4–5%. The novel fragments detected may have arisen either due to heterozygosity in one or other parent, or as a result of a deletion event following the hybridization. Around one half of the cDNA-AFLP fragments were common to both parents, about 30% were specific to the female parent, and somewhat under 20% specific to the male parent; the remainder (2.9-4.7%) of the hybrids’ fragments were not present in either parent’s profile. The MSAP fingerprinting demonstrated that the hybridization event also reduced the amount of global cytosine methylation, since > 50% of the parental fragments were methylated, while the corresponding frequencies for the two hybrids were 48.5% and 50.4%.ConclusionsCombining two different Asteraceae genomes via hybridization clearly induced a range of genomic and epigenomic alterations, some of which had an effect on the transcriptome. The rapid genomic and transcriptomic alterations induced by hybridization may accelerate the evolutionary process among progenies.