Multilocus System Research Articles

Analysis of the Genetic Diversity and Mating System of the Endangered Plant Keteleeria davidiana var. calcarea

Keteleeria davidiana var. calcarea is an endangered plant endemic to China. It is mainly distributed in the karst areas of the Guangxi and Guizhou Provinces. It is characterized by small populations and intermittent distribution. This study aimed to explore the genetic diversity and mating system of wild populations of Keteleeria davidiana var. calcarea in fragmented habitats. To achieve this, we genotyped 46 maternal trees and 214 progenies from four fragmented populations of Keteleeria davidiana var. calcarea using nine pairs of microsatellite primers. The genetic diversity of Keteleeria davidiana var. calcarea (Ho = 0.68, He = 0.63) was lower than that of the species overall but higher than that of other Keteleeria plants. The incidence of unbiased expected heterozygosity (uHe) and allelic richness (Ar) was higher in the maternal generation than in the progeny. This suggests that the genetic diversity of the progeny was lower than that of the maternal generation. Keteleeria davidiana var. calcarea is divided into four populations, but there is significant genetic exchange between the populations according to STRUCTURE and gene flow analyses. The multilocus mating system analysis (MLTR) results indicate that the multilocus outcrossing rate (tm) was 0.902, the single-locus outcrossing rate (ts) was 0.606, the bimaternal inbreeding coefficient (tm-ts) was 0.295, and the coefficient of inbreeding depression(δ) was 0.904. These results suggest a certain degree of selfing and inbreeding in Keteleeria davidiana var. calcarea. To prevent problems associated with inbreeding and conserve the genetic diversity of Keteleeria davidiana var. calcarea, we recommend establishing seed gardens, using artificial pollination, and employing asexual propagation techniques for conservation intervention.

Eradication of avian leukosis virus subgroups J and K in broiler cross chickens by selection against infected birds using multilocus PCR.

The avian leukosis virus (ALV) is a serious threat to sustainable and economically viable commercial poultry management world-wide. Active infections can result in more than 20% flock loss, resulting in significant economic damage. ALV detection and elimination from flocks and breeding programs is complicated by high sequence variability and the presence of endogenous virus copies which show up as false positives in assays. Previously-developed approaches to virus detection are either too labor-intensive to implement on an industrial scale or suffer from high false negative or positive rates. We developed a novel multi-locus multiplex quantitative real-time PCR system to detect viruses belonging to the J and K genetic subgroups that are particularly prevalent in our region. We used this system to eradicate ALV from our broiler breeding program comprising thousands of individuals. Our approach can be generalized to other ALV subgroups and other highly genetically diverse pathogens.

Multi-locus transcranial magnetic stimulation system for electronically targeted brain stimulation

BackgroundTranscranial magnetic stimulation (TMS) allows non-invasive stimulation of the cortex. In multi-locus TMS (mTMS), the stimulating electric field (E-field) is controlled electronically without coil movement by adjusting currents in the coils of a transducer. ObjectiveTo develop an mTMS system that allows adjusting the location and orientation of the E-field maximum within a cortical region. MethodsWe designed and manufactured a planar 5-coil mTMS transducer to allow controlling the maximum of the induced E-field within a cortical region approximately 30 mm in diameter. We developed electronics with a design consisting of independently controlled H-bridge circuits to drive up to six TMS coils. To control the hardware, we programmed software that runs on a field-programmable gate array and a computer. To induce the desired E-field in the cortex, we developed an optimization method to calculate the currents needed in the coils. We characterized the mTMS system and conducted a proof-of-concept motor-mapping experiment on a healthy volunteer. In the motor mapping, we kept the transducer placement fixed while electronically shifting the E-field maximum on the precentral gyrus and measuring electromyography from the contralateral hand. ResultsThe transducer consists of an oval coil, two figure-of-eight coils, and two four-leaf-clover coils stacked on top of each other. The technical characterization indicated that the mTMS system performs as designed. The measured motor evoked potential amplitudes varied consistently as a function of the location of the E-field maximum. ConclusionThe developed mTMS system enables electronically targeted brain stimulation within a cortical region.

Agile TMS: A multi-locus system for rapid and automatic spatial targeting and mapping

Agile TMS: A multi-locus system for rapid and automatic spatial targeting and mapping

Development of a Multi-Locus TMS System

Complete resection of symptomatic supratentorial cavernoma (SCA) and removal of the surrounding gliotic area is recommended to minimize the risk of persistent seizures or (re)bleeding. Surgery of SCA located in an eloquent area, can carry out severe postoperative neurological morbidity. We report a study aimed to assess feasibility, extent of resection and outcome after surgical removal of CA by cortico-subcortical intraoperative brain stimulation (ioBS) in the awake patient.Six patients diagnosed of symptomatic SCA located on an eloquent area and operated on while awake under local anaesthesia ioBS, were included. Preoperative planning included neuropsychologic assessment of language-related functions, sociocognitive functions and executive functions. Intraoperatively, we recorded the results achieved in the planned neuropsychological tasks when stimulation was applied (cortical and subcortical). Postoperative control 3D MRI was scheduled at 1 month after surgery to calculate extent of resection. Neuropsychological assessment at 6 months after surgery was performed in all cases.Six patients (5 females, 1 male) aged 24–48 years were included in our study. Locations of the lesions were right insular (n = 1), left insular (n = 1), left temporo-insular (n = 1), left temporal (n = 2) and left frontal (n = 1). In all patients, positive findings were obtained during ioBS. In 5 patients, complete surgical resection was achieved. Two patients had postoperative transient neurological deficits, one case of hemiparesis, one case of dysnomia, both cleared over a 6-month period. Clinical follow-up revealed that all patients experienced complete recovery from preoperative symptoms within a year and five patients with seizures showed marked improvement and eventually quit antiepileptic drugs. Neuropsychological assessment at 6 months provided normal results compared to preoperative baseline in all domains.Our study suggests that ioBS in the awake surgery of symptomatic SCA located in eloquent areas, allows to increase the rate of complete resection, minimizing postoperative neurological and neuropsychological deficit, and improving postoperative seizures control.La resección completa de los cavernomas supratentoriales (SCA) sintomáticos, incluyendo el área gliótica perilesional, es el tratamiento de elección para evitar la persistencia de crisis y el resangrado. La cirugía de los SCA localizados en áreas elocuentes puede asociar graves complicaciones neurológicas. Presentamos un estudio cuyo objetivo es documentar la viabilidad de la estimulación corticosubcortical intraoperatoria (ioBS) en el paciente despierto y su impacto en el grado de exéresis y el resultado clínico final.Incluimos 6 pacientes diagnosticados de SCA sintomático localizado en área elocuente, que fueron intervenidos mediante ioBS en el paciente despierto. El estudio preoperatorio incluyó una valoración neuropsicológica de funciones lingüísticas, sociocognitivas y ejecutivas. Durante la realización de la ioBS en el paciente despierto registramos los resultados obtenidos por los pacientes en las tareas neuropsicológicas planificadas. El grado de exéresis se estimó en una RM realizada un mes tras la cirugía. A los 6 meses de la cirugía se realizó una evaluación neuropsicológica de control.Cinco mujeres y un hombre con edades comprendidas entre los 24 y 48 años fueron incluidos en el estudio. Las localizaciones de los cavernomas fueron insular derecha (n = 1), insular izquierda (n = 1), temporo-insular izquierda (n = 1), temporal izquierda (n = 2) y frontal izquierda (n = 1). En todos los pacientes se encontraron hallazgos tras la ioBS. Se obtuvo una exéresis completa en 5 casos. Dos pacientes presentaron déficit neurológico transitorio, un caso de hemiparesia y un caso de disnomia, que mejoró a los 6 meses. El seguimiento clínico mostró que todos los pacientes presentaron al cabo de un año una recuperación completa de los síntomas por los que fueron diagnosticados. Los 5 pacientes con crisis de inicio pudieron dejar los fármacos antiepilépticos. La evaluación neuropsicológica a los 6 meses de la cirugía mostró una evolución normal en todos los dominios estudiados.Nuestro estudio sugiere que la ioBS con paciente despierto en la cirugía de SCA sintomático en área elocuente permite conseguir resecciones completas, reduciendo el riesgo de déficit neurológico y neuropsicológico posquirúrgico y mejorando el control de las crisis epilépticas.

Multi-Locus Sequence Typing of Ehrlichia chaffeensis Reveals Extensive Genotypic Variation across the United States.

Ehrlichia chaffeensis causes human monocytic ehrlichiosis, and its principal vector is the Amblyomma americanum tick. The most frequently identified cases of ehrlichiosis come from the southeastern and south central states of the United States. In this study, a molecular typing system was developed that allows for the genetic differentiation of E. chaffeensis isolates. This multi-locus typing system included sequencing and analyzing intergenic regions ECH0033-ECH0035 and ECH0217-ECH0218, plus, variable genes variable length PCR target, 28-kDa, 120-kDa, and hemE. We examined a total of 31 unique isolates from humans and white-tailed deer, and eight DNA samples extracted from infected A. americanum collected from multiple states. This is the largest evaluation of E. chaffeensis isolates and their genotypes. Our findings show that when sequences of all six loci were concatenated and compared, the 39 samples could be separated into 23 genotypes and further grouped into six phylogenetic clades. The data in this study show no clear pattern between the geographic alignment with the genetic differentiation between the strains. As a result, this poses a challenge to understanding the spread of E. chaffeensis in the United States. Interestingly, our findings indicate that multiple strains from distant geographic origins share the same mutations, which suggests that the strains are being moved from one site to another by their hosts or vectors. In addition, we are seeing a northward shift in the lone star tick distribution in the United States. Last, some data also suggest minimal genetic mutations have occurred over time among strains that are within geographical proximity.

A large-scale systematic framework of Chinese snakes based on a unified multilocus marker system

Snakes are one of the most diverse groups of terrestrial vertebrates, with approximately 3500 extant species. A robust phylogeny and taxonomy of snakes is crucial for us to know, study and protect them. For a large group such as snakes, broad-scale phylogenetic reconstructions largely rely on data integration. Increasing the compatibility of the data from different researches is thus important, which can be facilitated by standardization of the loci used in systematic analyses. In this study, we proposed a unified multilocus marker system for snake systematics by conflating 5 mitochondrial markers, 19 vertebrate-universal nuclear protein coding (NPC) markers and 72 snake-specific noncoding intron markers. This marker system is an addition to the large squamate conserved locus set (SqCL) for studies preferring a medium-scale data set. We applied this marker system to over 440 snake samples and constructed the currently most comprehensive systematic framework of the snakes in China. Robust snake phylogenetic relationships were recovered at both deep and shallow evolutionary depths, demonstrating the usefulness of this multilocus marker system. Discordance was revealed by a parallel comparison between the snake tree based on the multilocus marker system and that based on only the mitochondrial loci, highlighting the necessity of using multiple types of markers to better understand the snake evolutionary histories. The divergence times of different snake groups were estimated with the nuclear data set. Our comprehensive snake tree not only confirms many important nodes inferred in previous studies but also contributes new insights into many snake phylogenetic relationships. Suggestions are made for the current Chinese snake taxonomy.

Development of a multi-locus sequence typing system helps reveal the evolution of Cardinium hertigii, a reproductive manipulator symbiont of insects

BackgroundCardinium is an intracellular bacterial symbiont in the phylum Bacteroidetes that is found in many different species of arthropods and some nematodes. This symbiont is known to be able to induce three reproductive manipulation phenotypes, including cytoplasmic incompatibility. Placing individual strains of Cardinium within a larger evolutionary context has been challenging because only two, relatively slowly evolving genes, 16S rRNA gene and Gyrase B, have been used to generate phylogenetic trees, and consequently, the relationship of different strains has been elucidated in only its roughest form.ResultsWe developed a Multi Locus Sequence Typing (MLST) system that provides researchers with three new genes in addition to Gyrase B for inferring phylogenies and delineating Cardinium strains. From our Cardinium phylogeny, we confirmed the presence of a new group D, a Cardinium clade that resides in the arachnid order harvestmen (Opiliones). Many Cardinium clades appear to display a high degree of host affinity, while some show evidence of host shifts to phylogenetically distant hosts, likely associated with ecological opportunity. Like the unrelated reproductive manipulator Wolbachia, the Cardinium phylogeny also shows no clear phylogenetic signal associated with particular reproductive manipulations.ConclusionsThe Cardinium phylogeny shows evidence of diversification within particular host lineages, and also of host shifts among trophic levels within parasitoid-host communities. Like Wolbachia, the relatedness of Cardinium strains does not necessarily predict their reproductive phenotypes. Lastly, the genetic tools proposed in this study may help future authors to characterize new strains and add to our understanding of Cardinium evolution.

Specific clones of Trichomonas tenax are associated with periodontitis.

Trichomonas tenax, an anaerobic protist difficult to cultivate with an unreliable molecular identification, has been suspected of involvement in periodontitis, a multifactorial inflammatory dental disease affecting the soft tissue and bone of periodontium. A cohort of 106 periodontitis patients classified by stages of severity and 85 healthy adult control patients was constituted. An efficient culture protocol, a new identification tool by real-time qPCR of T. tenax and a Multi-Locus Sequence Typing system (MLST) based on T. tenax NIH4 reference strain were created. Fifty-three strains of Trichomonas sp. were obtained from periodontal samples. 37/106 (34.90%) T. tenax from patients with periodontitis and 16/85 (18.80%°) T. tenax from control patients were detected by culture (p = 0.018). Sixty of the 191 samples were tested positive for T. tenax by qPCR, 24/85 (28%) controls and 36/106 (34%) periodontitis patients (p = 0.089). By combining both results, 45/106 (42.5%) patients were positive by culture and/or PCR, as compared to 24/85 (28.2%) controls (p = 0.042). A link was established between the carriage in patients of Trichomonas tenax and the severity of the disease. Genotyping demonstrates the presence of strain diversity with three major different clusters and a relation between disease strains and the periodontitis severity (p<0.05). More frequently detected in periodontal cases, T. tenax is likely to be related to the onset or/and evolution of periodontal diseases.

Genotyping of Triticum ssp. Hexaploid Species Samples with ISSR-Markers

The genetic improvement of soft wheat (Triticum aestivum L.) and other hexaploid wheat species (T. spelta, T. spherococcum, T. petropavlovskyi, and T. compactum) is important. This can be done by transferring genes of interest (resistance to biotic and abiotic factors). For this purpose, phylogenetic connections of the studied genotypes should be investigated. This problem can be solved by using a multilocus system based on ISSR markers. The marker system is highly polymorphic and convenient for analysis. The study presents the results of a comparison of 20 different hexaploid wheat genotypes based on ISSR markers. In addition, the level of polymorphism was determined and a dendrogram reflecting the phylogenetic connections between the studied genotypes was constructed. It was shown that the species located next to each other according to the systematics turned out to be more remote after the ISSR-marker analysis, and, vice versa, the species that were systematically more distant showed a higher level of kinship.

Current state of knowledge on Wolbachia infection among Coleoptera: a systematic review.

BackgroundDespite great progress in studies on Wolbachia infection in insects, the knowledge about its relations with beetle species, populations and individuals, and the effects of bacteria on these hosts, is still unsatisfactory. In this review we summarize the current state of knowledge about Wolbachia occurrence and interactions with Coleopteran hosts.MethodsAn intensive search of the available literature resulted in the selection of 86 publications that describe the relevant details about Wolbachia presence among beetles. These publications were then examined with respect to the distribution and taxonomy of infected hosts and diversity of Wolbachia found in beetles. Sequences of Wolbachia genes (16S rDNA, ftsZ) were used for the phylogenetic analyses.ResultsThe collected publications revealed that Wolbachia has been confirmed in 204 beetle species and that the estimated average prevalence of this bacteria across beetle species is 38.3% and varies greatly across families and genera (0–88% infected members) and is much lower (c. 13%) in geographic studies. The majority of the examined and infected beetles were from Europe and East Asia. The most intensively studied have been two groups of herbivorous beetles: Curculionidae and Chrysomelidae. Coleoptera harbor Wolbachia belonging to three supergroups: F found in only three species, and A and B found in similar numbers of beetles (including some doubly infected); however the latter two were most prevalent in different families. A total of 59% of species with precise data were found to be totally infected. Single infections were found in 69% of species and others were doubly- or multiply-infected. Wolbachia caused numerous effects on its beetle hosts, including selective sweep with host mtDNA (found in 3% of species), cytoplasmic incompatibility (detected in c. 6% of beetles) and other effects related to reproduction or development (like male-killing, possible parthenogenesis or haplodiploidy induction, and egg development). Phylogenetic reconstructions for Wolbachia genes rejected cospeciation between these bacteria and Coleoptera, with minor exceptions found in some Hydraenidae, Curculionidae and Chrysomelidae. In contrast, horizontal transmission of bacteria has been suspected or proven in numerous cases (e.g., among beetles sharing habitats and/or host plants).DiscussionThe present knowledge about Wolbachia infection across beetle species and populations is very uneven. Even the basic data about infection status in species and frequency of infected species across genera and families is very superficial, as only c. 0.15% of all beetle species have been tested so far. Future studies on Wolbachia diversity in Coleoptera should still be based on the Multi-locus Sequence Typing system, and next-generation sequencing technologies will be important for uncovering Wolbachia relations with host evolution and ecology, as well as with other, co-occurring endosymbiotic bacteria.

DNA Isolation and Optimization of ISSR-PCR Reaction System in Oryza sativa L.

Inter simple sequence repeats (ISSRs) have been utilized widely for molecular markers in analyzing the genetic diversity and phylogenetic and regions in the genome flanked by microsatellite sequences. PCR amplification of these regions using a single primer yields multiple amplification products that can be used as a dominant multilocus marker system for the study of genetic variation in various organisms. For this study provides, DNA isolation, adjusting in six factors (Buffer, MgCl2, dNTPs, ISSR primers, Template DNA and Taq polymerase ) at six levels, and optimization of PCR temperature for the ISSR reaction was 60-45 °C, primers screening on indica rice ( Oryza sativa ). In this research, simple method of DNA isolation by using seedling. The objective of the present investigation was to assess the optimizations and quantification. Has been shown that stalk enhanced the maximum value of genomic. The results show that 100 ISSR primers were examined as well as, 56 ISSR primers was productively amplified. Optimum components for PCR reactions were 5.0 μl of 5X PCR Buffer, 1.5 μl of 25mM MgCl2, 1 μl of 10 mM dNTP, 1 μl of 10 Μm ISSR primers, 2 μl Template DNA, and 0.1 μl of 5 units/ml Taq polymerase. Based on this study, has brought out some information on the relationship between these ISSR primers will be applied further for molecular profiling as well as response evaluation in rice varieties

Quantifying the potential and flux landscapes of multi-locus evolution

Exploration of multi-locus evolution is critically important for understanding evolutionary dynamics. Recombination and epistasis lead to complex evolutionary dynamics. Quantifying the stability and function of such multi-locus evolutionary systems globally is a long-standing challenge for evolutionary biologists. The conventional Wright, Fisher and quasi-linkage equilibrium (QLE) theories can only be applied to highly restricted, simplified and special evolutionary scenarios. In this study, we developed a non-equilibrium potential and flux landscape theory to explore the multi-locus evolution beyond Wright, Fisher and the QLE. We found that the intrinsic potential landscape as a Lyapunov function under the zero noise limit can be used to describe the global stability of a deterministic multi-locus system. We identified and quantified two driving forces responsible for multi-locus evolution: the underlying landscape and the curl flux. We studied the evolution of different cases under recombination, epistasis and mutation. Recombination, frequency-dependent selection and mutation can give rise to non-zero curl flux. In particular, we investigated the dynamics of a simple example of a two-locus system. We explored the underlying potential landscape which shows stable basins attracting the system down to valleys and rotating curl flux, which aids the global communication under recombination, epistasis and mutation. Discontinuities in the first derivative of the non-equilibrium free energy functional shows the non-equilibrium phase transition as the recombination rate increases. We also explored the effect of epistasis on mono-stability and bi-stability evolution. Mutation may drive the system far from equilibrium and be another source of non-zero probability flux. Entropy production rate can quantify energy consumption or dissipation. We explained the origin of the Red Queen hypothesis for endless evolution using the curl flux. Our landscape and flux framework can be applied more generally to multi-locus evolutionary systems experiencing recombination and epistasis.

Developmental and internal validation of a novel 13 loci STR multiplex method for Cannabis sativa DNA profiling

Marijuana (Cannabis sativa L.) is a plant cultivated and trafficked worldwide as a source of fiber (hemp), medicine, and intoxicant. The development of a validated method using molecular techniques such as short tandem repeats (STRs) could serve as an intelligence tool to link multiple cases by means of genetic individualization or association of cannabis samples. For this purpose, a 13 loci STR multiplex method was developed, optimized, and validated according to relevant ISFG and SWGDAM guidelines. The STR multiplex consists of 13 previously described C. sativa STR loci: ANUCS501, 9269, 4910, 5159, ANUCS305, 9043, B05, 1528, 3735, CS1, D02, C11, and H06. A sequenced allelic ladder consisting of 56 alleles was designed to accurately genotype 101 C. sativa samples from three seizures provided by a U.S. Customs and Border Protection crime lab. Using an optimal range of DNA (0.5-1.0ng), validation studies revealed well-balanced electropherograms (inter-locus balance range: 0.500-1.296), relatively balanced heterozygous peaks (mean peak height ratio of 0.83 across all loci) with minimal artifacts and stutter ratio (mean stutter of 0.021 across all loci). This multi-locus system is relatively sensitive (0.13ng of template DNA) with a combined power of discrimination of 1 in 55 million. The 13 STR panel was found to be species specific for C. sativa; however, non-specific peaks were produced with Humulus lupulus. The results of this research demonstrate the robustness and applicability of this 13 loci STR system for forensic DNA profiling of marijuana samples.

Selective sweep of Wolbachia and parthenogenetic host genomes - the example of the weevil Eusomus ovulum.

Most parthenogenetic weevil species are postulated to have originated via hybridization, but Wolbachia has also been speculated to play a role via the induction of parthenogenesis. Here, we examine the molecular diversity of Wolbachia and parthenogenetic host genomes. The host species studied here, Eusomus ovulum, is known to be exclusively parthenogenetic and triploid. The E. ovulum populations that we examined had a low genetic diversity of mitochondrial (cytochrome oxidase I gene) and nuclear markers (internal transcribed spacer 2 and elongation factor 1-α gene), and they all were infected by only single bacteria strains (genotyped for five genes according to the multilocus sequence typing system). We found significant signs of linkage disequilibrium and a lack of recombination amongst all of the examined genomes (bacteria and host), which strongly indicates a selective sweep. The lack of heterozygosity in host nuclear genes, missing bisexual populations and selective sweep between the parthenogenetic host and bacteria genomes suggest that parthenogenesis in this species could have originated as a result of infection rather than hybridization. However, the finding that highly similar Wolbachia strains are also present in other parthenogenetic weevils from the same habitat suggests the opposite scenario: bacteria may have infected the already parthenogenetic lineage and taken advantage of the host's unisexual reproduction.

Evaluation of a 13-loci STR multiplex system for Cannabis sativa genetic identification.

Marijuana (Cannabis sativa) is the most commonly used illicit substance in the USA. The development of a validated method using Cannabis short tandem repeats (STRs) could aid in the individualization of samples as well as serve as an intelligence tool to link multiple cases. For this purpose, a modified 13-loci STR multiplex method was optimized and evaluated according to ISFG and SWGDAM guidelines. A real-time PCR quantification method for C. sativa was developed and validated, and a sequenced allelic ladder was also designed to accurately genotype 199 C. sativa samples from 11 U.S. Customs and Border Protection seizures. Distinguishable DNA profiles were generated from 127 samples that yielded full STR profiles. Four duplicate genotypes within seizures were found. The combined power of discrimination of this multilocus system is 1 in 70 million. The sensitivity of the multiplex STR system is 0.25 ng of template DNA. None of the 13 STR markers cross-reacted with any of the studied species, except for Humulus lupulus (hops) which generated unspecific peaks. Phylogenetic analysis and case-to-case pairwise comparison of 11 cases using F st as genetic distance revealed the genetic association of four groups of cases. Moreover, due to their genetic similarity, a subset of samples (N = 97) was found to form a homogeneous population in Hardy-Weinberg and linkage equilibrium. The results of this research demonstrate the applicability of this 13-loci STR system in associating Cannabis cases for intelligence purposes.

Genetic identity of common buckwheat (Fagopyrum esculentum Moench) landraces locally cultivated in the Alps

The conservation of landraces in Europe is challenging because very often they have already disappeared or cannot be properly identified, which in turn prevents any possibility for their utilization. This work deals with the collection of molecular and historical data to identify and study the original landraces of common buckwheat (Fagopyrum esculentum Moench), locally cultivated in Northern Italy (Valtellina) and to date surviving among other commercial varieties, recently introduced in the same areas of the Alps. As plant materials of F. esculentum, we analyzed a number of Italian accessions along with two foreign accessions from Poland and Nepal, for a total of 174 individuals. Molecular investigations were based on a set of eight nuclear SSR marker loci. The mean observed heterozygosity over all accessions was equal to Ho = 0.466, being significantly lower than the expected heterozygosity (He = 0.764). A major finding was the recognition of a marked inbreeding rate (Fit = 0.387) and a reduced fixation index (Fst = 0.061), indicating that most genetic variation is found within populations. A significant overall gene flow among accessions was found (Nm = 3.846). Results indicated that only two of the examined accessions, the so-called “Nustran” and “Curunin”, could be considered, authentic Valtellina landraces. On the basis of results, we successfully developed a multi-locus marker system and identified a number of co-dominant marker alleles suitable for genetic traceability and authenticity certification of a “Nustran” and a “Curunin” autochthonous landraces of Valtellina and its food derivatives (i.e., Pizzoccheri, Polenta taragna).

Long-distance pollen dispersal ensures genetic connectivity of the low-density tree species, Eurycorymbus cavaleriei, in a fragmented karst forest landscape

Karst forest in southwest China represents one of the world’s most spectacular examples of tropical-subtropical karst landscapes and harbours high floristic richness and endemism. Many plant species tend to occur in naturally isolated patches due to the edaphic and topographic heterogeneity and are suffering an increasing stress of habitat deterioration due to both extensive human activities and global climate change in the area. Here Eurycorymbus cavaleriei was selected as a representative insect-pollinated plant species with low density to assess the pollen dispersal and mating patterns of such limestone associated trees. Paternity analysis revealed substantial long-distance pollination events, with a high level of pollen immigration (27.9–41.9 %) from outside the study sites over distance of at least 4,000 m. Pollen dispersal best fits an exponential power model with a fat-tailed (b < 1) feature and the average pollen dispersal distance was over 2,000 m inferred by the twogener analysis. Despite the dioecious nature, a small amount of biparental inbreeding and moderate correlated paternity were detected based on the multilocus mating system analysis. Relatively few pollen donors (Nep = 3.5–7.0) and substantial variance in male reproductive success (21–24 % of males were responsible for 52–58 % of pollinations) were detected. These results have important implications for the genetic conservation and management of E. cavaleriei. Extensive pollen dispersal provides evidence for substantial intra- and/or inter-patch pollinator movement, which seems promising for E. cavaleriei and other trees with generalized pollination system in karst forest landscape.

Clonality, recombination, and hybridization in the plumbing-inhabiting human pathogen Fusarium keratoplasticum inferred from multilocus sequence typing.

BackgroundRecent work has shown that Fusarium species and genotypes most commonly associated with human infections, particularly of the cornea (mycotic keratitis), are the same as those most commonly isolated from plumbing systems. The species most dominant in plumbing biofilms is Fusarium keratoplasticum, a cosmopolitan fungus known almost exclusively from animal infections and biofilms. To better understand its diversity and population dynamics, we developed and utilized a nine-locus sequence-based typing system to make inferences about clonality, recombination, population structure, species boundaries and hybridization.ResultsHigh levels of genetic diversity and evidence for recombination and clonality were detected among 75 clinical and 156 environmental isolates of Fusarium keratoplasticum. The multilocus sequence typing system (MLST) resolved 111 unique nine-locus sequence types (STs). The single locus bifactorial determinants of mating compatibility (mating types MAT1-1 and MAT1-2), were found in a ratio of 70:30. All but one of the 49 isolates of the most common ST (FSSC 2d-2) came from human infections, mostly of the cornea, and from biofilms associated with contact lenses and plumbing surfaces. Significant levels of phylogenetic incongruence were found among loci. Putative clonal relationships among genotypes were estimated, showing a mixture of large clonal complexes and unrelated singletons. Discordance between the nuclear ribosomal rRNA and other gene genealogies is consistent with introgression of ribosomal RNA alleles of phylogenetic species FSSC 9 into F. keratoplasticum. No significant population subdivision based on clinical versus non-clinical sources was found.ConclusionsIncongruent phylogenetic trees and the presence of both mating types within otherwise identical STs were observed, providing evidence for sexuality in F. keratoplasticum. Cryptic speciation suggested in a published three-locus MLST system was not supported with the addition of new loci, but evidence of introgression of ribosomal RNA genes from another strongly supported phylogenetic species (FSSC 9), also known from plumbing systems and human infections, was detected in two isolates. Overall, F. keratoplasticum is a diverse and geographically unstructured species with a mixed clonal and recombinant life history.

A multi-locus approach to barcoding in the Anopheles strodei subgroup (Diptera: Culicidae)

BackgroundThe ability to successfully identify and incriminate pathogen vectors is fundamental to effective pathogen control and management. This task is confounded by the existence of cryptic species complexes. Molecular markers can offer a highly effective means of species identification in such complexes and are routinely employed in the study of medical entomology. Here we evaluate a multi-locus system for the identification of potential malaria vectors in the Anopheles strodei subgroup.MethodsLarvae, pupae and adult mosquitoes (n = 61) from the An. strodei subgroup were collected from 21 localities in nine Brazilian states and sequenced for the COI, ITS2 and white gene. A Bayesian phylogenetic approach was used to describe the relationships in the Strodei Subgroup and the utility of COI and ITS2 barcodes was assessed using the neighbor joining tree and “best close match” approaches.ResultsBayesian phylogenetic analysis of the COI, ITS2 and white gene found support for seven clades in the An. strodei subgroup. The COI and ITS2 barcodes were individually unsuccessful at resolving and identifying some species in the Subgroup. The COI barcode failed to resolve An. albertoi and An. strodei but successfully identified approximately 92% of all species queries, while the ITS2 barcode failed to resolve An. arthuri and successfully identified approximately 60% of all species queries. A multi-locus COI-ITS2 barcode, however, resolved all species in a neighbor joining tree and successfully identified all species queries using the “best close match” approach.ConclusionsOur study corroborates the existence of An. albertoi, An. CP Form and An. strodei in the An. strodei subgroup and identifies four species under An. arthuri informally named A-D herein. The use of a multi-locus barcode is proposed for species identification, which has potentially important utility for vector incrimination. Individuals previously found naturally infected with Plasmodium vivax in the southern Amazon basin and reported as An. strodei are likely to have been from An. arthuri C identified in this study.