Single Molecular Markers Research Articles

Characterization of Genetic Diversity and Variation in Pathogenicity of the Rice False Smut Pathogen Ustilaginoidea virens from a Single Source.

Rice false smut, caused by Ustilaginoidea virens, is one of the most destructive fungal diseases in rice-growing countries. Studies of the genetic diversity, evolution, and pathogenicity of U. virens can provide more information for disease control and cultivar breeding. Contrary to previous studies on the genetic diversity of different geographical populations of U. virens, this study analyzed the genetic variation of U. virens from different panicles of the same rice cultivar in a field in Yunnan Province using single nucleotide polymorphism molecular markers. A total of 183 polymorphic loci and five haplotypes, hap_1 to hap_5, were identified based on the 1,350-bp combined DNA fragment of 127 isolates, showing some genetic diversity. Hap_1 and hap_3 had the highest occurrence, indicating they were the dominant haplotypes in the field. Further analysis showed that most rice panicles could be coinfected by different haplotypes, and even a few spikelets could be coinfected by multiple haplotypes. The phylogeny indicated that all isolates were divided into five genetic groups. Groups I, II, and III clustered together and were distinguished from Groups IV and V. Significant genetic variations in five pairwise comparisons of panicle populations, accounting for 72.45% of the total variation, were found according to FST values. This variation might be caused by different field microenvironments and the uneven distribution of inoculum sources. An unweighted pair-group method with arithmetic means dendrogram and the population structure revealed that the genetic composition of the isolates collected from YN1, YN2, and YN4, which were dominated by the same genetic subgroup, was different from that collected from YN3. Finally, genetic recombination was found in 11 isolates; hap_2 and hap_5, probably as genetic recombination progenies produced by sexual hybridization between hap_1 and hap_3, acquired a greater virulence than their ancestors according to population structure and pathogenicity analyses. These results will help us understand the genetic diversity, evolution, and infection process of U. virens and aid in the development of more effective management strategies for rice false smut, including new cultivars with improved resistance.

Screening of stable resistant accessions and identification of resistance loci to Barley yellow mosaic virus disease.

BackgroundThe disease caused by Barley yellow mosaic virus (BaYMV) infection is a serious threat to autumn-sown barley (Hordeum vulgare L.) production in Europe, East Asia and Iran. Due to the rapid diversification of BaYMV strains, it is urgent to discover novel germplasm and genes to assist breeding new varieties with resistance to different BaYMV strains, thus minimizing the effect of BaYMV disease on barley cropping.MethodsA natural population consisting of 181 barley accessions with different levels of resistance to BaYMV disease was selected for field resistance identification in two separate locations (Yangzhou and Yancheng, Jiangsu Province, China). Additive main effects and multiplicative interaction (AMMI) analysis was used to identify accessions with stable resistance. Genome-wide association study (GWAS) of BaYMV disease resistance was broadly performed by combining both single nucleotide polymorphisms (SNPs) and specific molecular markers associated with the reported BaYMV disease resistance genes. Furthermore, the viral protein genome linked (VPg) sequences of the virus were amplified and analyzed to assess the differences between the BaYMV strains sourced from the different experimental sites.ResultsSeven barley accessions with lower standardized Area Under the Disease Progress Steps (sAUDPS) index in every environment were identified and shown to have stable resistance to BaYMV disease in each assessed location. Apart from the reported BaYMV disease resistance genes rym4 and rym5, one novel resistance locus explaining 24.21% of the phenotypic variation was identified at the Yangzhou testing site, while two other novel resistance loci that contributed 19.23% and 19.79% of the phenotypic variation were identified at the Yancheng testing site, respectively. Further analysis regarding the difference in the VPg sequence of the predominant strain of BaYMV collected from these two testing sites may explain the difference of resistance loci differentially identified under geographically distinct regions. Our research provides novel genetic resources and resistance loci for breeding barley varieties for BaMYV disease resistance.

Provenance of Mytilus food products in Europe using SNP genetic markers

Mussels of the Mytilus genus or blue mussels are cultured extensively worldwide. According to FAO statistics in 2019, the main cultured species in order of annual production are M. chilensis, M. edulis and M. galloprovincialis. Due to their morphological similarity, blue mussels may be incorrectly assigned to the species leading to the occurrence of inconsistencies in the food labeling. Molecular markers in mono-locus or multi-locus approaches have often been used for species identification.This paper aimed to assess the information about the species and geographical origin labelled on mussel products available in European stores and markets in 2011–2015 using a multi-locus approach based on Single Nucleotide Polymorphisms (SNPs). A total of 491 Mytilus spp. individuals from 21 food samples purchased mainly in Italy, Spain, Poland and Germany, were genotyped using a 54 SNP panel. Most of the declared information on the labels of food products was consistent with the results obtained with genetic tools (85%). However in-complete information, such as missing commercial designation, scientific name, or geographic origin was found in 11 samples. Also, incorrect information about the species origin of a few samples of fresh mussels imported from other countries to the Mediterranean was observed in fishmongers' shops. The results consistently revealed a division of the European food samples into two species: M. edulis and M. galloprovincialis (Atlantic and Mediterranean forms). The presence of M. trossulus in the food samples was not observed. Our results confirm that traditionally used single molecular diagnostic markers can provide only preliminary information on the taxonomic status of Northern hemisphere mussels included in food products. A multi-locus approach based on a set of multiple SNPs is needed for correct label assessment. The results demonstrate that a reduced panel of 34 SNP loci can be widely used to confirm the geographical origin and the identity of Mytilus taxa in food samples, verify data on food labels, and monitor the mussel trade in Europe.

Genetic Diversity of Trypanosoma cruzi in Panama Inferred by Multi-locus Sequence Typing of Mitochondrial Genes.

The objective of this study was to provide information on Trypanosoma cruzi genetic diversity among isolates obtained from different biological sources circulating in endemic areas of Panama. Initial discrete typing units (DTUs) assignment was performed evaluating three single locus molecular markers (mini-exon, heat shock protein 60 and glucose-6-phosphate isomerase genes). Further diversity within TcI lineages was explored using a multi-locus sequence typing approach with six maxicircle genes. Haplotype network analysis and evolutionary divergency estimations were conducted to investigate the genetic relatedness between Panamanian TcI isolates and isolates from different endemic regions in the Americas. Our molecular approach validated that TcI is the predominant DTU circulating in Panama across different hosts and vector species, but also confirmed the presence of TcIII and TcVI circulating in the country. The phylogenetic tree topography for most Panamanian TcI isolates displayed a high level of genetic homogeneity between them. The haplotype network analysis inferred a higher genetic diversity within Panamanian TcI isolates, displaying eight different haplotypes circulating in endemic regions of the country, and revealed geographical structuring among TcI from different endemic regions in the Americas. This study adds novelty on the genetic diversity of T. cruzi circulating in Panama and complements regional phylogeographic studies regarding intra-TcI variations.

GPTransformer: A Transformer-Based Deep Learning Method for Predicting Fusarium Related Traits in Barley.

Fusarium head blight (FHB) incited by Fusarium graminearum Schwabe is a devastating disease of barley and other cereal crops worldwide. Fusarium head blight is associated with trichothecene mycotoxins such as deoxynivalenol (DON), which contaminates grains, making them unfit for malting or animal feed industries. While genetically resistant cultivars offer the best economic and environmentally responsible means to mitigate disease, parent lines with adequate resistance are limited in barley. Resistance breeding based upon quantitative genetic gains has been slow to date, due to intensive labor requirements of disease nurseries. The production of a high-throughput genome-wide molecular marker assembly for barley permits use in development of genomic prediction models for traits of economic importance to this crop. A diverse panel consisting of 400 two-row spring barley lines was assembled to focus on Canadian barley breeding programs. The panel was evaluated for FHB and DON content in three environments and over 2 years. Moreover, it was genotyped using an Illumina Infinium High-Throughput Screening (HTS) iSelect custom beadchip array of single nucleotide polymorphic molecular markers (50 K SNP), where over 23 K molecular markers were polymorphic. Genomic prediction has been demonstrated to successfully reduce FHB and DON content in cereals using various statistical models. Herein, we have studied an alternative method based on machine learning and compare it with a statistical approach. The bi-allelic SNPs represented pairs of alleles and were encoded in two ways: as categorical (–1, 0, 1) or using Hardy-Weinberg probability frequencies. This was followed by selecting essential genomic markers for phenotype prediction. Subsequently, a Transformer-based deep learning algorithm was applied to predict FHB and DON. Apart from the Transformer method, a Residual Fully Connected Neural Network (RFCNN) was also applied. Pearson correlation coefficients were calculated to compare true vs. predicted outputs. Models which included all markers generally showed marginal improvement in prediction. Hardy-Weinberg encoding generally improved correlation for FHB (6.9%) and DON (9.6%) for the Transformer network. This study suggests the potential of the Transformer based method as an alternative to the popular BLUP model for genomic prediction of complex traits such as FHB or DON, having performed equally or better than existing machine learning and statistical methods.

Development of specific single nucleotide polymorphism molecular markers for Angelica gigas Nakai

Development of specific single nucleotide polymorphism molecular markers for <i>Angelica gigas</i> Nakai

High Sequence Divergence but Limited Architectural Rearrangements in Organelle Genomes of Cyanophora (Glaucophyta) Species.

Cyanophora is the glaucophyte model taxon. Following the sequencing of the nuclear genome of C.paradoxa, studies based on single organelle and nuclear molecular markers revealed previously unrecognized species diversity within this glaucophyte genus. Here, we present the complete plastid (ptDNA) and mitochondrial (mtDNA) genomes of C.kugrensii, C.sudae, and C.biloba. The respective sizes and coding capacities of both ptDNAs and mtDNAs are conserved among Cyanophora species with only minor differences due to specific gene duplications. Organelle phylogenomic analyses consistently recover the species C.kugrensii and C.paradoxa as a clade and C.sudae and C.biloba as a separate group. The phylogenetic affiliations of the four Cyanophora species are consistent with architectural similarities shared at the organelle genomic level. Genetic distance estimations from both organelle sequences are also consistent with phylogenetic and architecture evidence. Comparative analyses confirm that the Cyanophora mitochondrial genes accumulate substitutions at 3-fold higher rates than plastid counterparts, suggesting that mtDNA markers are more appropriate to investigate glaucophyte diversity and evolutionary events that occur at a population level. The study of complete organelle genomes is becoming the standard for species delimitation and is particularly relevant to study cryptic diversity in microbial groups.

Structure and evolution of the complete mitochondrial genome of the freshwater drum, Aplodinotus grunniens (Actinopterygii: Perciformes: Sciaenidae)

Background. Overfishing and habitat degradation caused a decline of populations of many fish species belonging to the speciose family Sciaenidae. A reliable taxonomic framework is a prerequisite for implementing effective stock management and conservation measures, but phylogeny and taxonomy of the Sciaenidae remain poorly resolved. As traditionally used morphological and single gene-based molecular markers carry a too limited phylogenetic signal for the task, mitochondrial phylogenomics may be a more suitable tool. The freshwater drum, Aplodinotus grunniens Rafinesque, 1819, is one of the few Sciaenidae species that live in freshwater habitats, which makes it an important model for studying the phylogeny and evolution of Sciaenidae. Materials and methods. We sequenced and characterized its mitogenome, and reconstructed the phylogeny of Sciaenidae using mitogenomes of 28 species. Results. The architecture of the mitogenome (16487 bp in length) is standard for this family, and three typical elements were identified in the control region: extended termination associated sequences, central conserved region, and conserved sequence block. Poor availability of sciaenid mitogenomes (especially those belonging to different lineages) prevented us from resolving the phylogeny of this family with confidence. Notably, our results indicate that Larimichthys and Collichthys species may belong to a single genus, and we suspect that the mitogenome of Chrysochir aureus (Richardson, 1846) has been misidentified taxonomically, and urge its resequencing. Conclusion. The sequencing of additional mitogenomes belonging to non-represented and poorly represented lineages is needed to facilitate the understanding of phylogeny and taxonomy of Sciaenidae.

A multigene phylogeny to infer the evolutionary history of Chaetocerotaceae (Bacillariophyta)

The diatom family Chaetocerotaceae (Bacillariophyta) is common in the marine plankton worldwide, especially in coastal areas and upwelling zones. Its defining character constitutes hollow processes, called setae, which emerge from the valves of the vegetative cells. The family comprises two extant genera: Bacteriastrum and Chaetoceros. Current systematics is based on morphological features of vegetative cells and resting spores and is summarised in a classification scheme subdividing Bacteriastrum in two sections, Isomorpha and Sagittata, and Chaetoceros in three subgenera: Hyalochaete, Chaetoceros (Phaeoceros) and Bacteriastroidea, and further into 22 sections. Phylogenies inferred from single molecular markers (18S and partial 28S rDNA) show only partial topological agreement and many poorly or unresolved basal ramifications. Since classification should not only satisfy practical needs but also reflect well-supported evolutionary relationships of the taxa under investigation, we inferred a multigene phylogeny of the family Chaetocerotaceae amplifying five genes of 100 strains encompassing six Bacteriastrum and 60 Chaetoceros species. We also compared the phylogenetic signal of nuclear, plastid and mitochondrial compartments to ascertain if the inferred tree topologies were congruent. Our results provided a robust multigene phylogeny of the family Chaetocerotaceae, offering a solid framework to test the validity of the traditional taxonomical classification. The genera Bacteriastrum and Chaetoceros were resolved as sister clades, whilst the subgenus Hyalochaete was found to be paraphyletic. Consequently, we rejected the subdivision in subgenera and only considered sections. Most of the already recognised sections were found to be monophyletic. We emended one section, rejected seven and erected three new ones. As a consequence of our proposed changes, all the sections investigated are supported by morphological and molecular characters alike. Thus, a natural classification is feasible for this important and very diverse marine planktonic family.

Molecular classification of diffuse gliomas.

In this study we assessed whether gliomas could be subdivided into different molecular subtypes by immunohistochemistry (IHC) reminiscent of those first described by Verhaak et al. in 2010 (classical, proneural, mesenchymal and neural). We also evaluated the prognostic significance of single molecular factors and searched for significant correlations between markers. In this study, we included 146 patients with glioblastomas (GBMs) and 26 with diffuse astrocytomas (DAs). The glioma samples were tested for PDGFRA, IDH1 R132H, CD44, p53, Ki-67, p21 and p27 expression. We found that gliomas could be subdivided into molecular subtypes by IHC. Fifty per cent of GBMs were of the proneural subtype, 18.5% of mesenchymal subtype and 31.5% were not otherwise classified. However, most of the DAs (92.3%) belonged to the proneural subtype. No prognostic role was found for the molecular subtypes, but predictive roles were noted. Both proneural and mesenchymal molecular subtypes showed a benefit from the addition of chemotherapy and radiotherapy; however, the mesenchymal subtype showed a greater response. Interestingly, the mesenchymal subtype did not receive any benefit from the addition of radiotherapy compared with palliative management and surgery alone. Regarding single molecular markers, only IDH1 R132H was found to have a prognostic role for GBMs. There was a trend towards better survival in tumours with lower PDGFRA expression (p = 0.066). In DAs, PDGFRA and Ki-67 expression had prognostic roles. The following statistically significant correlations were found in GBMs: Ki-67/p53, Ki-67/p27 and p53/PDGFRA; in DAs: p53/PDGFRA, CD44/PDGFRA, and p21/PDGFRA.

Three new Diplozoidae mitogenomes expose unusual compositional biases within the Monogenea class: implications for phylogenetic studies

BackgroundAs the topologies produced by previous molecular and morphological studies were contradictory and unstable (polytomy), evolutionary relationships within the Diplozoidae family and the Monogenea class (controversial relationships among the Discocotylinea, Microcotylinea and Gastrocotylinea suborders) remain unresolved. Complete mitogenomes carry a relatively large amount of information, sufficient to provide a much higher phylogenetic resolution than traditionally used morphological traits and/or single molecular markers. However, their implementation is hampered by the scarcity of available monogenean mitogenomes. Therefore, we sequenced and characterized mitogenomes belonging to three Diplozoidae family species, and conducted comparative genomic and phylogenomic analyses for the entire Monogenea class.ResultsTaxonomic identification was inconclusive, so two of the species were identified merely to the genus level. The complete mitogenomes of Sindiplozoon sp. and Eudiplozoon sp. are 14,334 bp and 15,239 bp in size, respectively. Paradiplozoon opsariichthydis (15,385 bp) is incomplete: an approximately 2000 bp-long gap within a non-coding region could not be sequenced. Each genome contains the standard 36 genes (atp8 is missing). G + T content and the degree of GC- and AT-skews of these three mitogenome (and their individual elements) were higher than in other monogeneans. nad2, atp6 and nad6 were the most variable PCGs, whereas cox1, nad1 and cytb were the most conserved. Mitochondrial phylogenomics analysis, conducted using concatenated amino acid sequences of all PCGs, indicates that evolutionary relationships of the three genera are: (Eudiplozoon, (Paradiplozoon, Sindiplozoon)); and of the three suborders: (Discocotylinea, (Microcotylinea, Gastrocotylinea)). These intergeneric relationships were also supported by the skewness and principal component analyses.ConclusionsOur results show that nad2, atp6 and nad6 (fast-evolving) would be better candidates than cox1 (slow-evolving) for species identification and population genetics studies in Diplozoidae. Nucleotide bias and codon and amino acid usage patterns of the three diplozoid mitogenomes are more similar to cestodes and trematodes than to other monogenean flatworms. This unusual mutational bias was reflected in disproportionately long branches in the phylogram. Our study offsets the scarcity of molecular data for the subclass Polyopisthocotylea to some extent, and might provide important new insights into the evolutionary history of the three genera and three suborders.

How does marker choice affect your diet analysis: comparing genetic markers and digestion levels for diet metabarcoding of tropical-reef piscivores

Tropical reefs are highly diverse ecosystems, and reliable biomonitoring, through diet metabarcoding, is needed to understand present and future trophic relationships in this changing habitat. Several studies have assessed the reliability and effectiveness of single molecular markers; however, a cross-marker validation has rarely been performed. This study identified crucial properties for 12S rDNA, 16S rDNA and COI metabarcoding in tropical-reef piscivores (Plectropomus spp.). In addition, three new versatile primer sets for 16S were designed in silico for metabarcoding of reef fish. Results showed that COI was overall better at recovering true diversity because of a well-supported database. Second, optimal 16S amplicon sizes ranged between 160 and 440 base pairs for full diversity recovery, with increased species detection for the 270-base pairs region. Finally, blocking of predator-specific COI sequences was not equally effective in all host species, potentially introducing bias when diet compositions are directly compared. In conclusion, this novel study showed that marker success for prey identification is highly dependent on the reference database, taxonomic scope, DNA quality, amplicon length and sequencing platform. Results suggest that COI, complemented with 16S, yields the best outcome for diet metabarcoding in reef piscivores. Findings in this paper are relevant to other piscivores and other metabarcoding applications.

Urinary Protein Markers for the Detection and Prognostication of Urothelial Carcinoma.

Bladder cancer diagnosis and surveillance is mainly based on cystoscopy and urine cytology. However, both methods have significant limitations; urine cytology has a low sensitivity for low-grade tumors, while cystoscopy is uncomfortable for the patients. Therefore, in the last decade urine analysis was the subject of intensive research resulting in the identification of many potential biomarkers for the detection, surveillance, or prognostic stratification of bladder cancer. Current trends move toward the development of multiparametric models to improve the diagnostic accuracy compared with single molecular markers. Recent technical advances for high-throughput and more sensitive measurements have led to the development of multiplex assays showing potential for more efficient tools toward future clinical application. In this review, we focus on the findings of urinary protein research in the context of detection and prognostication of bladder cancer. Furthermore, we provide an up-to-date overview on the recommendations for the quality evaluation of published studies as well as for the conduction of future urinary biomarker studies.

First interspecific genetic linkage map for Castanea sativa x Castanea crenata revealed QTLs for resistance to Phytophthora cinnamomi.

The Japanese chestnut (Castanea crenata) carries resistance to Phytophthora cinnamomi, the destructive and widespread oomycete causing ink disease. The European chestnut (Castanea sativa), carrying little to no disease resistance, is currently threatened by the presence of the oomycete pathogen in forests, orchards and nurseries. Determining the genetic basis of P. cinnamomi resistance, for further selection of molecular markers and candidate genes, is a prominent issue for implementation of marker assisted selection in the breeding programs for resistance. In this study, the first interspecific genetic linkage map of C. sativa x C. crenata allowed the detection of QTLs for P. cinnamomi resistance. The genetic map was constructed using two independent, control-cross mapping populations. Chestnut populations were genotyped using 452 microsatellite and single nucleotide polymorphism molecular markers derived from the available chestnut transcriptomes. The consensus genetic map spans 498,9 cM and contains 217 markers mapped with an average interval of 2.3 cM. For QTL analyses, the progression rate of P. cinnamomi lesions in excised shoots inoculated was used as the phenotypic metric. Using non-parametric and composite interval mapping approaches, two QTLs were identified for ink disease resistance, distributed in two linkage groups: E and K. The presence of QTLs located in linkage group E regarding P. cinnamomi resistance is consistent with a previous preliminary study developed in American x Chinese chestnut populations, suggesting the presence of common P. cinnamomi defense mechanisms across species. Results presented here extend the genomic resources of Castanea genus providing potential tools to assist the ongoing and future chestnut breeding programs.

Assessment of genetic variation in Linum L. using SPAR markers

Flax (Linum usitatissimum subsp. usitatissimum) is an important crop plant cultivated both for its fiber and seed oil content. Uniform cultivation and selection practice may lead to genetic erosion and reduced genetic variability of Linum cultivars, which in turn makes the crop vulnerable to diseases and pathogens. Therefore we performed a genetic diversity analysis of few populations including both Linum usitatissimum subsp. usitatissimum L. and L. Bienne L. We used a combination of single primer amplification reaction markers, including RAPD, ISSR, and RAMPO molecular markers for genetic variability study. In general, all three molecular markers revealed a low degree of genetic variability in the materials studied; however, L. bienne had a higher level of genetic variability. Clustering and network analyses grouped the studied plants inter-mixed and did not separate the studied species or populations. STRUCTURE plot and K-Means clustering also identified these populations as genetically homogenous. These results indicate the need for introducing new linseed cultivars into the country for future hybridization and breeding programmes.

Popmarker: Identifying Phylogenetic Markers at the Population Level.

As phylogenomic approach becomes a common practice for constructing true bacterial phylogenies, it has become apparent that single molecular markers such as 16S ribosomal DNA often lead to misclassification of species. In this study, we present a program called Popmarker that uses the true species phylogeny and identifies a minimum set of molecular markers reflecting the bacterial evolution history and phylogenetic relationship at the resolution of populations. Popmarker ranks the proteome according to the correlation of whole species tree or subtree branch length against orthologous sequence distances. We demonstrate that 5 proteins of 2 top ranks achieve the same resolution as concatenation of 2203 single-copy orthologous genes and the right species classification as well as correct split of the 2 groups of Vibrio campbellii. The top-ranking genes selected by Popmarker are candidates that lead to speciation and are useful in distinguishing close related species in microbiome study.

Prognostic relevance of miRNA-155 methylation in anaplastic glioma.

The outcome of patients with anaplastic gliomas varies considerably depending on single molecular markers, such as mutations of the isocitrate dehydrogenase (IDH) genes, as well as molecular classifications based on epigenetic or genetic profiles. Remarkably, 98% of the RNA within a cell is not translated into proteins. Of those, especially microRNAs (miRNAs) have been shown not only to have a major influence on physiologic processes but also to be deregulated and prognostic in malignancies.To find novel survival markers and treatment options we performed unbiased DNA methylation screens that revealed 12 putative miRNA promoter regions with differential DNA methylation in anaplastic gliomas. Methylation of these candidate regions was validated in different independent patient cohorts revealing a set of miRNA promoter regions with prognostic relevance across data sets. Of those, miR-155 promoter methylation and miR-155 expression were negatively correlated and especially the methylation showed superior correlation with patient survival compared to established biomarkers.Functional examinations in malignant glioma cells further cemented the relevance of miR-155 for tumor cell viability with transient and stable modifications indicating an onco-miRNA activity. MiR-155 also conferred resistance towards alkylating temozolomide and radiotherapy as consequence of nuclear factor (NF)κB activation.Preconditioning glioma cells with an NFκB inhibitor reduced therapy resistance of miR-155 overexpressing cells. These cells resembled tumors with a low methylation of the miR-155 promoter and thus mir-155 or NFκB inhibition may provide treatment options with a special focus on patients with IDH wild type tumors.

Multi-gene-based phylogenetic analysis of oligotrich ciliates with emphasis on two dominant groups: Cyrtostrombidiids and strombidiids (Protozoa, Ciliophora)

Phylogenetic analyses of ciliated protists are frequently based on single molecular markers, usually the small subunit ribosomal RNA gene (SSU rDNA), despite the well-known limitations of this approach. Here, 78 new sequences of three linked genes (SSU rDNA, ITS1-5.8S rDNA-ITS2, LSU rDNA) were characterized and applied to phylogenetic analyses of oligotrichs (s. str.). It was found that: (1) three taxa, that is tontoniids, pelagostrombidiids and cyrtostrombidiids should be split from the family Strombidiidae (s. l.), which supports Agatha’s classification based on morphological characters; (2) the families Tontoniidae and Cyrtostrombidiidae are both monophyletic whereas Strombidiidae is polyphyletic; (3) the positions of the families Cyrtostrombidiidae and Pelagostrombidiidae varied in different trees although with low support values; (4) the close relationship between Varistrombidium and Apostrombidium is confirmed, which updates the evolutionary hypothesis for oligotrichs based on ciliary patterns; and (5) two relatively stable clades were found in the family Strombidiidae.

Sequencing of whole plastid genomes and nuclear ribosomal DNA of Diospyros species (Ebenaceae) endemic to New Caledonia: many species, little divergence.

Background and Aims Some plant groups, especially on islands, have been shaped by strong ancestral bottlenecks and rapid, recent radiation of phenotypic characters. Single molecular markers are often not informative enough for phylogenetic reconstruction in such plant groups. Whole plastid genomes and nuclear ribosomal DNA (nrDNA) are viewed by many researchers as sources of information for phylogenetic reconstruction of groups in which expected levels of divergence in standard markers are low. Here we evaluate the usefulness of these data types to resolve phylogenetic relationships among closely related Diospyros species.Methods Twenty-two closely related Diospyros species from New Caledonia were investigated using whole plastid genomes and nrDNA data from low-coverage next-generation sequencing (NGS). Phylogenetic trees were inferred using maximum parsimony, maximum likelihood and Bayesian inference on separate plastid and nrDNA and combined matrices.Key Results The plastid and nrDNA sequences were, singly and together, unable to provide well supported phylogenetic relationships among the closely related New Caledonian Diospyros species. In the nrDNA, a 6-fold greater percentage of parsimony-informative characters compared with plastid DNA was found, but the total number of informative sites was greater for the much larger plastid DNA genomes. Combining the plastid and nuclear data improved resolution. Plastid results showed a trend towards geographical clustering of accessions rather than following taxonomic species.Conclusions In plant groups in which multiple plastid markers are not sufficiently informative, an investigation at the level of the entire plastid genome may also not be sufficient for detailed phylogenetic reconstruction. Sequencing of complete plastid genomes and nrDNA repeats seems to clarify some relationships among the New Caledonian Diospyros species, but the higher percentage of parsimony-informative characters in nrDNA compared with plastid DNA did not help to resolve the phylogenetic tree because the total number of variable sites was much lower than in the entire plastid genome. The geographical clustering of the individuals against a background of overall low sequence divergence could indicate transfer of plastid genomes due to hybridization and introgression following secondary contact.

HER-2, EGFR, Cox-2 and Ki67 expression in lymph node metastasis of canine mammary carcinomas: Association with clinical-pathological parameters and overall survival

Studies about canine mammary tumors based on single molecular markers probably cannot accurately account for the heterogeneity of this disease, and the investigation of multiple molecular alterations in primary tumors and their metastases, in conjunction, has assumed great importance for the understanding of mammary tumor progression. In the present study, we selected 54 primary mammary carcinomas with lymph node metastasis (T1,2,3N1M0), 29 primary mammary carcinomas without metastasis (T1,2,3N0M0), and 25 canine lymph nodes metastasis to evaluate the immunohistochemical expression of HER-2, EGFR, Cox-2 and Ki67 and its association with clinical-pathological parameters and overall survival. Our results found a concordance between the expression of HER-2 (K coefficient: 0.250), Cox-2 (K coefficient: 0.571), and Ki67 (K coefficient: 0.397) and a discordance between EGFR expression (K coefficient: −0.195) in primary mammary carcinomas and paired lymph node metastasis. Furthermore, a high Ki67 index (>24%), large tumor size and the presence of angiolymphatic invasion in canine primary mammary carcinoma with lymph node metastasis plus the presence of extracapsular extension in lymph nodes metastasis were also related to worse prognoses and shorter overall survival (P<0.05). In conclusion, our study demonstrates that primary mammary carcinomas with high expression of HER-2, Cox-2 and Ki67 also show high expression of these markers in paired lymph node metastasis. Moreover, the expression of these molecular markers in lymph nodes metastasis did not demonstrate a prognostic relevance.