A multi-marker nuclear genotyping approach was performed on larval and adult specimens of Anisakis spp. (N = 689) collected from fish and cetaceans in allopatric and sympatric areas of the two ...species Anisakis pegreffii and Anisakis simplex (s. s.), in order to: (1) identify specimens belonging to the parental taxa by using nuclear markers (allozymes loci) and sequence analysis of a new diagnostic nuclear DNA locus (i.e. partial sequence of the EF1 α−1 nDNA region) and (2) recognize hybrid categories. According to the Bayesian clustering algorithms, based on those markers, most of the individuals (N = 678) were identified as the parental species i.e. A. pegreffii or A. simplex (s. s.), whereas a smaller portion (N = 11) were recognized as F1 hybrids. Discordant results were obtained when using the polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLPs) of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) on the same specimens, which indicated the occurrence of a large number of ‘hybrids’ both in sympatry and allopatry. These findings raise the question of possible misidentification of specimens belonging to the two parental Anisakis and their hybrid categories derived from the application of that single marker (i.e. PCR–RFLPs analysis of the ITS of rDNA). Finally, Bayesian clustering, using allozymes and EF1 α−1 nDNA markers, has demonstrated that hybridization between A. pegreffii and A. simplex (s. s.) is a contemporary phenomenon in sympatric areas, while no introgressive hybridization takes place between the two species.
Abstract
The genus Metopograpsus H. Milne Edwards, 1853 is widespread throughout the Indo-West Pacific and currently consists of seven species that can only be separated by minor morphological ...differences. Therefore, it represents a good example for the usefulness of genetic analyses for identification and classification. In order to obtain phylogenetic information at both lower and higher evolutionary levels, it is best to use a combination of mitochondrial and nuclear molecular markers. Here we present for the first time a molecular phylogeny based on a relative long fragment of the 28S rRNA nuclear gene for the genus Metopograpsus, after application of newly developed primers. Our data suggest an alternative intrageneric speciation order, with M. thukuhar and M. cannicci holding a basal position and a monophyletic grouping of M. frontalis, M. oceanicus and M. quadridentatus, which differs from prior phylogenetic reconstructions. Previously recognized intraspecific phylogeographic patterns in M. latifrons and M. quadridentatus could not be confirmed, due to limited variability of this conserved nuclear gene and due to an incomplete geographic coverage of the corresponding species. In contrast, the previously indicated phylogenetic subdivision within the formerly widespread species M. thukuhar, which led to the recent description of M. cannicci, is here supported.
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•Another matamata species is described as new to science.•Chelus orinocensis is genetically and morphologically significantly different.•Chelus orinocensis and C. fimbriata diverged ...approximately 13 million years ago.•Chelus orinocensis lives in the Orinoco, Río Negro, and Essequibo drainages.•Chelus fimbriata sensu stricto occurs in the Amazon and Mahury drainages.
The matamata is one of the most charismatic turtles on earth, widely distributed in northern South America. Debates have occurred over whether or not there should be two subspecies or species recognized due to its geographic variation in morphology. Even though the matamata is universally known, its natural history, conservation status and biogeography are largely unexplored. In this study we examined the phylogeographic differentiation of the matamata based on three mitochondrial DNA fragments (2168 bp of the control region, cytochrome oxidase subunit I, and the cytochrome b gene), one nuclear genomic DNA fragment (1068 bp of the R35 intron) and 1661 Single Nucleotide Polymorphisms (SNPs). Our molecular and morphological analyses revealed the existence of two distinct, genetically deeply divergent evolutionary lineages of matamatas that separated in the late Miocene (approximately 12.7 million years ago), corresponding well to the time when the Orinoco Basin was established. As a result of our analyses, we describe the genetically and morphologically highly distinct matamata from the Orinoco and Río Negro Basins and the Essequibo drainage as a species new to science (Chelus orinocensis sp. nov.). Chelus fimbriata sensu stricto is distributed in the Amazon Basin and the Mahury drainage. Additionally, the analyses revealed that each species displays phylogeographic differentiation. For C. orinocensis, there is moderate mitochondrial differentiation between the Orinoco and the Río Negro. For C. fimbriata, there is more pronounced differentiation matching different river systems. One mitochondrial clade was identified from the Amazon, Ucayali, and Mahury Rivers, and another one from the Madeira and Jaci Paraná Rivers. The C. orinocensis in the Essequibo and Branco Rivers have haplotypes that constitute a third clade clustering with C. fimbriata. Phylogenetic analyses of the R35 intron and SNP data link the matamatas from the Essequibo and Branco with the new species, suggesting past gene flow and old mitochondrial introgression. Chelus orinocensis is collected for the pet trade in Colombia and Venezuela. However, neither the extent of the harvest nor its impact are known. Hence, it is crucial to gather more information and to assess its exploitation throughout its distribution range to obtain a better understanding of its conservation status and to design appropriate conservation and management procedures.
La matamata es una de las tortugas más carismáticas del mundo, ampliamente distribuida en el norte de Sudamérica. Debido a su variación morfológica geográfica, se debate sobre el reconocimiento de dos subespecies o especies. A pesar de que la matamata es universalmente conocida, su historia natural, estado de conservación y biogeografía han sido muy poco estudiados. En este estudio examinamos la diferenciación filogeográfica de las matamatas en base a tres fragmentos de ADN mitocondrial (2168 pb de la región de control, la subunidad I del citocromo oxidasa y el gen del citocromo b), un fragmento de ADN genómico nuclear (1068 pb del intrón R35) y 1661 polimorfismos de nucleótido único (SNPs). Nuestros análisis moleculares y morfológicos revelaron la existencia de dos linajes evolutivos distintos de matamatas, genéticamente divergentes que se separaron en el Mioceno tardio (hace aproximadamente 12.7 millones de años), correspondiendo al tiempo en que se estableció la cuenca del Orinoco. Como resultado de nuestros análisis, describimos las genéticamente y morfológicamente distintas matamatas de las cuencas del Orinoco, Río Negro y Essequibo como una especie nueva para la ciencia (Chelus orinocensis sp. nov.). Chelus fimbriata sensu stricto se distribuye en la cuenca del Amazonas y en el drenaje del Mahury. Adicionalmente, los análisis revelaron que cada especie muestra diferenciación filogeográfica. Para C. orinocensis, hay una moderada diferenciación mitocondrial entre el Orinoco y el Río Negro. Para C. fimbriata, hay una diferenciación más pronunciada, concordando con los diferentes sistemas fluviales. Se identificó un clado de los ríos Amazonas, Ucayali y Mahury y otro de los ríos Madeira y Jaci Paraná. Las C. orinocensis de los ríos Essequibo y Branco tienen haplotipos que constituyen un tercer clado que se agrupa con C. fimbriata. Los análisis filogenéticos del intrón R35 y los datos de SNP asocian las matamatas de Essequibo y Branco con la nueva especie, sugiriendo flujo de genes pasado e introgresión mitocondrial antigua. Chelus orinocensis se colecta para el comercio de mascotas en Colombia y Venezuela. Sin embargo, ni se conoce el alcance de las colectas ni su impacto. Por lo tanto, es crucial recopilar más información y evaluar su explotación en todo su rango de distribución, comprender mejor su estado de conservación y para diseñar acciones apropiadas de conservación y manejo.
Eighty seven women with benign breast lesion, 120 patients with breast cancer (BC) and one hundred controls were included in the study. Quantification of mtDNA and nDNA was done by qPCR. Global DNA ...methylation was measured using ELISA. Circulating cell-free nDNA and mtDNA were significantly elevated in BC and benign breast lesions patients. Global methylation was significantly low in BC patients. Combining the studied parameters in one panel, nDNA/mtDNA/hypomethylation, improved their sensitivity in detecting BC to reach 92.5%. Circulating cell-free nDNA, mtDNA and global DNA hypomethylation can be used as diagnostic and prognostic markers for BC.
Mitochondria (MT), the major site of cellular energy production, are under dual genetic control by 37 mitochondrial DNA (mtDNA) genes and numerous nuclear genes (MT-nDNA). In the CHARGEmtDNA+ ...Consortium, we studied genetic associations of mtDNA and MT-nDNA associations with body mass index (BMI), waist-hip-ratio (WHR), glucose, insulin, HOMA-B, HOMA-IR, and HbA1c. This 45-cohort collaboration comprised 70,775 (insulin) to 170,202 (BMI) pan-ancestry individuals. Validation and imputation of mtDNA variants was followed by single-variant and gene-based association testing. We report two significant common variants, one in MT-ATP6 associated (p ≤ 5E−04) with WHR and one in the D-loop with glucose. Five rare variants in MT-ATP6, MT-ND5, and MT-ND6 associated with BMI, WHR, or insulin. Gene-based meta-analysis identified MT-ND3 associated with BMI (p ≤ 1E−03). We considered 2,282 MT-nDNA candidate gene associations compiled from online summary results for our traits (20 unique studies with 31 dataset consortia’s genome-wide associations GWASs). Of these, 109 genes associated (p ≤ 1E−06) with at least 1 of our 7 traits. We assessed regulatory features of variants in the 109 genes, cis- and trans-gene expression regulation, and performed enrichment and protein-protein interactions analyses. Of the identified mtDNA and MT-nDNA genes, 79 associated with adipose measures, 49 with glucose/insulin, 13 with risk for type 2 diabetes, and 18 with cardiovascular disease, indicating for pleiotropic effects with health implications. Additionally, 21 genes related to cholesterol, suggesting additional important roles for the genes identified. Our results suggest that mtDNA and MT-nDNA genes and variants reported make important contributions to glucose and insulin metabolism, adipocyte regulation, diabetes, and cardiovascular disease.
In spite of our awareness that the biosphere is represented to a greater extent in the oceans than elsewhere, little is still known about the genetic structure and phylogeography of most deep-sea ...fish species. Here, different questions regarding the phenotypic and genetic diversity of deep-sea morid cod Lepidion lepidion across its distribution area have been investigated. Principal Component Analyses of 17 different morphological measurements indicated no differences between Atlantic Ocean and Mediterranean Sea, but the presence of a latitudinal phenotypic gradient. The genetic analyses comprised three mitochondrial and two nuclear markers. Neutrality tests and mismatch distributions indicated events of demographic expansion in the western Mediterranean Sea and the northeastern Atlantic Ocean that should be interpreted with caution due to the limited sample size. In addition, two different nuclear lineages partially mixed have been detected, suggesting isolation with secondary contact between the Atlantic Ocean and the Mediterranean Sea. Moderate genetic structure was found in Aviles Canyon (NW Iberian Peninsula) showing limitations to gene flow and asymmetric migration, probably due to the oceanographic conditions in the area. The results suggest that genetic connectivity of L. lepidion is probably due to its pelagic stages.
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•Lepidion lepidion exists as two partially mixed Atlantic-Mediterranean lineages.•L. lepidion exhibits a phenotypic latitudinal gradient across its distribution area.•The Iberian Peninsula waters constituted glacial refugia for L. lepidion.
Liquid biopsy recently became a very promising diagnostic method that has several advantages over conventional invasive methods. Liquid biopsy may serve as a source of several important biomarkers ...including cell-free nucleic acids (cf-NAs). Cf-DNA is widely used in prenatal testing in order to characterize fetal genetic disorders. Analysis of cf-DNA may provide information about the mutation profile of tumor cells, while cell-free non-coding RNAs are promising biomarker candidates in the diagnosis and prognosis of cancer. Many of these markers have the potential to help clinicians in therapy selection and in the follow-up of patients. Thus, cf-NA-based diagnostics represent a new path in personalized medicine. Although several reviews are available in the field, most of them focus on a limited number of cf-NA types. In this review, we give an overview about all known cf-NAs including cf-DNA, cf-mtDNA and cell-free non-coding RNA (miRNA, lncRNA, circRNA, piRNA, YRNA, and vtRNA) by discussing their biogenesis, biological function and potential as biomarker candidates in liquid biopsy. We also outline possible future directions in the field.
This chapter introduces neutralized DNA (nDNA) as a novel design for the primers of PCR and RT-PCR by methylating phosphate groups of some oligonucleotides in their structures. It starts with an ...introduction of the nDNA which possesses an electrically chimeric neutral backbone as well as the proposed standards in designing nDNA as a novel primer for PCR and RT-PCR , concluded from various experimental results presented afterward. The primary content comprises empirical data from PCR to compare nDNA and unmodified DNA as primers in terms of ability to distinguish and amplify mismatch templates, activities of polymerase enzymes, melting temperature of double-stranded sequences, and the trials and discussions on various modified positions of the nDNA primers. In summary, nDNA exhibited outstanding performance as a primer for PCR and RT-PCR , compared to unmodified DNA, and is expected to be expanded in diverse applications which require enhanced specificity.
Age-related mitochondrial markers may facilitate the prognosis of artificial reproductive technology outcomes. In this report, we present our study concerning the ratio of cf-mtDNA/cf-nDNA, namely ...the amount of cell-free mitochondrial DNA relative to cell-free nuclear DNA, in the follicular fluid (FF) of women undergoing IVF, aiming to generate a molecular fingerprint of oocyte quality. The values of this ratio were measured and compared among three groups of women (101 in total): (A) 31 women with polycystic ovary syndrome (PCOS), (B) 34 women younger than 36 years, and (C) 36 women older than 35 years of age. Real-time quantitative PCR (qPCR) was performed to quantify the ratio by using nuclear- and mitochondrial-specific primers and analyzed for potential correlation with age and pregnancy rate. Our analysis showed that the level of FF-cf-mtDNA was lower in the group of advanced-age women than in the groups of PCOS and non-PCOS women. Moreover, a significant positive correlation between FF-cf-mtDNA and the number of mature (MII) oocytes was observed. Collectively, the data show that the relative ratio of cf- mtDNA to cf-nDNA content in human FF can be an effective predictor for assessing the corresponding oocyte's age-related performance in IVF.
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