RESUMEN
Naive CD4+ T cells in specific pathogen-free (SPF) mice are characterized by transcriptional heterogeneity and subpopulations distinguished by the expression of quiescence, the extracellular matrix (ECM) and cytoskeleton, type I interferon (IFN-I) response, memory-like, and T cell receptor (TCR) activation genes. We demonstrate that this constitutive heterogeneity, including the presence of the IFN-I response cluster, is commensal independent insofar as being identical in germ-free and SPF mice. By contrast, Nippostrongylus brasiliensis infection altered this constitutive heterogeneity. Naive T cell-intrinsic transcriptional changes acquired during helminth infection correlated with and accounted for decreased immunization response to an unrelated antigen. These compositional and functional changes were dependent variables of helminth infection, as they disappeared at the established time point of its clearance in mice. Collectively, our results indicate that the naive T cell pool is subject to dynamic transcriptional changes in response to certain environmental cues, which in turn permutes the magnitude of the immune response.
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Linfocitos T CD4-Positivos , Nippostrongylus , Animales , Ratones , Linfocitos T CD4-Positivos/inmunología , Nippostrongylus/inmunología , Infecciones por Strongylida/inmunología , Infecciones por Strongylida/parasitología , Organismos Libres de Patógenos Específicos , Transcripción Genética , Receptores de Antígenos de Linfocitos T/inmunología , Receptores de Antígenos de Linfocitos T/metabolismo , Helmintiasis/inmunología , Interferón Tipo I/metabolismo , Interferón Tipo I/inmunología , Ratones Endogámicos C57BL , Activación de Linfocitos/inmunologíaRESUMEN
Transcriptomics analyses play pivotal roles in understanding the complex regulatory networks that govern cellular processes. The abundance of rRNAs, which account for 80%-90% of total RNA in eukaryotes, limits the detection and investigation of other transcripts. While mRNAs and long noncoding RNAs have poly(A) tails that are often used for positive selection, investigations of poly(A)- RNAs, such as circular RNAs, histone mRNAs, and small RNAs, typically require the removal of the abundant rRNAs for enrichment. Current approaches to deplete rRNAs for downstream molecular biology investigations are hampered by restrictive RNA input masses and high costs. To address these challenges, we developed rRNA Removal by RNaseH (rRRR), a method to efficiently deplete rRNAs from a wide range of human, mouse, and rat RNA inputs and of varying qualities at a cost 10- to 20-fold cheaper than other approaches. We used probe-based hybridization and enzymatic digestion to selectively target and remove rRNA molecules while preserving the integrity of non-rRNA transcripts. Comparison of rRRR to two commercially available approaches showed similar rRNA depletion efficiencies and comparable off-target effects. Our developed method provides researchers with a valuable tool for investigating gene expression and regulatory mechanisms across a wide range of biological systems at an affordable price that increases the accessibility for researchers to enter the field, ultimately advancing our understanding of cellular processes.
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ARN Ribosómico , ARN Ribosómico/genética , ARN Ribosómico/metabolismo , Animales , Humanos , Ratones , Ratas , Ribonucleasa H/metabolismo , Ribonucleasa H/genética , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Migration and invasion enhancer 1 (MIEN1) overexpression characterizes several cancers and facilitates cancer cell migration and invasion. Leveraging conserved immunoreceptor tyrosine-based activation motif and prenylation motifs within MIEN1, we identified potent anticancer peptides. Among them, bioactive peptides LA3IK and RP-7 induced pronounced transcriptomic and protein expression changes at sub-IC50 concentrations. The peptides effectively inhibited genes and proteins driving cancer cell migration, invasion, and epithelial-mesenchymal transition pathways, concurrently suppressing epidermal growth factor-induced nuclear factor kappa B nuclear translocation in metastatic breast cancer cells. Specifically, peptides targeted the same signal transduction pathway initiated by MIEN1. Molecular docking and CD spectra indicated the formation of MIEN1-peptide complexes. The third-positioned isoleucine in LA3IK and CVIL motif in RP-7 were crucial for inhibiting breast cancer cell migration. This is evident from the limited migration inhibition observed when MDA-MB-231 cells were treated with scrambled peptides LA3IK SCR and RP-7 SCR. Additionally, LA3IK and RP-7 effectively suppressed tumor growth in an orthotopic breast cancer model. Notably, mice tolerated high intraperitoneal (ip) peptide doses of 90 mg/Kg well, surpassing significantly lower doses of 5 mg/Kg intravenously (iv) and 30 mg/Kg intraperitoneally (ip) used in both in vivo pharmacokinetic studies and orthotopic mouse model assays. D-isomers of LA3IK and RP-7 showed enhanced anticancer activity compared to their L-isomers. D-LA3IK remained stable in mouse plasma for 24 h with 75% remaining, exhibiting superior pharmacokinetic properties over D/L-RP-7. In summary, our findings mark the first report of short peptides based on MIEN1 protein sequence capable of inhibiting cancer signaling pathways, effectively impeding cancer progression both in vitro and in vivo.
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Péptidos y Proteínas de Señalización Intracelular , Proteínas de Neoplasias , Animales , Ratones , Movimiento Celular/genética , Proliferación Celular , Transición Epitelial-Mesenquimal , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Simulación del Acoplamiento Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Transducción de Señal , Humanos , Línea Celular , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologíaRESUMEN
Salt is frequently introduced in ecosystems, where it acts as a pollutant. This study examined how changes in salinity affect the survival and development of zebrafish from the two-cell to the blastocyst stage and from the blastocyst to the larval stage. Control zebrafish embryos were cultured in E3 medium containing 5 mM Sodium Chloride (NaCl), 0.17 mM Potassium Chloride (KCL), 0.33 mM Calcium Chloride (CaCl2), and 0.33 mM Magnesium Sulfade (MgSO4). Experiments were conducted using increasing concentrations of each individual salt at 5×, 10×, 50×, and 100× the concentration found in E3 medium. KCL, CaCl2, and MgSO4 did not result in lethal abnormalities and did not affect early embryo growth at any of the concentrations tested. Concentrations of 50× and 100× NaCl caused embryonic death in both stages of development. Concentrations of 5× and 10× NaCl resulted in uninflated swim bladders in 12% and 65% of larvae, compared to 4.2% of controls, and caused 1654 and 2628 genes to be differentially expressed in blastocysts, respectively. The ATM signaling pathway was affected, and the Sonic Hedgehog pathway genes Shh and Ptc1 implicated in swim bladder development were downregulated. Our findings suggest that increased NaCl concentrations may alter gene expression and cause developmental abnormalities in animals found in affected ecosystems.
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Proteínas Hedgehog , Perciformes , Animales , Proteínas Hedgehog/genética , Cloruro de Sodio/farmacología , Agua , Pez Cebra/genética , Cloruro de Calcio , Ecosistema , Cloruro de Sodio Dietético , Larva/genética , Expresión GénicaRESUMEN
BACKGROUND: Collective cell migration underlies many essential processes, including sculpting organs during embryogenesis, wound healing in the adult, and metastasis of cancer cells. At mid-oogenesis, Drosophila border cells undergo collective migration. Border cells round up into a small group at the pre-migration stage, detach from the epithelium and undergo a dynamic and highly regulated migration at the mid-migration stage, and stop at the oocyte, their final destination, at the post-migration stage. While specific genes that promote cell signaling, polarization of the cluster, formation of protrusions, and cell-cell adhesion are known to regulate border cell migration, there may be additional genes that promote these distinct active phases of border cell migration. Therefore, we sought to identify genes whose expression patterns changed during border cell migration. RESULTS: We performed RNA-sequencing on border cells isolated at pre-, mid-, and post-migration stages. We report that 1,729 transcripts, in nine co-expression gene clusters, are temporally and differentially expressed across the three migration stages. Gene ontology analyses and constructed protein-protein interaction networks identified genes expected to function in collective migration, such as regulators of the cytoskeleton, adhesion, and tissue morphogenesis, but also uncovered a notable enrichment of genes involved in immune signaling, ribosome biogenesis, and stress responses. Finally, we validated the in vivo expression and function of a subset of identified genes in border cells. CONCLUSIONS: Overall, our results identified differentially and temporally expressed genetic networks that may facilitate the efficient development and migration of border cells. The genes identified here represent a wealth of new candidates to investigate the molecular nature of dynamic collective cell migrations in developing tissues.
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Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Redes Reguladoras de Genes , Oogénesis/genética , Movimiento Celular/genética , Perfilación de la Expresión Génica , Drosophila melanogaster/genéticaRESUMEN
Seasonal infection rates of individual viruses are influenced by synergistic or inhibitory interactions between coincident viruses. Endemic patterns of SARS-CoV-2 and influenza infection overlap seasonally in the Northern hemisphere and may be similarly influenced. We explored the immunopathologic basis of SARS-CoV-2 and influenza A (H1N1pdm09) interactions in Syrian hamsters. H1N1 given 48 h prior to SARS-CoV-2 profoundly mitigated weight loss and lung pathology compared to SARS-CoV-2 infection alone. This was accompanied by the normalization of granulocyte dynamics and accelerated antigen-presenting populations in bronchoalveolar lavage and blood. Using nasal transcriptomics, we identified a rapid upregulation of innate and antiviral pathways induced by H1N1 by the time of SARS-CoV-2 inoculation in 48 h dual-infected animals. The animals that were infected with both viruses also showed a notable and temporary downregulation of mitochondrial and viral replication pathways. Quantitative RT-PCR confirmed a decrease in the SARS-CoV-2 viral load and lower cytokine levels in the lungs of animals infected with both viruses throughout the course of the disease. Our data confirm that H1N1 infection induces rapid and transient gene expression that is associated with the mitigation of SARS-CoV-2 pulmonary disease. These protective responses are likely to begin in the upper respiratory tract shortly after infection. On a population level, interaction between these two viruses may influence their relative seasonal infection rates.
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COVID-19 , Subtipo H1N1 del Virus de la Influenza A , Gripe Humana , Cricetinae , Animales , Humanos , COVID-19/patología , Mesocricetus , SARS-CoV-2 , Gripe Humana/patología , Pulmón , Modelos Animales de EnfermedadRESUMEN
Expression of the long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) correlates with tumor progression and metastasis in many tumor types. However, the impact and mechanism of action by which MALAT1 promotes metastatic disease remain elusive. Here, we used CRISPR activation (CRISPRa) to overexpress MALAT1/Malat1 in patient-derived lung adenocarcinoma (LUAD) cell lines and in the autochthonous K-ras/p53 LUAD mouse model. Malat1 overexpression was sufficient to promote the progression of LUAD to metastatic disease in mice. Overexpression of MALAT1/Malat1 enhanced cell mobility and promoted the recruitment of protumorigenic macrophages to the tumor microenvironment through paracrine secretion of CCL2/Ccl2. Ccl2 up-regulation was the result of increased global chromatin accessibility upon Malat1 overexpression. Macrophage depletion and Ccl2 blockade counteracted the effects of Malat1 overexpression. These data demonstrate that a single lncRNA can drive LUAD metastasis through reprogramming of the tumor microenvironment.
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Adenocarcinoma del Pulmón , Quimiocina CCL2 , Neoplasias Pulmonares , Metástasis de la Neoplasia , ARN Largo no Codificante , Microambiente Tumoral , ARN Largo no Codificante/genética , Microambiente Tumoral/inmunología , Animales , Ratones , Humanos , Neoplasias Pulmonares/inmunología , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/secundario , Adenocarcinoma del Pulmón/inmunología , Adenocarcinoma del Pulmón/genética , Adenocarcinoma del Pulmón/patología , Quimiocina CCL2/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/inmunología , Inflamación/inmunología , Inflamación/genética , Macrófagos/inmunologíaRESUMEN
Responses of cells to stimuli are increasingly discovered to involve the binding of sequence-specific transcription factors outside of known target genes. We wanted to determine to what extent the genome-wide binding and function of a transcription factor are shaped by the cell type versus the stimulus. To do so, we induced the Heat Shock Response pathway in two different cancer cell lines with two different stimuli and related the binding of its master regulator HSF1 to nascent RNA and chromatin accessibility. Here, we show that HSF1 binding patterns retain their identity between basal conditions and under different magnitudes of activation, so that common HSF1 binding is globally associated with distinct transcription outcomes. HSF1-induced increase in DNA accessibility was modest in scale, but occurred predominantly at remote genomic sites. Apart from regulating transcription at existing elements including promoters and enhancers, HSF1 binding amplified during responses to stimuli may engage inactive chromatin.
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Proteínas de Unión al ADN , Neoplasias , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Factores de Transcripción/metabolismo , Respuesta al Choque Térmico/genética , Cromatina/genética , Neoplasias/genéticaRESUMEN
Multicellular evolution is a major transition associated with momentous diversification of multiple lineages and increased developmental complexity. The volvocine algae comprise a valuable system for the study of this transition, as they span from unicellular to undifferentiated and differentiated multicellular morphologies despite their genomes being similar, suggesting multicellular evolution requires few genetic changes to undergo dramatic shifts in developmental complexity. Here, the evolutionary dynamics of six volvocine genomes were examined, where a gradual loss of genes was observed in parallel to the co-option of a few key genes. Protein complexes in the six species exhibited novel interactions, suggesting that gene loss could play a role in evolutionary novelty. This finding was supported by gene network modeling, where gene loss outpaces gene gain in generating novel stable network states. These results suggest gene loss, in addition to gene gain and co-option, may be important for the evolution developmental complexity.
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Evolución Biológica , FilogeniaRESUMEN
Leptomeningeal carcinomatosis (LC) occurs when tumor cells spread to the cerebrospinal fluid-containing leptomeninges surrounding the brain and spinal cord. LC is an ominous complication of cancer with a dire prognosis. Although any malignancy can spread to the leptomeninges, breast cancer, particularly the HER2+ subtype, is its most common origin. HER2+ breast LC (HER2+ LC) remains incurable, with few treatment options, and the molecular mechanisms underlying proliferation of HER2+ breast cancer cells in the acellular, protein, and cytokine-poor leptomeningeal environment remain elusive. Therefore, we sought to characterize signaling pathways that drive HER2+ LC development as well as those that restrict its growth to leptomeninges. Primary HER2+ LC patient-derived ("Lepto") cell lines in coculture with various central nervous system (CNS) cell types revealed that oligodendrocyte progenitor cells (OPC), the largest population of dividing cells in the CNS, inhibited HER2+ LC growth in vitro and in vivo, thereby limiting the spread of HER2+ LC beyond the leptomeninges. Cytokine array-based analyses identified Lepto cell-secreted GMCSF as an oncogenic autocrine driver of HER2+ LC growth. LC/MS-MS-based analyses revealed that the OPC-derived protein TPP1 proteolytically degrades GMCSF, decreasing GMCSF signaling and leading to suppression of HER2+ LC growth and limiting its spread. Finally, intrathecal delivery of neutralizing anti-GMCSF antibodies and a pan-Aurora kinase inhibitor (CCT137690) synergistically inhibited GMCSF and suppressed activity of GMCSF effectors, reducing HER2+ LC growth in vivo. Thus, OPC suppress GMCSF-driven growth of HER2+ LC in the leptomeningeal environment, providing a potential targetable axis. SIGNIFICANCE: This study characterizes molecular mechanisms that drive HER2+ leptomeningeal carcinomatosis and demonstrates the efficacy of anti-GMCSF antibodies and pan-Aurora kinase inhibitors against this disease.
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Comunicación Autocrina , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Factor Estimulante de Colonias de Granulocitos y Macrófagos/metabolismo , Carcinomatosis Meníngea/secundario , Transducción de Señal , Animales , Línea Celular Tumoral , Proliferación Celular/genética , Supervivencia Celular , Modelos Animales de Enfermedad , Expresión Génica , Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Humanos , Carcinomatosis Meníngea/diagnóstico , Ratones , Proteínas Oncogénicas/genética , Proteínas Oncogénicas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Receptor ErbB-2/metabolismo , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
The pentameric B subunit of cholera toxin (CtxB) is an efficient mucosal adjuvant for vaccines. We report the expression of a chimeric protein comprising the synthetic cholera toxin B subunit fused at its C-terminal with rabies surface glycoprotein (G protein) in tobacco plants. The approximately 80.3 kDa fusion polypeptide expressed at 0.4% of the total soluble protein in leaves of the selected transgenic lines. The fusion protein formed a approximately 403 kDa pentameric protein which was functionally active in binding to GM1 receptor. The plant-made protein had a higher affinity for GM1 receptor than the native bacterial CtxB. The pentameric fusion protein was recognized by the anti-cholera toxin as well as anti-rabies antibodies. Its immuno-protective ability against rabies remains to be examined.
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Antígenos Virales/genética , Toxina del Cólera/genética , Glicoproteínas/genética , Multimerización de Proteína , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas del Envoltorio Viral/genética , Vacunas contra el Cólera/inmunología , Pliegue de Proteína , Vacunas Antirrábicas/inmunología , Proteínas Recombinantes de Fusión/genética , Nicotiana/genéticaRESUMEN
Environmental conditions play an important role in the emergence of genetic variations in natural populations. We identified genome-wide patterns of nucleotide variations in the coding regions of natural Arabidopsis thaliana populations. These populations originated from 700 m to 3400 m a.m.s.l. in the Western Himalaya. Using a pooled RNA-Seq approach, we identified the local and global level population-specific SNPs. The biological functions of the SNP-containing genes were primarily related to the high light intensity prevalent at high-altitude regions. The novel SNPs identified in these genes might have arisen de novo in these populations. In another approach, the FSTs of SNP-containing genes were correlated with the corresponding climatic factors. 'Radiation in the growing season' was the only environmental factor found to be strongly correlated with the gene-level FSTs. In both the approaches, the high light intensity was identified as the primary abiotic stress associated with the variations in these populations. The differential gene expression analysis between field and controlled condition grown plants also showed high light intensity as the primary abiotic stress, particularly for the high altitude populations. Our results provide a genome-wide perspective of nucleotide variations in populations along altitudinal gradient and their putative role in emergence of these variations.
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Arabidopsis/clasificación , Arabidopsis/efectos de la radiación , Variación Genética , Luz , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Clima , ADN de Plantas/genética , Exposición a Riesgos Ambientales , Genes de Plantas , Genoma de Planta , Geografía , Polimorfismo de Nucleótido Simple , Selección GenéticaRESUMEN
The natural genetic variation within a plant species is primarily a consequence of its phylogeography and evolutionary history. This variation largely determines its present-day population structure. Arabidopsis thaliana, as a model plant, has been studied in great detail including its probable origin, local as well as global genetic diversity pattern, population structure, adaptation, etc. However, no such studies have so far been reported from the Indian Himalayan region. Here, we describe a comprehensive study on the genetic diversity and population structure of A. thaliana from an altitudinal range of 700-3400 m above mean sea level the highest altitudinal range reported so far. We also compare these populations with previously reported worldwide populations. A total of 48 accessions representing six populations were analysed using 19 microsatellites and 11 chloroplast markers. Genetic diversity analysis indicated populations to be highly diverse and comparable with worldwide populations. STRUCTURE, principal coordinate and isolation by distance (IBD) analyses showed that genetic variation in different populations is structured at geographical and altitudinal level. Further analyses indicate that these populations are genetically distinct from the rest of the world populations. Different parameters of the demographic expansion model support a rapid expansion. Based on mismatch distribution, the initial time of expansion of west Himalayan populations was found to be about 130 000 years. Bayesian analysis of divergence time indicated that these populations have a long evolutionary history in this region. Based on the results of genetic diversity parameters, demographic expansion and divergence time estimation, it appears that west Himalayan populations may be the source of the west-east expansion model.
RESUMEN
BACKGROUND: DNA barcoding as a tool for species identification has been successful in animals and other organisms, including certain groups of plants. The exploration of this new tool for species identification, particularly in tree species, is very scanty from biodiversity-rich countries like India. rbcL and matK are standard barcode loci while ITS, and trnH-psbA are considered as supplementary loci for plants. METHODOLOGY AND PRINCIPAL FINDINGS: Plant barcode loci, namely, rbcL, matK, ITS, trnH-psbA, and the recently proposed ITS2, were tested for their efficacy as barcode loci using 300 accessions of tropical tree species. We tested these loci for PCR, sequencing success, and species discrimination ability using three methods. rbcL was the best locus as far as PCR and sequencing success rate were concerned, but not for the species discrimination ability of tropical tree species. ITS and trnH-psbA were the second best loci in PCR and sequencing success, respectively. The species discrimination ability of ITS ranged from 24.4 percent to 74.3 percent and that of trnH-psbA was 25.6 percent to 67.7 percent, depending upon the data set and the method used. matK provided the least PCR success, followed by ITS2 (59. 0%). Species resolution by ITS2 and rbcL ranged from 9.0 percent to 48.7 percent and 13.2 percent to 43.6 percent, respectively. Further, we observed that the NCBI nucleotide database is poorly represented by the sequences of barcode loci studied here for tree species. CONCLUSION: Although a conservative approach of a success rate of 60-70 percent by both ITS and trnH-psbA may not be considered as highly successful but would certainly help in large-scale biodiversity inventorization, particularly for tropical tree species, considering the standard success rate of plant DNA barcode program reported so far. The recommended matK and rbcL primers combination may not work in tropical tree species as barcode markers.
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Código de Barras del ADN Taxonómico/métodos , ADN Espaciador Ribosómico/genética , Genes de Plantas/genética , Sitios Genéticos/genética , Árboles/clasificación , Árboles/genética , Clima Tropical , Secuencia de Bases , Variación Genética , Geografía , India , Datos de Secuencia Molecular , Filogenia , Estándares de Referencia , Comunicaciones por Satélite , Especificidad de la EspecieRESUMEN
BACKGROUND: DNA barcoding refers to the use of short DNA sequences for rapid identification of species. Genetic distance or character attributes of a particular barcode locus discriminate the species. We report an efficient approach to analyze short sequence data for discrimination between species. METHODOLOGY AND PRINCIPAL FINDINGS: A new approach, Oligonucleotide Frequency Range (OFR) of barcode loci for species discrimination is proposed. OFR of the loci that discriminates between species was characteristic of a species, i.e., the maxima and minima within a species did not overlap with that of other species. We compared the species resolution ability of different barcode loci using p-distance, Euclidean distance of oligonucleotide frequencies, nucleotide-character based approach and OFR method. The species resolution by OFR was either higher or comparable to the other methods. A short fragment of 126 bp of internal transcribed spacer region in ribosomal RNA gene was sufficient to discriminate a majority of the species using OFR. CONCLUSIONS/SIGNIFICANCE: Oligonucleotide frequency range of a barcode locus can discriminate between species. Ability to discriminate species using very short DNA fragments may have wider applications in forensic and conservation studies.
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Dermatoglifia del ADN/métodos , Sitios Genéticos/genética , Oligodesoxirribonucleótidos/genética , Animales , ADN Espaciador Ribosómico/genética , Hongos/clasificación , Hongos/genética , Mutación INDEL , Internet , Filogenia , Plantas/clasificación , Plantas/genética , Especificidad de la EspecieRESUMEN
BACKGROUND: The concept of DNA barcoding for species identification has gained considerable momentum in animals because of fairly successful species identification using cytochrome oxidase I (COI). In plants, matK and rbcL have been proposed as standard barcodes. However, barcoding in complex genera is a challenging task. METHODOLOGY AND PRINCIPAL FINDINGS: We investigated the species discriminatory power of four reportedly most promising plant DNA barcoding loci (one from nuclear genome--ITS, and three from plastid genome--trnH-psbA, rbcL and matK) in species of Indian Berberis L. (Berberidaceae) and two other genera, Ficus L. (Moraceae) and Gossypium L. (Malvaceae). Berberis species were delineated using morphological characters. These characters resulted in a well resolved species tree. Applying both nucleotide distance and nucleotide character-based approaches, we found that none of the loci, either singly or in combinations, could discriminate the species of Berberis. ITS resolved all the tested species of Ficus and Gossypium and trnH-psbA resolved 82% of the tested species in Ficus. The highly regarded matK and rbcL could not resolve all the species. Finally, we employed amplified fragment length polymorphism test in species of Berberis to determine their relationships. Using ten primer pair combinations in AFLP, the data demonstrated incomplete species resolution. Further, AFLP analysis showed that there was a tendency of the Berberis accessions to cluster according to their geographic origin rather than species affiliation. CONCLUSIONS/SIGNIFICANCE: We reconfirm the earlier reports that the concept of universal barcode in plants may not work in a number of genera. Our results also suggest that the matK and rbcL, recommended as universal barcode loci for plants, may not work in all the genera of land plants. Morphological, geographical and molecular data analyses of Indian species of Berberis suggest probable reticulate evolution and thus barcode markers may not work in this case.