Oligonucleotide Frequencies of Barcoding Loci Can Discriminate Species across Kingdoms

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

Universal Plant DNA Barcode Loci May Not Work in Complex Groups: A Case Study with Indian Berberis Species

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

High altitude population of Arabidopsis thaliana is more plastic and adaptive under common garden than controlled condition

Abstract Background Population differentiation and their adaptation to a particular environment depend on their ability to respond to a new environment. This, in turn is governed to an extent, by the degree of phenotypic plasticity exhibited by the populations. The populations of same species inhabiting different climatic conditions may differ in their phenotypic plasticity. Himalayan populations of Arabidopsis thaliana originating from a steep altitude are exposed to different climatic conditions ranging from sub-tropical to temperate. Thus they might have experienced different selection pressures during evolution and may respond differently under common environmental condition. Results Phenotypic plasticity and differentiation of natural populations of A. thaliana grown under common garden and controlled conditions were determined. A total of seventeen morphological traits, their plasticity, association between traits and environment were performed using 45 accessions from three populations. Plants from different altitudes differed in phenotypes, their selection and fitness under two conditions. Under both the conditions lower altitude population was characterized by higher leaf count and larger silique than higher and middle altitude population. Flowering time of high altitude population increased while that of low and medium altitude decreased under controlled condition compared to open field. An increase in seed weight and germination was observed for all the population under open field than controlled. Rosette area was under divergent selection in both the condition. The heritability of lower altitude population was the highest under both the conditions, where as it was the least for higher altitude population further indicating that the high altitude populations are more responsive towards phenotypic changes under new environmental conditions. Ninety-nine percent of variability in traits and their plasticity co-varied with the altitude of their origin. The population of high altitude was more plastic and differentiated as compared to the lower altitude one. Conclusions Arabidopsis thaliana population native to different altitudes of the west Himalaya responds differently when grown under common environments. The success of high altitude population is more in common garden than the controlled conditions. The significant variability in phenotype and its association with altitude of origin predicts for non-random genetic differentiation among the populations

L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

13 juin 19401940/06/13 (A41,N14419)

Molecular Cloning and Functional Identification of a Ribosome Inactivating/Antiviral Protein from Leaves of Post-Flowering Stage of Celosia Cristata and Its Expression in E. Coli

A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC 50 values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC 50 at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV)

Molecular cloning and functional identification of a ribosome inactivating/antiviral protein from leaves of post-flowering stage of Celosia cristata and its expression in E. coli

A full-length cDNA clone, encoding a ribosome inactivating/antiviral protein (RIP/AVP) was isolated from the cDNA library of post-flowering stage of Celosia cristata leaves. The full-length cDNA consisted of 1015 nucleotides, with an open reading frame encoding 283 amino acids. The deduced amino acid sequence had a putative active site domain conserved in other ribosome inactivating/antiviral proteins (RIPs/AVPs). The coding region of the cDNA was amplified by polymerase chain reaction (PCR), cloned and expressed in Escherichia coli as recombinant protein of 72 kDa. The expressed fusion product was confirmed by Western analysis and purification by affinity chromatography. Both the recombinant protein (reCCP-27) and purified expressed protein (eCCP-27) inhibited translation in rabbit reticulocytes showing IC<SUB>50</SUB> values at 95 ng and 45 ng, respectively. The native purified nCCP-27 has IC<SUB>50</SUB> at 25 ng. The purified product also showed N-glycosidase activity towards tobacco ribosomes and antiviral activity towards tobacco mosaic virus (TMV) and sunnhemp rosette virus (SRV)

Identification and Expression Analyses of miRNAs from Two Contrasting Flower Color Cultivars of Canna by Deep Sequencing.

miRNAs are endogenous small RNA (sRNA) that play critical roles in plant development processes. Canna is an ornamental plant belonging to family Cannaceae. Here, we report for the first time the identification and differential expression of miRNAs in two contrasting flower color cultivars of Canna, Tropical sunrise and Red president. A total of 313 known miRNAs belonging to 78 miRNA families were identified from both the cultivars. Thirty one miRNAs (17 miRNA families) were specific to Tropical sunrise and 43 miRNAs (10 miRNA families) were specific to Red president. Thirty two and 18 putative new miRNAs were identified from Tropical sunrise and Red president, respectively. One hundred and nine miRNAs were differentially expressed in the two cultivars targeting 1343 genes. Among these, 16 miRNAs families targeting 60 genes were involved in flower development related traits and five miRNA families targeting five genes were involved in phenyl propanoid and pigment metabolic processes. We further validated the expression analysis of a few miRNA and their target genes by qRT-PCR. Transcription factors were the major miRNA targets identified. Target validation of a few randomly selected miRNAs by RLM-RACE was performed but was successful with only miR162. These findings will help in understanding flower development processes, particularly the color development in Canna

Purification, characterization and cloning of antiviral/ribosome inactivating protein from Amaranthus tricolor leaves

An antiviral protein (AVP), imparting high level of resistance against sunnhemp rosette virus (SRV) was purified from the dried leaves of Amaranthus tricolor. The purified protein (AAP-27) exhibited not, vert, ~98% inhibition of local lesion formation at a concentration range of not, vert, ~30 μg ml−1. The protein was found to be highly basic glycoprotein monomer (pI not, vert, ~9.8) of Mr 27 kDa, with neutral sugar content of 4%. The purified protein exhibited N-glycosidase and RNase activities. We have also isolated full-length cDNA clone, encoding this protein designated as A. tricolor antiviral protein-1 (AAP-1). Two primers, one designed on the basis of N-terminal sequence of the purified protein and the other from the conserved active peptides of other AVPs/RIPs were used for PCR amplification of double stranded cDNA, isolated from the leaves of A. tricolor. The amplified fragment was used as a probe for library screening. The isolated full-length cDNA consisted of 1058 nucleotides with an open reading frame encoding a polypeptide of 297 amino acids. The deduced amino acid sequence of AAP-1 has a putative active domain conserved in other AVPs/RIPs and shows varying homology to the RIPs from other plant species

Rabies glycoprotein fused with B subunit of cholera toxin expressed in tobacco plants folds into biologically active pentameric protein

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 ⋍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 ⋍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