Intraspecific Sequence Diversity Research Articles

Assessment of genetic diversity in germplasm of Guinea grass (Panicum maximum Jacq.)

Mucuna pruriens is a nutritionally and pharmaceutically important underutilized legume belonging to genus Mucuna of Leguminosae family. Extensive intraspecific diversity and adverse morphological features among the members have led to taxonomical confusions in the species. A total of 12 Mucuna pruriens accessions including its two botanical varieties, var. pruriens and var. utilis with six accessions each were analyzed to characterize the intraspecific diversity among them using the three barcode markers viz. ITS2, matK and trnHpsbA. Extensive intraspecific sequence diversity was observed in the trnHpsbA region, where as ITS2 and matK exhibited significant conserved regions. Such intraspecific sequence diversity is a limitation in adapting trnHpsbA as barcode as it may lead to misinterpretations, but in the present study an attempt has been made to extrapolate this diversity to explain within the species variations among Mucuna pruriens genotypes. Intraspecific sequence variations exhibited by trnHpsbA sequences were 83% in correlation with morphological features. The between groups mean distance of var. pruriens and var. utilis was found to be 0.346. Results of this study will complement with the existing molecular, morphological, ytological and biochemical markers towards Velvet bean improvement programs.

Genomics and immunity of the Mediterranean mussel Mytilus galloprovincialis in a changing environment

The Mediterranean mussel (Mytilus galloprovincialis) is a marine invasive species cultured all over the world. Mussels are an appreciated resource in local aquaculture enterprises because of their robust production and resilience that translates into a reliable economic value. So far, no massive mortalities have been reported in natural or cultured populations of this species. In the last years, the knowledge about its immune system has greatly improved but there are still many questions to be answered. One of them is why mussels, with their high filtering activity, are able to be exposed to a high number of potential pathogens without getting infected and without developing an elevated inflammatory response.The sequencing of the mussel genome has revealed a very complex organization with high heterozygosity, abundance of repetitive sequences and extreme intraspecific sequence diversity among individuals, mainly in immune related genes. Among those genes, antimicrobial peptides are the most expressed gene families in mussels, highly polymorphic and with antimicrobial effect against molluscs pathogens, but also against pathogens of lower vertebrates and humans. The combination of a complex genome with the adaptation of mussel immune system to a changing environment could explain this high variability, not only in immune-related genes, but also in the functional response among individuals sampled in the same location and date.

Molecular characterisation of plant parasitic nematode Longidorus poessneckensis Altherr, 1974 (Nematoda: Longidoridae)

The plant parasitic nematode Longidorus poessneckensis found in Austria, the Czech Republic, Germany, Poland and the Slovak Republic was molecularly characterized. Mitochondrial genes encoding cytochrome c oxidase subunit I (COI) and nicotinamide dehydrogenase subunit 4 (nad4), the D2 and D3 expansion segments of 28S rRNA and Internal transcribed spacer 1 (ITS1) rRNA were sequenced for 16 L. poessneckensis populations. Six haplotypes of COI and five haplotypes of nad4 were determined. Nucleotide intraspecific variation was up to 17.1% for the partial sequenced COI gene and up to 17.7% for the partial sequenced nad4 gene, the latter being the highest up to date known intraspecific variation in this genus. The analyses of multiple amino acid sequence alignments of mitochondrial genes revealed low variability (0–2.4%) for COI gene and high divergence (0–7.6%) for nad4 gene. Intraspecific sequence diversity for the D2-D3 of 28S rRNA gene was up to 1.2% and for ITS1 rRNA gene was up to 1.6%. It has been hypothesized, that during the Last Glacial Maximum, L. poessneckensis populations probably persisted in refuge areas in the Carpathian Mountains and subsequently expanding from these areas and colonizing other European regions.

Characterization of Brazilian populations of Pratylenchus brachyurus using morphological and molecular analyses

In Brazil, Pratylenchus brachyurus damages economic important crops and it is managed with non-chemical methods such as crop rotation or resistant cultivars. However, the variability in reproduction and damaging effects observed among P. brachyurus populations could limit the efficacy and long lasting effect of nematode control practices. Twelve populations of P. brachyurus from different geographical areas of Brazil were analyzed morphologically and molecularly. The morphological and morphometric characters of the populations studied were variable, but fitted those of P. brachyurus. Assemblages of populations with different host preferences and reproductive capabilities were revealed by PCA of these populations using selected morphological characters. Phylogenetic analysis using ITS-rDNA sequences distinguished statistically supported groups of sequences that matched those of other populations of P. brachyurus deposited in the GenBank, confirming the identity of populations here studied. Intraspecific sequence diversity was observed among the populations. These variant sequences suggest the presence of microheterogeneity between ITS-1 copies in individuals. The findings of this study did not provide evidence of cryptic species in the P. brachyurus populations studied. The variability within and among populations of P. brachyurus observed in this work must be taken into consideration in breeding programs for resistance to this nematode.

PyroClean: Denoising Pyrosequences from Protein-Coding Amplicons for the Recovery of Interspecific and Intraspecific Genetic Variation

High-throughput parallel sequencing is a powerful tool for the quantification of microbial diversity through the amplification of nuclear ribosomal gene regions. Recent work has extended this approach to the quantification of diversity within otherwise difficult-to-study metazoan groups. However, nuclear ribosomal genes present both analytical challenges and practical limitations that are a consequence of the mutational properties of nuclear ribosomal genes. Here we exploit useful properties of protein-coding genes for cross-species amplification and denoising of 454 flowgrams. We first use experimental mixtures of species from the class Collembola to amplify and pyrosequence the 5′ region of the COI barcode, and we implement a new algorithm called PyroClean for the denoising of Roche GS FLX pyrosequences. Using parameter values from the analysis of experimental mixtures, we then analyse two communities sampled from field sites on the island of Tenerife. Cross-species amplification success of target mitochondrial sequences in experimental species mixtures is high; however, there is little relationship between template DNA concentrations and pyrosequencing read abundance. Homopolymer error correction and filtering against a consensus reference sequence reduced the volume of unique sequences to approximately 5% of the original unique raw reads. Filtering of remaining non-target sequences attributed to PCR error, sequencing error, or numts further reduced unique sequence volume to 0.8% of the original raw reads. PyroClean reduces or eliminates the need for an additional, time-consuming step to cluster reads into Operational Taxonomic Units, which facilitates the detection of intraspecific DNA sequence variation. PyroCleaned sequence data from field sites in Tenerife demonstrate the utility of our approach for quantifying evolutionary diversity and its spatial structure. Comparison of our sequence data to public databases reveals that we are able to successfully recover both interspecific and intraspecific sequence diversity.

Authentication of Chinese Crude Drug Gecko by DNA Barcoding

Gekko gecko, an animal used as a valued traditional Chinese medicine, has been widely used for over 2000 years. Due to localized habitat destruction, the amount of G. gecko has dramatically decreased in recent years. As a result, more and more adulterants have been detected in the traditional medicine, which has resulted in a chaotic market. Therefore, a correct identification method is badly needed. In this study, we employed a new molecular method of DNA barcoding for discriminating gecko from its adulterants. Fifty-seven specimens of gecko and its adulterants were collected as test samples. The full-barcode and mini-barcode sequences of these specimens were separately amplified and sequenced separately. Together with other published barcode sequences, we detected that the intra-specific sequence diversity was far lower than the inter-specific diversity in G. gecko and its adulterants (3% compared with 35% in full-length barcode; 4% compared with 33.5% in mini-barcode). These results showed that both the full-length and mini-barcodes were effective for identifying gecko, which suggested that the DNA barcode could be an effective and powerful tool for identifying the Chinese crude drug gecko.

Comparative phylogenies of Burkholderia, Ralstonia, Comamonas, Brevundimonas and related organisms derived from rpoB, gyrB and rrs gene sequences

Phylogenetic analysis of strains from Burkholderia, Ralstonia, Cupriavidus, Comamonas, Delftia, Acidovorax, Brevundimonas, Herbaspirillum huttiense and “ Pseudomonas butanovora” was performed based on the protein-coding genes rpoB and gyrB and on the 16S rRNA-coding gene rrs. Overall, the phylogenies deduced from the three genes were concordant among themselves and with current taxonomy. However, a few differences among individual gene phylogenies were noted. For example, the separation of Cupriavidus from Ralstonia was not supported in the rpoB tree, as Ralstonia was nested within Cupriavidus. Similarly, the separation of Delftia from Comamonas was not supported in the gyrB tree. Based on rrs and rpoB, the genus Burkholderia contained four groups: (i) the B. cepacia complex, (ii) the B. pseudomallei- B. thailandensis group, (iii) a 6-species group including B. caledonica and B. glathei and (iv) the B. plantarii- B. glumae- B. gladioli group. However, B. caribensis and B. glathei stood as a fifth group based on gyrB. It appears that a phylogeny cannot be reliably based on a single gene. Using rpoB and gyrB, better separation of closely related species was obtained compared to rrs, indicating the potential of these two genes for identification and species definition. Nevertheless, intraspecific sequence diversity will need to be determined to fully establish the value of these genes for strain identification.

Population subdivision in Europe's great bustard inferred from mitochondrial and nuclear DNA sequence variation.

A continent-wide survey of sequence variation in mitochondrial (mt) and nuclear (n) DNA of the endangered great bustard (Otis tarda) was conducted to assess the extent of phylogeographic structure in a morphologically monotypic bird. DNA sequence variation in a combined 809 bp segment of the mtDNA genome from 66 individuals from the last six breeding regions showed relatively low levels of intraspecific sequence diversity (n = 0.32%) but significant differences in the regional distribution of 11 haplotypes (phiST = 0.49). Despite their exceptional potential for dispersal, a complete and long-term historical separation between the populations from the Iberian Peninsula (Spain) and mainland Europe (Hungary, Slovakia, Germany, and Russia) was demonstrated. Divergence between populations based on a 3-bp insertion-deletion polymorphism within the intron region of the nuclear CHD-Z gene was geographically concordant with the primary subdivision identified within the mtDNA sequences. Inferred aspects of phylogeography were used to formulate conservation recommendations for this endangered species.

Molecular variation in trypanosomes

Several species of the genus Trypanosoma cause parasitic diseases of considerable medical and veterinary importance throughout Africa, Asia and the Americas. These parasites exhibit considerable intra-species genetic diversity and variation, which has complicated their taxonomic classification. This diversity and variation can be defined at the level of both the genome and of individual genes. The nuclear genome shows considerable inter- and intra-species plasticity in terms of chromosome number and size (molecular karyotype). The mitochondrial (kDNA) genome also varies considerably between species, especially in terms of minicircle size and organization. There is also considerable intra-specific sequence diversity in minicircles and within the Variable Region of the maxicircle. Restriction enzyme analysis of this diversity has lead to the concept of ‘schizodemes’. At the gene level, isoenzyme analysis has proven very useful for strain and isolate identification, with the classification into numerous ‘zymodemes’. Considerable antigenic diversity has also been identified in T. cruzi and T. brucei, with the development of ‘serodemes’ in the latter. In addition to this inter-strain diversity, African trypanosomes ( T. brucei, T. congolense, and T. vivax) exhibit the phenomenon of antigenic variation, where individual parasites are able to express any one of hundreds of different copies of the Variant Surface Glycoprotein gene at any particular time. The molecular mechanisms underlying antigenic variation are now understood in considerable detail. The implication of this molecular diversity and variation are discussed in terms of trypanosome taxonomy and disease control.

Theileria parva: genomic DNA studies reveal intra-specific sequence diversity.

Theileria parva parva piroplasm DNA was purified from 11 different infections of cattle with 6 different East African isolates of the parasite. Total DNA was also prepared from bovine lymphoblastoid cells infected with schizonts of one of the isolates. Two of the infections were with cloned parasites. The DNA was of high molecular weight and free from protein and RNA, but some of the samples contained a proportion of bovine DNA. The 6 samples least contaminated with bovine DNA had a mean 'melting' temperature (Tm) of 84 degrees C and a mean GC content of 31.3%. Reassociation kinetics gave an estimate of 1.2 x 10(7) base pairs for the genome of T. parva. Repetitive restriction fragments were cloned from two samples, separated from the recombinant vectors and used as probes to demonstrate RFLPs between isolates. Discrimination into five groups was achieved. Schizont and piroplasm DNAs from the same isolate gave identical RFLPs, and one of the cloned parasites appeared to be a sub-population selected from a mixed-infection field isolate. Comparison of RFLPs with monoclonal antibody profiles suggested that neither technique yet provides discrimination between all the isolates which may comprise a strain. The importance of DNA probes for studying the epidemiology of theileriosis and for control programs is discussed.