Utility of iPBS retrotransposons markers for molecular characterization of African Gnetum species

Abstract

Species of African Gnetum are lianas used as vegetables, medicines and for generating income. Despite the taxonomic confusion, identification of new species and diverse morphological characters in African Gnetum, molecular markers on these plants are lacking. However, the inter-primer binding site (iPBS) retrotransposons markers could be simple and excellent molecular markers for African Gnetum. The objective of this study was to determine the efficiency of iPBS markers in detecting genetic differentiation in African Gnetum species. A set of 21 iPBS markers were analysed on 14 accessions including G. africanum Welw., G. buchholzianum Engl. and the recently identified species G. latispicum. Six best selected primers generated 103 bands in G. africanum, 95 in G. buchholzianum and 24 in G. latispicum. Cluster analysis divided the accessions into two major groups. The first group contained all the accessions of G. africanum, whereas the second group was further divided in two subgroups representing accessions of G. buchholzianum and G. latispicum. Additionally, the Jaccard similarity coefficient indicated a close relationship between accessions of G. buchholzianum and G. latispicum. The iPBS marker system revealed genetic differentiation within African Gnetum and could be useful for evaluating genetic diversity, conservation, taxonomy and evolution studies.     

Drosophila genomes and the development of affordable molecular markers for species genotyping

The recent completion of genome sequencing of 12 species of Drosophila has provided a powerful resource for hypothesis testing, as well as the development of technical tools. Here we take advantage of genome sequence data from two closely related species of Drosophila, Drosophila simulans and Drosophila sechellia, to quickly identify candidate molecular markers for genotyping based on expected insertion or deletion (indel) differences between species. Out of 64 candidate molecular markers selected along the second and third chromosome of Drosophila, 51 molecular markers were validated using PCR and gel electrophoresis. We found that the 20% error rate was due to sequencing errors in the genome data, although we cannot rule out possible indel polymorphisms. The approach has the advantage of being affordable and quick, as it only requires the use of bioinformatics tools for predictions and a PCR and agarose gel based assay for validation. Moreover, the approach could be easily extended to a wide variety of taxa with the only limitation being the availability of complete or partial genome sequence data.     

Genetic analyses of Oryza species by molecular markers for chloroplast genomes

Summary Relationships in a wide range of Oryza species (13 species) were analyzed using the large subunits (LS) of Fraction I protein (Rubisco) and the Bam HI restriction patterns of chloroplast DNA (ctDNA) as molecular markers. Four types of LS were detected by isoelectrofocusing with and without S-carboxymethylation. The close relation between AA and CCDD genome species was suggested by analyses of LS and ctDNA. Intraspecific variation in O. latifolia was detected at the levels of both LS and ctDNA. The LS of the BB, BBCC, and CC genomes and FF (O. brachyantha) were not distinguishable, although the native Rubisco of the latter was slightly different from those of the first three. It was also shown that O. australiensis, the only EE genome species, might have evolved differently than the other Oryza species.     

Mitochondrial genomes in the Hymenoptera and their utility as phylogenetic markers

Abstract.  In this study, we assessed the ability of mitochondrial genome sequences to recover a test phylogeny of five hymenopteran taxa from which phylogenetic relationships are well accepted. Our analyses indicated that the test phylogeny was well recovered in all nucleotide Bayesian analyses when all the available holometabolan (i.e. outgroup) taxa were included, but only in Bayesian analyses excluding third codon positions when only the hymenopteran representatives and a single outgroup were included. This result suggests that taxon sampling of the outgroup might be as important as taxon sampling of the ingroup when recovering hymenopteran phylogenetic relationships using whole mitochondrial genomes. Parsimony analyses were more sensitive to both taxon sampling and the analytical model than Bayesian analyses, and analyses using the protein dataset did not recover the test phylogeny. In general, mitochondrial genomes did not resolve the position of the Hymenoptera within the Holometabola with confidence, suggesting that an increased taxon sampling, both within the Holometabola and among outgroups, is necessary.     

Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species

We report the identification and characterization of the major repeats in the centromeric and peri-centromeric heterochromatin of Brassica rapa. The analysis involved the characterization of 88 629 bacterial artificial chromosomes (BAC) end sequences and the complete sequences of two BAC clones. We identified centromere-specific retrotransposons of Brassica (CRB) and various peri-centromere-specific retrotransposons (PCRBr). Three copies of the CRB were identified in one BAC clone as nested insertions within a tandem array of 24 copies of a 176 bp centromeric repeat, CentBr. A complex mosaic structure consisting of nine PCRBr elements and large blocks of 238 bp degenerate tandem repeats (TR238) were found in or near a derivative of 5S-25S rDNA sequences. The chromosomal positions of selected repeats were determined using in situ hybridization. These revealed that CRB is a major component of all centromeres in three diploid Brassica species and their allotetraploid relatives. However, CentBr was not detected in the most distantly related of the diploid species analyzed, B. nigra. PCRBr and TR238 were found to be major components in the peri-centromeric heterochromatin blocks of four chromosomes of B. rapa. These repetitive elements were not identified in B. oleracea or B. nigra, indicating that they are A-genome-specific. GenBank accession numbers: KBrH001P13 (AC 166739); KBrH015B20 (AC 166740); end sequences of KBrH BAC library (CW 978640 - CW 988843); end sequences of KBrS BAC library (DU 826965 - DU 835595); end sequences of KBrB BAC library (DX 010661 - DX 083363).     

Characterization of chromosome-specific S-SAP markers and their use in studying genetic diversity in Aegilops species.

The short interspersed nuclear element (SINE), Au, was used to develop sequence-specific amplified polymorphism (S-SAP) markers for U- and M-genome chromosomes. The markers were localized using Triticum aestivum (wheat)-- Aegilops geniculata and wheat-- Aegilops biuncialis disomic chromosome addition lines. Thirty-seven markers distributed over 6 U and 6 M chromosomes were produced. A genetic diversity study carried out on 37 accessions from Ae. biuncialis, Ae. comosa, Ae. geniculata, and Ae. umbellulata suggested that Ae. biuncialis have arisen from its diploid ancestors more recently than Ae. geniculata. Several earlier studies indicated that the M genomes in polyploid Aegilops species had accumulated substantial rearrangements, whereas the U genomes remained essentially unmodified. However, this cannot be attributed to the preferential insertion of retroelements into the M genome chromosomes. Fourteen markers from a total of 8 chromosomes were sequenced; 3 markers were similar to known plant genes, 1 was derived from a long terminal repeat (LTR) retrotransposon, and 10 markers did not match to any known DNA sequences, suggesting that they were located in the highly variable intergenic regions.     

LTR retrotransposons from genomes of Aspergillus fumigatus and A. nidulans

Fungi Aspergillus spp. are able to infect all tissues and organs and often cause invasive mycosis (aspergillosis), which is usually a fatal disease, especially in the patients with compromised immune system. Microbiological monitoring of these infectious agents is necessary in modem medical facilities. Mobile elements can be used as markers for identification of species and strains of Aspergillus found indoors as well as in aspergillosis diagnostics. Genomic sequences of two representative Aspergillus species, A. fumigatus and A. nidulans, were analysed in silico in order to detect LTR retrotransposons. We found considerable differences in the composition of retrotransposon families between two studied species. One of the detected families, which is present in both studied Aspergillus species, is phylogenetically quite different from all other known fungal retrotransposons. The majority of elements are represented by damaged copies. Nevertheless, we describe for the first time allegedly non-damaged LTR copies that contain intact ORFs and could be active.     

Genome-wide identification of intron fragment insertion mutations and their potential use as SCAR molecular markers in the soybean

Introns often have a high probability of mutation as a result of DNA insertions and deletions (indels). In this study, 503 introns with exon-derived insertions were identified using a comprehensive search of the soybean genome. Of the 375 pairs of PCR primer sets designed for the loci in question, 161 primer sets amplified length polymorphism among nine soybean varieties and were identified as soybean gene-intron-driven functional sequence characterized amplified region (SCAR) markers. These SCAR markers are distributed among all 20 of the soybean chromosomes, and they developed from numerous genes involved in various physiological and biochemical processes that influence important agronomic traits of the soybean. The development of these novel gene-driven functional SCAR markers was fast and cost effective, and their use will facilitate molecular-assisted breeding of the soybean.     

Characterization of the complete mitochondrial genomes of five Eimeria species from domestic chickens

Chicken coccidiosis caused by members of the genus Eimeria causes significant economic losses worldwide. In the present study we sequenced the complete mitochondrial DNA (mtDNA) sequences of six Eimeria species and analyzed features of their gene contents and genome organizations. The complete mt genomes of E. acervulina, E. brunetti, E. maxima, E. necatrix, E. tenella and E. praecox were 6179bp, 6148bp, 6169bp, 6214bp, 6213bp and 6174bp in size, respectively. All of the mt genomes consist of 3 genes for proteins (cox1, cox3, and cytb), 12 gene fragments for the large subunit (LSU) rRNA, and 7 gene fragments for the small subunit (SSU) rRNA, but no transfer RNA genes. The organization of the mt genomes is similar to that of Plasmodium, but distinct from Babesia and Theileria. The putative direction of translation for 3 genes (cox1, cox3, and cytb) was the same in all six Eimeria species. The contents of A+T of the mt genomes were 65.35% for E. acervulina, 65.43% for E. brunetti, 64.53% for E. maxima, 65.04% for E. necatrix, 64.98% for E. tenella and 65.59% for E. praecox. The AT bias has a significant effect on both the codon usage pattern and amino acid composition of proteins. Phylogenetic analyses using concatenated nucleotide sequences of the 2 protein-coding genes (cytb and cox1), with three different computational algorithms (Bayesian analysis, maximum parsimony and maximum likelihood), all revealed distinct groups with high statistical support, indicating that the six Eimeria spp. represent six distinct but closely-related species. These data provide novel mtDNA markers for studying the molecular epidemiology and population genetics of the six Eimeria spp., and should have implications for the molecular diagnosis, prevention and control of coccidiosis in domestic chickens.     

Glutamine synthetase sequence evolution in the mycobacteria and their use as molecular markers for Actinobacteria speciation

Background  

Although the gene encoding for glutamine synthetase (gln A) is essential in several organisms, multiple glnA copies have been identified in bacterial genomes such as those of the phylum Actinobacteria, notably the mycobacterial species. Intriguingly, previous reports have shown that only one copy (gln A1) is essential for growth in M. tuberculosis, while the other copies (gln A2, gln A3 and gln A4) are not.     

Characterization of Gypsophila species and commercial hybrids with nuclear whole-genome and cytoplasmic molecular markers

The genus Gypsophila contains about 150 annual and perennial flowering plant species native to the temperate regions of Europe and Asia. Nowadays Gypsophila species are present worldwide as garden ornamental plants. Although Gypsophila is one of the most economically important ornamental crops, little is known about its genetic variability and the relationships among the different wild species, cultivars, and commercial hybrids. The aim of our work was to analyze genetic distances among 5 wild species and 13 commercial hybrids of Gypsophila with similar phenotypes but unknown origin. For this purpose, we have used amplified fragment length polymorphism, target region amplification polymorphism, and inter simple sequence repeat whole-genome markers and chloroplast simple sequence repeat (cpSSR), targeting chloroplast DNA. Nuclear markers were found to distinguish all the analyzed samples while cpSSR markers were found to discriminate the different wild species, but could not sufficiently separate the commercial hybrids. This notwithstanding, the data obtained allowed us to cluster the commercial hybrids into different sub-groups and to determine the relationships with the putative species of origin.     

Identification of thermophilic species by the amino acid compositions deduced from their genomes

The global amino acid compositions as deduced from the complete genomic sequences of six thermophilic archaea, two thermophilic bacteria, 17 mesophilic bacteria and two eukaryotic species were analysed by hierarchical clustering and principal components analysis. Both methods showed an influence of several factors on amino acid composition. Although GC content has a dominant effect, thermophilic species can be identified by their global amino acid compositions alone. This study presents a careful statistical analysis of factors that affect amino acid composition and also yielded specific features of the average amino acid composition of thermophilic species. Moreover, we introduce the first example of a 'compositional tree' of species that takes into account not only homologous proteins, but also proteins unique to particular species. We expect this simple yet novel approach to be a useful additional tool for the study of phylogeny at the genome level.     

Evolutionary dynamics of the LTR retrotransposons roo and rooA inferred from twelve complete Drosophila genomes

Background  

Roo is the most abundant retrotransposon in the fruit fly Drosophila melanogaster. Its evolutionary origins and dynamics are thus of special interest for understanding the evolutionary history of Drosophila genome organization. We here study the phylogenetic distribution and evolution of roo, and its highly diverged relative rooA in 12 completely sequenced genomes of the genus Drosophila.     

Gypsy-like retrotransposons in Pyrenophora: an abundant and informative class of molecular markers.

This paper describes the development of S-SAP (sequence-specific amplified polymorphism) using a primer derived from the LTR (long terminal repeat) of the Pyggy retrotransposon isolated from Pyrenophora graminea. Fragments were amplified by S-SAP from different Pyrenophora spp., indicating the presence of Pyggy-like sequences in these genomes. The bands were highly polymorphic between isolates and the number of bands differed by as much as 10-fold between species, demonstrating the potential of this method for genetic analysis in fungi. The phylogenetic relationship among the isolates as deduced using S-SAP data is presented, and shows evidence of genetic exchange between P. graminea and P. teres.     

Molecular analysis of highly repeated genome fractions in Solanum and their use as markers for the characterization of species and cultivars

Summary Highly repeated DNA of potato (Solanum sp.) was characterized by cloning various major repeated elements of the nuclear genome. The percentage of the nuclear genome of the specific fractions and the restriction enzyme patterns were determined in order to show the distribution and organization of the respective repeats in the genome of Solanum tuberosum cultivars, dihaploid breeding lines and in wild species of Solanum. Several of the clones obtained were represented in a high copy number but showed no informative RFLP patterns. More information was gained from restriction satellite repeats. The clone pR1T320 was found to contain satellite repeats (360 bp in length) that are proportionally present in the genome of all Solanum species at frequencies, between 0.5% and 2.6% and which are differently organized. This repeat was also found in the genera Lycopersicon, Datura and Nicotiana. With various restriction enzymes characteristic RFLP patterns were detected. A more or less genus-specific element for Solanum was the 183-bp repeat (clone pSA287; between 0.2–0.4% of the nuclear genome) that was present in the majority of the Solanum species analyzed except S. kurtzianum, S. bulbocastanum and S. pinnatisectum. In a few wild species (prominently in S. kurtzianum, S. demissum and S. acaule) a specific repeat type was detected (clone pSDT382; repeat length approximately 370 bp) that could be used to trace the wild species introduced into S. tuberosum cultivars. The repeats analyzed together with the 18S, 5.8S and 25S ribosomal DNA (1.9–5.2%, corresponding to 1800-5500 rDNA copies) comprised approximately 4–7% of the Solanum genome.     

Citrus phylogeny and genetic origin of important species as investigated by molecular markers

Citrus phylogeny was investigated using RAPD, SCAR and cpDNA markers. The genotypes analyzed included 36 accessions belonging to Citrus together with 1 accession from each of the related genera Poncirus, Fortunella, Microcitrus and Eremocitrus. Phylogenetic analysis with 262 RAPDs and 14 SCARs indicated that Fortunella is phylogenetically close to Citrus while the other three related genera are distant from Citrus and from each other. Within Citrus, the separation into two subgenera, Citrus and Papeda, designated by Swingle, was clearly observed except for C. celebica and C. indica. Almost all the accessions belonging to subgenus Citrus fell into three clusters, each including 1 genotype that was considered to be a true species. Different phylogenetic relationships were revealed with cpDNA data. Citrus genotypes were separated into subgenera Archicitrus and Metacitrus, as proposed by Tanaka, while the division of subgenera Citrus and Papeda disappeared. C. medica and C. indica were quite distant from other citrus as well from related genera. C. ichangensis appeared to be the ancestor of the mandarin cluster, including C. tachibana. Lemon and Palestine sweet lime were clustered into the Pummelo cluster led by C. latipes. C. aurantifolia was located in the Micrantha cluster. Furthermore, genetic origin was studied on 17 cultivated citrus genotypes by the same molecular markers, and a hybrid origin was hypothesized for all the tested genotypes. The assumptions are discussed with respect to previous studies; similar results were obtained for the origin of orange and grapefruit. Hybrids of citron and sour orange were assumed for lemon, Palestine sweet lime, bergamot and Volkamer lemon, while a citron × mandarin hybrid was assumed for Rangpur lime and Rough lemon. For Mexican lime our molecular data indicated C. micrantha to be the female parent and C. medica as the male one. Received: 5 October 1999 / Accepted: 3 November 1999