From stop to start: tandem gene arrangement, copy number and trans-splicing sites in the dinoflagellate Amphidinium carterae

Dinoflagellate genomes present unique challenges including large size, modified DNA bases, lack of nucleosomes, and condensed chromosomes. EST sequencing has shown that many genes are found as many slightly different variants implying that many copies are present in the genome. As a preliminary survey of the genome our goal was to obtain genomic sequences for 47 genes from the dinoflagellate Amphidinium carterae. A PCR approach was used to avoid problems with large insert libraries. One primer set was oriented inward to amplify the genomic complement of the cDNA and a second primer set would amplify outward between tandem repeats of the same gene. Each gene was also tested for a spliced leader using cDNA as template. Almost all (14/15) of the highly expressed genes (i.e. those with high representation in the cDNA pool) were shown to be in tandem arrays with short intergenic spacers, and most were trans-spliced. Only two moderately expressed genes were found in tandem arrays. A polyadenylation signal was found in genomic copies containing the sequence AAAAG/C at the exact polyadenylation site and was conserved between species. Four genes were found to have a high intron density (>5 introns) while most either lacked introns, or had only one to three. Actin was selected for deeper sequencing of both genomic and cDNA copies. Two clusters of actin copies were found, separated from each other by many non-coding features such as intron size and sequence. One intron-rich gene was selected for genomic walking using inverse PCR, and was not shown to be in a tandem repeat. The first glimpse of dinoflagellate genome indicates two general categories of genes in dinoflagellates, a highly expressed tandem repeat class and an intron rich less expressed class. This combination of features appears to be unique among eukaryotes.     

General-use polymerase chain reaction primers for amplification and direct sequencing of enolase, a single-copy nuclear gene, from different animal phyla

In contrast to mitochondrial DNA, remarkably few general-use primer sets are available for single-copy nuclear genes across animal phyla. Here, we present a primer set that yields a c. 364-bp coding fragment of the metabolic gene enolase, which includes an intron in some taxa. In species where introns are absent or have few insertions/deletions, the amplified fragment can be sequenced directly for phylogenetic or population analysis. Between-species variation in the coding region occurs widely at third codon positions, even between closely related taxa, making the fragment useful for species-level systematics. In low gene-flow species, the primers may also be of use for population genetics, as intraspecific polymorphisms occur at several silent positions in the taxa examined.     

Development of primer sets designed for use with the PCR to amplify conserved genes from filamentous ascomycetes.

We constructed nine sets of oligonucleotide primers on the basis of the results of DNA hybridization of cloned genes from Neurospora crassa and Aspergillus nidulans to the genomes of select filamentous ascomycetes and deuteromycetes (with filamentous ascomycete affiliations). Nine sets of primers were designed to amplify segments of DNA that span one or more introns in conserved genes. PCR DNA amplification with the nine primer sets with genomic DNA from ascomycetes, deuteromycetes, basidiomycetes, and plants revealed that five of the primer sets amplified a product only from DNA of the filamentous ascomycetes and deuteromycetes. The five primer sets were constructed from the N. crassa genes for histone 3, histone 4, beta-tubulin, and the plasma membrane ATPase. With these five primer sets, polymorphisms were observed in both the size of and restriction enzyme sites in the amplified products from the filamentous ascomycetes. The primer sets described here may provide useful tools for phylogenetic studies and genome analyses in filamentous ascomycetes and deuteromycetes (with ascomycete affiliations), as well as for the rapid differentiation of fungal species by PCR.     

Genetic relationships among Stylosanthes species as revealed by sequence-tagged site markers

 Nineteen sequence-tagged site (STS) primer pairs were designed on coding and non-coding regions in nine published Stylosanthes genes, which were mostly derived from cDNA. Direct sequencing of PCR products derived from genomic DNA allowed us to identify introns and to design specific primers flanking these introns. The use of 24 STS primer pairs for the detection of intra- and inter-specific variation in Stylosanthes based on size differences was tested on a core set of Stylosanthes species. Based on these results, 20 STS markers were selected to determine genetic relationships among 63 genotypes representing 24 Stylosanthes species. A total of 148 alleles were amplified and analyzed, resulting in a genetic similarity value ranging from 0.62 to 0.98 among the species. Based on cluster analysis, three main groups and three subgroups were determined, and most of the species were classified unambiguously. Alloploid species were recognized by the occurrence of more than one allele per STS marker, indicating fixed heterozygosity. Sixteen STS markers were useful for the identification of genotypes within a species. Inter-species relationships, as revealed by STS, were in general agreement with previous morphological and molecular relationship studies. These STS markers are useful as an additional tool for the identification of species, subspecies and genotypes in Stylosanthes, with a view to plant conservation and breeding. Received: 2 June 1998 / Accepted: 28 October 1998     

Genome-specific primer sets for starch biosynthesis genes in wheat

Common wheat (Triticum aestivum L., 2n=6x=42) is an allohexaploid composed of three closely related genomes, designated A, B, and D. Genetic analysis in wheat is complicated, as most genes are present in triplicated sets located in the same chromosomal regions of homoeologous chromosomes. The goal of this report was to use genomic information gathered from wheat–rice sequence comparison to develop genome-specific primer sets for five genes involved in starch biosynthesis. Intron locations in wheat were inferred through the alignment of wheat cDNA sequences with rice genomic sequence. Exon-anchored primers, which amplify across introns, allowed the sequencing of introns from the three genomes for each gene. Sequence variation within introns among the three wheat genomes provided the basis for genome-specific primer design. For three genes, ADP-glucose pyrophosphorylase (Agp-L), sucrose transporter (SUT), and waxy (Wx), genome-specific primer sets were developed for all three genomes. Genome-specific primers were developed for two of the three genomes for Agp-S and starch synthase I (SsI). Thus, 13 of 15 possible genome-specific primer sets were developed using this strategy. Seven genome-specific primer combinations were used to amplify alleles in hexaploid wheat lines for sequence comparison. Three single nucleotide polymorphisms (SNPs) were identified in a comparison of 5,093 bp among a minimum of ten wheat accessions. Two of these SNPs could be converted into cleaved amplified polymorphism sequence (CAPS) markers. Our results indicated that the design of genome-specific primer sets using intron-based sequence differences has a high probability of success, while the identification of polymorphism among alleles within a genome may be a challenge.     

Characterisation of type-I thionin loci from the A, B, D and R genomes of wheat and rye

 DNA sequences encoding type-I thionins were isolated from Triticum aestivum L. cv ‘Chinese Spring’ using PCR with consensus primers. Blunt-end cloning, sequencing and PCR-based chromosome assignment of these fragments uncovered the three orthologous sequences corresponding to the single-copy genes at the Pur-1 loci on each of the group-1 chromosomes. Comparison with two previously published cDNA sequences revealed the presence of two introns that contain most of the polymorphic nucleotide sites. The observed orthologous DNA sequence variation among Pur-1 loci, encoded by each of the A, B and D genomes, enabled us to establish interlocus relationships and to construct locus-specific primer sets. Analogously, the Pur-R1 sequence from rye was isolated, and a locus-specific primer pair was constructed as well. Hence, four locus-specific primer sets are now available as molecular markers for the homoeologous 1AL, 1BL, 1DL and 1RL chromosome arms. Amplification from several diploid and tetraploid wheat species showed that the primers can be used as molecular tools for studying wheat phylogeny. Received: 30 January 1997 / Accepted: 23 June 1997     

Multiple origins of fungal group I introns located in the same position of nuclear SSU rRNA gene

The archiascomycetous fungus Protomyces pachydermus has two group I introns within the nuclear small subunit (nSSU) rRNA gene. One of these introns has an internal open reading frame (ORF) that encodes a predicted protein of 228 amino acid residues. On the other hand, Protomyces macrosporus has two group I introns that insert at the same positions as P. pachydermus, which have no ORF. Each alignment was constructed with Protomyces group I introns located in the same position and other introns retrieved by the BLAST Search. Each phylogenetic tree based on the alignment shows that Protomyces introns are monophyletic but the relationships among fungal introns do not reflect on the fungal phylogeny. Therefore, it is suggested that two different horizontal transfers of group I introns occurred at the early stage of Protomyces species diversification. Received: 11 June 1997 / Accepted: 2 September 1997     

Sixteen novel microsatellite markers developed for Dendrocalamus sinicus (Poaceae), the strongest woody bamboo in the world

? Premise of the study: Microsatellite primers were developed in the strongest and most economically important bamboo species, Dendrocalamus sinicus, to investigate its genetic variability. ? Methods and Results: Using the Fast Isolation by AFLP of Sequences COntaining Repeats (FIASCO) protocol, 16 polymorphic primer sets were identified within 24 individuals from two geographic locations. The number of alleles per locus ranged from two to four, with a mean of 2.6. The observed and expected heterozygosities varied from 0 to 1.000 and from 0.311 to 0.754, respectively. The cross-amplification of the 16 primer pairs was tested in four other Dendrocalamus species, and nine to 13 (56.3-81.3%) primer pairs were successfully amplified. ? Conclusions: These markers will be useful for studies on the genetic diversity of D. sinicus, which may facilitate conservation of this species.     

Nuclear DNA-based markers for plant evolutionary biology

While DNA-based markers can provide a wealth of information for the study of plant evolutionary biology, progress is limited by the lack of primers available for PCR. To overcome this limitation, we outline a protocol for developing oligonucleotide primers targeting regions of low copy-number nuclear genes. This protocol is intended to lead to universally useful primer sets. To test our approach, we designed eight primer sets and tested their abilities to amplify targets from representatives of each dicot and one monocot subclass. Five of the eight primer sets amplified targets from at least five of the seven taxa and thus exhibited broad taxonomic usefulness; the remaining primers were rather specific, however, and amplified targets from at most three taxa. In only one primer-taxon combination was a complex multiple-banded amplification produced. Overall, the protocol outlined proved quite useful at identifying broadly applicable primers targeted to low copy-number nuclear genes. Wider application of this approach should be effective at greatly increasing the amount of genetic information available for a diversity of plant nuclear genomes.     

Molecular phylogeny of New World sheath-tailed bats (Emballonuridae: Diclidurini) based on loci from the four genetic transmission systems in mammals

Multiple unlinked loci are surveyed in a methodological approach for mammalian systematics that uses genes from the four pathways of genetic transmission: mitochondrial, autosomal, and X and Y sex chromosomes. Each of these components has different properties, such as effective population size, mutation rate, and recombination, that result in a robust hypothesis of evolutionary history. The utility of this experimental design is tested with bats in the family Emballonuridae and the hypothesis that the New World taxa are monophyletic. Parsimony and Bayesian analyses of the individual data sets give generally congruent topologies with high bootstrap proportions and posterior probabilities for monophyletic clades representing species and genera. The mitochondrial gene has significantly faster rates of substitution, higher levels of homoplasy, and a greater degree of saturation than the nuclear genes that contributed to the loss of phylogenetic signal at deeper branches of the tree. However, there is better resolution and support for the more slowly evolving nuclear introns including a New World clade, indicating a single origin of emballonurid bats in the Neotropics (tribe Diclidurini). One novel subtribe has a hard basal polytomy that is unresolved for all of the nuclear partitions, suggesting a rapid burst of evolution during the diversification of genera.  © 2008 The Linnean Society of London, Biological Journal of the Linnean Society , 2008, 93 , 189–209.     

Gene-specific universal mammalian sequence-tagged sites: Application to the canine genome

We are developing a genetic map of the dog based partly upon markers contained within known genes. In order to facilitate the development of these markers, we have used polymerase chain reaction (PCR) primers designed to conserved regions of genes that have been sequenced in at least two species. We have refined the method for designing primers to maximize the number that produce successful amplifications across as many mammalian species as possible. We report the development of primer sets for 11 loci in detail:CFTR, COL10A1, CSFIR, CYP1A1, DCN1, FES, GHR, GLB1, PKLR, PVALB, andRB1. We also report an additional 75 primer sets in the appendices. The PCR products were sequenced to show that the primers amplify the expected canine genes. These primer sets thus define a class of gene-specific sequence-tagged sites (STSs). There are a number of uses for these STSs, including the rapid development of various linkage tools and the rapid testing of genomic and cDNA libraries for the presence of their corresponding genes. Six of the eleven gene targets reported in detail have been proposed to serve as “anchored reference loci” for the development of mammalian genetic maps [O'Brien, S. J.,et al., Nat. Genet. 3:103, 1993]. The primer sets should cover a significant portion of the canine genome for the development of a linkage map. In order to determine how useful these primer sets would be for the other genome projects, we tested the 11 primer sets on the DNA from species representing five mammalian orders. Eighty-four percent of the gene-species combinations amplified successfully. We have named these primer sets “universal mammalian sequence-tagged sites” because they should be useful for many mammalian genome projects.     

Development of Type I Genetic Markers from Expressed Sequence Tags in Highly Polymorphic Species

Expressed sequence tag (EST) databases provide a primary source of nuclear DNA sequences for genetic marker development in non-model organisms. To date, the process has been relatively inefficient for several reasons: 1) priming site polymorphism in the template leads to inferior or erratic amplification; 2) introns in the target amplicon are too large and/or numerous to allow effective amplification under standard screening conditions; and 3) at least occasionally, a PCR primer straddles an exon–intron junction and is unable to bind to genomic DNA template. The first is only a minor issue for species or strains with low heterozygosity but becomes a significant problem for species with high genomic variation, such as marine organisms with extremely large effective population sizes. Problems arising from unanticipated introns are unavoidable but are most pronounced in intron-rich species, such as vertebrates and lophotrochozoans. We present an approach to marker development in the Pacific oyster Crassostrea gigas, a highly polymorphic and intron-rich species, which minimizes these problems, and should be applicable to other non-model species for which EST databases are available. Placement of PCR primers in the 3′ end of coding sequence and 3′ UTR improved PCR success rate from 51% to 97%. Almost all (37 of 39) markers developed for the Pacific oyster were polymorphic in a small test panel of wild and domesticated oysters.     

Transfer of sequence tagged site PCR markers between wheat and barley.

Transfer of mapping information between related species has facilitated the development of restriction fragment length polymorphism (RFLP) maps in the cereals. Sequence tagged site (STS) primer sets for use in the polymerase chain reaction may be developed from mapped RFLP clones. For this study, we mapped 97 STS primer sets to chromosomes in wheat and barley to determine the potential transferability of the primer sets and the degree of correspondence between RFLP and STS locations. STS products mapped to the same chromosome group in wheat and barley 75% of the time. RFLP location predicted STS location 69% of the time in wheat and 56% of the time in barley. Southern hybridizations showed that most primer sets amplified sequences homologous to the RFLP clone, although additional sequences were often amplified that did not hybridize to the RFLP clone. Nontarget sequences were often amplified when primer sets were transferred across species. In general, results suggest a good probability of success in transferring STSs between wheat and barley, and that RFLP location can be used to predict STS location. However, transferability of STSs cannot be assumed, suggesting a need for recombinational mapping of STS markers in each species as new primer sets are developed. Key words : sequence tagged sites, PCR, wheat, barley.     

Development of Rapidly Evolving Intron Markers to Estimate Multilocus Species Trees of Rodents

One of the major challenges in the analysis of closely related species, speciation and phylogeography is the identification of variable sequence markers that allow the determination of genealogical relationships in multiple genomic regions using coalescent and species tree approaches. Rodent species represent nearly half of the mammalian diversity, but so far no systematic study has been carried out to detect suitable informative markers for this group. Here, we used a bioinformatic pipeline to extract intron sequences from rodent genomes available in databases and applied a series of filters that allowed the identification of 208 introns that adequately fulfilled several criteria for these studies. The main required characteristics of the introns were that they had the maximum possible mutation rates, that they were part of single-copy genes, that they had an appropriate sequence length for amplification, and that they were flanked by exons with suitable regions for primer design. In addition, in order to determine the validity of this approach, we chose ten of these introns for primer design and tested them in a panel of eleven rodent species belonging to different representative families. We show that all these introns can be amplified in the majority of species and that, overall, 79% of the amplifications worked with minimum optimization of the annealing temperature. In addition, we confirmed for a pair of sister species the relatively high level of sequence divergence of these introns. Therefore, we provide here a set of adequate intron markers that can be applied to different species of Rodentia for their use in studies that require significant sequence variability.     

Refining DNA Barcoding Coupled High Resolution Melting for Discrimination of 12 Closely Related Croton Species

DNA barcoding coupled high resolution melting (Bar-HRM) is an emerging method for species discrimination based on DNA dissociation kinetics. The aim of this work was to evaluate the suitability of different primer sets, derived from selected DNA regions, for Bar-HRM analysis of species in Croton (Euphorbiaceae), one of the largest genera of plants with over 1,200 species. Seven primer pairs were evaluated (matK, rbcL1, rbcL2, rbcL3, rpoC, trnL and ITS1) from four plastid regions, matK, rbcL, rpoC, and trnL, and the nuclear ribosomal marker ITS1. The primer pair derived from the ITS1 region was the single most effective region for the identification of the tested species, whereas the rbcL1 primer pair gave the lowest resolution. It was observed that the ITS1 barcode was the most useful DNA barcoding region overall for species discrimination out of all of the regions and primers assessed. Our Bar-HRM results here also provide further support for the hypothesis that both sequence and base composition affect DNA duplex stability.     

PCR primers for an aldolase-B intron in acanthopterygian fishes

Background  

Nuclear DNA sequences provide genetic information that complements studies using mitochondrial DNA. Some 'universal' primer sets have been developed that target introns within protein-coding loci, but many simultaneously amplify introns from paralogous loci. Refining existing primer sets to target a single locus could circumvent this problem.     

Nucleotide Sequences of DNA Fragments of Encephalitozoon cuniculi Amplified by Polymerase Chain Reaction with Primers Regarded as Specific for Echinococcus

Encephalitozoon -like spores were separated from a human echinococcal liver lesion, which was caused by Echinococcus multilocularis. They were found to fall into the species Encephalitozoon cuniculi , which was shown to have En. cunniculi specific DNA by way of polymerase chain reaction (PCR). We also used PCR to genetically discriminate between the En. cuniculi spores and the Ec. multilocularis larvae. Two primer sets, known to be specific for Echinococcus , were examined. These primers were expected to work normally when the two quite different DNA preparations were tested as templates, i.e. only Echinococcus DNA could give a positive signal in the PCR tests. However, it was found that the two Echinococcus -specific primer sets could amplify not only EC. multilocularis DNA, but also En. cuniculi spore DNA. We then tried to determine the order of nucleotides in the Echinococcus -specific primers-amplified En. cuniculi PCR products and compared the determined sequences with those of Ec. multilocularis. The results clearly indicated that sequencing made little difference between En. cuniculi and Ec. multilocularis.     

Development of PCR primer sets for intron 1 of the low-copy gene LEAFY in Davalliaceae

? Premise of the study: Primers were designed for amplifying intron 1 of the single-copy nuclear LEAFY gene for species of Davalliaceae. ? Methods and Results: New primer sets were designed and successfully amplified for intron 1 of the LEAFY gene in 13 species representing the five genera of Davalliaceae. The orthology of these sequences was further confirmed by phylogenetic analyses. Site variation in LEAFY intron 1 sequences across genera of the Davalliaceae and among accessions of the Humata repens complex were 18% and 8%, respectively. Such variation was greater than that for the cpDNA atpB-rbcL intergenic spacer region across the same taxa and accessions. ? Conclusions: Using our newly designed primers, intron 1 of the LEAFY gene could be amplified for all species tested. In addition, this single-copy, biparentally inherited, and quickly evolving region showed considerable potential for addressing infraspecific-level questions.     

Properties of sequence-tagged-site primer sets influencing repeatability.

The polymerase chain reaction (PCR) has become a standard procedure in plant genetics, and is the basis for many emerging genomics approaches to mapping and gene identification. One advantage of PCR is that sequence information for primer sets can be exchanged between laboratories, obviating the need for exchange and maintenance of biological materials. Repeatability of primer sets, whereby the same products are amplified in different laboratories using the same primer set, is important to successful exchange and utilization. We have developed several hundred sequence-tagged site (STS) primer sets for wheat and barley. The ability of the primer sets to generate reproducible amplifications in other laboratories has been variable. We wished to empirically determine the properties of the primer sets that most influenced repeatability. A total of 96 primer sets were tested with four genomic DNA samples on each of four thermocyclers. All major bands were repeatable across all four thermocylers for approximately 50% of the primer sets. Characteristics most often associated with differences in repeatability included primer GC content and 3'-end stability of the primers. The propensity for primer-dimer formation was not a factor in repeatability. Our results provide empirical direction for the development of repeatable primer sets.     

Identification of group I introns within the SSU rDNA gene in species of Ceratocystiopsis and related taxa

During a recent phylogenetic study, group I introns were noted that interrupt the nuclear small subunit ribosomal RNA (SSU rDNA) gene in species of Ceratocystiopsis. Group I introns were found to be inserted at the following rDNA positions: S943, S989, and S1199. The introns have been characterized and phylogenetic analysis of the host gene and the corresponding intron data suggest that for S943 vertical transfer and frequent loss appear to be the most parsimonious explanation for the distribution of nuclear SSU rDNA introns among species of Ceratocystiopsis. The SSU rDNA data do suggest that a recent proposal of segregating the genus Ophiostoma sensu lato into Ophiostoma sensu stricto, Grosmannia, and Ceratocystiopsis has some merit but may need further amendments, as the SSU rDNA suggests that Ophiostoma s. str. may now represent a paraphyletic grouping.