SSCP-SNP-based conserved ortholog set (COS) markers for comparative genomics in cassava (Manihot esculenta crantz)

The ever increasing body of information on genomics and functional genomics from model plants, and new tools of comparative genomics, provide an opportunity to accelerate the development of molecular markers for increasing the efficiency of breeding of lesser studied crops, so-called “orphan crops.” Conserved ortholog set (COS) markers represent orthologous genes in widely divergent plant species, and are currently the principal tool of choice for comparative genomics. EST sequences of 3 drought tolerance related genes—chalcone synthase (CHS), dihydroflavonol-4-reductase (DHRF) and drought responsive element binding factor 1 (DREB-1) fromMusa sp—were used to identify cassava EST homologs that were then scanned against the Arabidopsis genome database to identify them as COS markers. The CHS and DHRF ESTs were demonstrated to be COS markers, while the DREB EST was shown to belong to a gene family. The three genes were evaluated as single strand conformation polymorphism—single nucleotide polymorphism (SSCP-SNP) markers in the parents of an F1 mapping population and subsequently in the progenies. The DHRF COS marker mapped to linkage group R of the female-derived map while the DREB-1 EST mapped at an end of the male-derived linkage group K. The CHS COS marker could not be mapped because it was not polymorphic in the parents of the mapping population. These new marker tools should accelerate the development of markers associated with genes controlling traits of agronomic interest via the candidate gene loci (CGL) QTL-mapping approach.     

Heterogeneity of intron presence/absence in Olifantiella sp. (Bacillariophyta) contributes to the understanding of intron loss

Although hypotheses have been proposed and developed to interpret the origins and functions of introns, substantial controversies remain about the mechanism of intron evolution. The availability of introns in the intermediate state is quite helpful for resolving this debate. In this study, a new strain of diatom (denominated as DB21‐1) was isolated and identified as Olifantiella sp., which possesses multiple types of 18S rDNAs (obtained from genomic DNA; lengths ranged from 2,056 bp to 2,988 bp). Based on alignments between 18S rDNAs and 18S rRNA (obtained from cDNA; 1,783 bp), seven intron insertion sites (IISs) located in the 18S rDNA were identified, each of which displayed the polymorphism of intron presence/absence. Specific primers around each IIS were designed to amplify the introns and the results indicated that introns in the same IIS varied in lengths, while terminal sequences were conserved. Our study showed that the process of intron loss happens via a series of successive steps, and each step could derive corresponding introns under intermediate states. Moreover, these results indicate that the mechanism of genomic deletion that occurs at DNA level can also lead to exact intron loss.     

EvolMarkers: a database for mining exon and intron markers for evolution, ecology and conservation studies

Recent innovations in next-generation sequencing have lowered the cost of genome projects. Nevertheless, sequencing entire genomes for all representatives in a study remains expensive and unnecessary for most studies in ecology, evolution and conservation. It is still more cost-effective and efficient to target and sequence single-copy nuclear gene markers for such studies. Many tools have been developed for identifying nuclear markers, but most of these have focused on particular taxonomic groups. We have built a searchable database, EvolMarkers, for developing single-copy coding sequence (CDS) and exon-primed-intron-crossing (EPIC) markers that is designed to work across a broad range of phylogenetic divergences. The database is made up of single-copy CDS derived from BLAST searches of a variety of metazoan genomes. Users can search the database for different types of markers (CDS or EPIC) that are common to different sets of input species with different divergence characteristics. EvolMarkers can be applied to any taxonomic group for which genome data are available for two or more species. We included 82 genomes in the first version of EvolMarkers and have found the methods to be effective across Placozoa, Cnidaria, Arthropod, Nematoda, Annelida, Mollusca, Echinodermata, Hemichordata, Chordata and plants. We demonstrate the effectiveness of searching for CDS markers within annelids and show how to find potentially useful intronic markers within the lizard Anolis.     

Exploitation of conserved intron scanning as a tool for molecular marker development in the Saccharum complex

This study investigated the potential of the conserved intron scanning approach to develop molecular markers for genes involved in lignin biosynthesis among members of the Saccharum complex and a distantly related Imperata species. Five intron-flanking primer sets targeting genes encoding five lignin biosynthetic enzymes??phenylalanine ammonia lyase (PAL), 4-coumarate coenzyme A ligase (4-CL), caffeoyl-CoA 3-O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR) and peroxidase (POX)??were designed based on the sequence analysis between sugarcane expressed sequence tags (ESTs) and Sorghum bicolor orthologs. Nucleotide sequence analyses of the amplicons of PAL, 4-CL and CCoAOMT orthologs revealed the presence of single nucleotide polymorphisms (SNPs) and insertions?Cdeletions (INDELs) in the target introns as well as (CT)-simple sequence repeats (SSRs) in CCoAOMT orthologs. The SSR marker screening against fifty-nine accessions of the Saccharum complex and an Imperata species confirmed that the identified SSR markers were highly polymorphic among Saccharum and Erianthus species. PCR-restriction fragment length polymorphism (PCR?CRFLP) and cleaved amplified polymorphic sequence (CAPS) marker screening of 4-CL and CCoAOMT orthologs developed genus-specific molecular markers that confirmed the intergeneric hybridization status of Saccharum Fiji hybrids. The current study showed that the conserved intron scanning strategy is applicable to multiple copy genes of polyploid monocots. The conserved intron scanning approach provides a novel way of investigating DNA polymorphisms among species within the Saccharum complex and has the potential to help in the development of marker-assisted selection in intergeneric hybrids.     

A general pipeline for the development of anchor markers for comparative genomics in plants

Background

The massive scale of microarray derived gene expression data allows for a global view of cellular function. Thus far, comparative studies of gene expression between species have been based on the level of expression of the gene across corresponding tissues, or on the co-expression of the gene with another gene.

Results

To compare gene expression between distant species on a global scale, we introduce the "expression context". The expression context of a gene is based on the co-expression with all other genes that have unambiguous counterparts in both genomes. Employing this new measure, we show 1) that the expression context is largely conserved between orthologs, and 2) that sequence identity shows little correlation with expression context conservation after gene duplication and speciation.

Conclusion

This means that the degree of sequence identity has a limited predictive quality for differential expression context conservation between orthologs, and thus presumably also for other facets of gene function.     

Lack of trnL intron in the thermoacidophilic red alga Galdieria sulphuraria (Galdieri) Merola

Abstract

The plastid trnC‐trnL(UAA)‐ilvH region from Galdieria sulphuraria was cloned and sequenced with the aim of verifying the absence of the trnL intron. The sequence alignment shows both the absence of a trnL intron and the colinearity of the whole region of the plastidial DNA of G. sulphuraria with that of the other thermoacidophilic red algae.     

The SSU rDNA coding region of a filose amoeba contains a group I intron lacking the universally conserved G at the 3'-terminus

We sequenced small subunit ribosomal DNA (rDNA) PCR-fragments of sizes 2.3 kb and 2.9 kb isolated from a culture of the red alga, Porphyra spiralis var. spiralis. Phylogenetic analysis of the 2.3-kb fragment showed that it encoded the sequence of a contaminant filose amoeba. The Nuclearia-like amoeba (named strain N-Por) was identified with scanning electron microscopy. Its rDNA sequence was positioned with strong bootstrap support within a diverse protist assemblage that includes filose amoebae, chlorarachniophytes, cercomonads, and Plasmodiophora brassicae. The rDNA of N-Por contained a group I intron at the conserved 943 position that remarkably, had a U at the 3'-terminus rather than the universally conserved G.     

Sequence-tagged-site (STS) markers of arbitrary genes: the utility of black spruce-derived STS primers in other conifers

 Sequence-tagged-site primers, previously developed based upon black spruce (Picea mariana) cDNA sequences, were tested for their ability to direct specific amplification in two individuals of each of 12 additional conifer species. Nearly all (95–97%) of the primers functioned well in congeneric trials, while a lower proportion (21–33%) scored positively in other Pinaceae genera. Outside of the Pinaceae, amplification of homologous products was not achieved. Products from the various species often differed in size from their homologs in black spruce. In one case a large difference in size was due to the lack of an intron in a jack pine product while in several other cases the differences were due to the presence or absence of large direct repeats in the DNA sequences. Length polymorphism was occasionally evident between the two individuals examined of a given species. We investigated marker polymorphism in detail in a panel of 15 white spruce (Picea glauca) trees. Allelic segregation among haploid megagametophytes was revealed directly at 16 loci by standard agarose-gel electrophoresis without any additional manipulation of amplification products. Polymorphisms observed at 12 of these loci were exclusively co-dominant. For this subset of 12 loci, the average number of alleles was 3.2 and the average observed heterozygosity was 0.37. Received: 10 April 1998 / Accepted: 22 April 1998     

Construction of a linkage map of Lentinula edodes (shiitake) with the HEGS (high-efficiency genome scanning) system: use of versatile AFLP and PCR-based gene markers

An improved linkage map of Lentinula edodes (shiitake) was constructed with an HEGS (high-efficiency genome scanning) system. Two hundred twenty-one HEGS-derived amplified fragment length polymorphism (AFLP-H) markers and 21 gene markers were developed and combined with 203 previously developed sequencer-derived AFLP markers (AFLP-S markers) and 3 mating factor loci (A, Bα, and Bβ) to construct a comprehensive linkage analysis. As a result, a novel linkage map with 166 markers including 2 mating factors (A and B), 10 HEGS-derived gene markers, 72 AFLP-H markers, and 82 AFLP-S markers was obtained. Of the total 448 markers, 273 could not be located on a linear map and thus were assigned to linkage groups as accessory markers. The map covers a total length of 1398.4 centimorgans (cM) with an average marker interval distance of 8.4 cM. The map consists of 11 linkage groups (LGs) in agreement with our previous map, and 7 LGs among them were found to contain branched linkages, which may be the result of reciprocal translocations representing dynamic reorganization of the shiitake genome. The previously reported linkage map was improved in terms of number of markers, marker density, linear order of markers, and total map length. Contribution no. 384 of the Tottori Mycological Institute     

Novel microsatellite markers for the saltmarsh sharp-tailed sparrow, Ammodramus caudacutus (Aves: Passeriformes)

We have developed eight high-quality microsatellite DNA loci for the saltmarsh sharp-tailed sparrow and one additional locus with evidence of null alleles. In a sample of 250-350 individuals, the average number of alleles per locus was 14.7 and average observed heterozygosity was 0.80. These loci were tested in three additional species of emberizid sparrows, indicating that more than half of the loci could be useful in other sparrows.     

DNA sequences of tobacco chloroplast genes for tRNASer (GGA), tRNAThr (UGU), tRNALeu (UAA), tRNAPhe (GAA): the tRNALeu gene contains a 503 bp intron

Summary The location and nucleotide sequence of tobacco chloroplast genes for tRNA^Ser (GGA), tRNA^Thr (UGU), tRNA^Leu (UAA) and tRNA^Phe (GAA) (trnS-GGA, trnT-UGU, trnL-UAA and trnF-GAA, respectively) have been determined. These genes are located in the 10 kbp BamHI fragment which lies in the middle of the large single-copy region of the chloroplast DNA. The gene order is trnS-trnT-trnL-trnF. The trnS, trnL and trnF are encoded on the same strand while the trnT on the opposite strand. The trnL contains a 503 bp intron like maize and broad bean trnL-UAAs.     

Discovery of a group I intron in the SSU rDNA of Ploeotia costata (Euglenozoa)

The gene coding for the small ribosomal subunit RNA of Ploeotia costata contains an actively splicing group I intron (Pco.S516) which is unique among euglenozoans. Secondary structure predictions indicate that paired segments P1-P10 as well as several conserved elements typical of group I introns and of subclass IC1 in particular are present. Phylogenetic analyses of SSU rDNA sequences demonstrate a well-supported placement of Ploeotia costata within the Euglenozoa; whereas, analyses of intron data sets uncover a close phylogenetic relation of Pco.S516 to S-516 introns from Acanthamoeba, Aureoumbra lagunensis (Stramenopila) and red algae of the order Bangiales. Discrepancies between SSU rDNA and intron phylogenies suggest horizontal spread of the group I intron. Monophyly of IC1 516 introns from Ploeotia costata, A. lagunensis and rhodophytes is supported by a unique secondary structure element: helix P5b possesses an insertion of 19 nt length with a highly conserved tetraloop which is supposed to take part in tertiary interactions. Neither functional nor degenerated ORFs coding for homing endonucleases can be identified in Pco.S516. Nevertheless, degenerated ORFs with His-Cys box motifs in closely related intron sequences indicate that homing may have occurred during evolution of the investigated intron group.     

Large scale development of selectively amplified microsatellite polymorphic loci (SAMPL) markers in spruce (Picea)

The spruce (Picea) species are ecologically and economically important in Canada. Highly informative markers with high multiplex ratios are needed to assist spruce genomics, genetics, and breeding programs. Selectively amplified microsatellite polymorphic loci (SAMPL) markers are highly suitable for these programs. We have developed, optimized, and characterized a set of 10 new SAMPL primers in combination with 16 MseI primers and resolved a large number of polymorphic SAMPL markers in spruce. The SAMPL primers were designed from the compound microsatellite repeats found in Norway spruce (Picea abies) and white spruce (Picea glauca). A total of 6313 polymorphic SAMPL makers were produced by 160 SAMPL–MseI primers combinations in eight progeny of a spruce mapping population.     

Eight polymorphic microsatellite loci markers for the barnacle Balanus amphitrite (syn. Amphibalanus amphitrite) Darwin 1854

Balanus amphitrite is a widespread species of barnacle. It is frequently studied, and of great importance to the marine coatings industry due to its significant abundance as a fouling organism on commercial shipping. We isolated and characterized eight highly polymorphic microsatellite loci, to aid in the determination of population genetic structure within this species. All loci showed considerable genetic variation with the number of alleles ranging from two to 14. Expected heterozygosity ranged from 0.74 to 0.98.     

New microsatellite markers for the snow crab Chionoecetes opilio (Brachyura: Majidae)

Nine novel microsatellite loci were isolated from Chionoecetes opilio by screening an enriched genomic library using nonradioactive polymerase chain reaction techniques, and the polymorphisms were examined to estimate genetic variability. The genetic variabilities varied depending on the locus. All loci were found to be polymorphic with an average of 9.7 alleles per locus (range 3–25). The observed and expected heterozygosities ranged from 0.80 to 0.98 and from 0.56 to 0.95, respectively. Five loci showed significant Hardy–Weinberg disequilibrium at the P < 0.05 level. The high variabilities revealed in this study suggest that these microsatellite loci should provide useful markers for genetic variation monitoring of C. opilio.     

The Princeton Protein Orthology Database (P-POD): a comparative genomics analysis tool for biologists

Many biological databases that provide comparative genomics information and tools are now available on the internet. While certainly quite useful, to our knowledge none of the existing databases combine results from multiple comparative genomics methods with manually curated information from the literature. Here we describe the Princeton Protein Orthology Database (P-POD, http://ortholog.princeton.edu), a user-friendly database system that allows users to find and visualize the phylogenetic relationships among predicted orthologs (based on the OrthoMCL method) to a query gene from any of eight eukaryotic organisms, and to see the orthologs in a wider evolutionary context (based on the Jaccard clustering method). In addition to the phylogenetic information, the database contains experimental results manually collected from the literature that can be compared to the computational analyses, as well as links to relevant human disease and gene information via the OMIM, model organism, and sequence databases. Our aim is for the P-POD resource to be extremely useful to typical experimental biologists wanting to learn more about the evolutionary context of their favorite genes. P-POD is based on the commonly used Generic Model Organism Database (GMOD) schema and can be downloaded in its entirety for installation on one's own system. Thus, bioinformaticians and software developers may also find P-POD useful because they can use the P-POD database infrastructure when developing their own comparative genomics resources and database tools.     

A sequence-based model accounts largely for the relationship of intron positions to protein structural features

Claims of intron-structure correlations have played a major role in debates surrounding split gene origins. In the formative (as opposed to disruptive or "insertional") model of split gene origins, introns represent the scars of chimaeric gene assembly. When analyzed retrospectively, formative introns should tend to fall between modular units, if such units exist, or at least to exhibit a preference for sites favorable to chimaera formation. However, there is another possible source of preferences: under a disruptive model of split gene origins, fortuitous intron-structure correlations may arise because the gain of introns is biased with respect to flanking nucleotide sequences. To investigate the extent to which a sequence-biased intron gain model may account for the present-day distribution of introns, data on over 10,000 introns in eukaryotic protein-coding genes were integrated with structural data from a set of 1,851 nonredundant protein chains. The positions of introns with respect to secondary structures, solvent accessibility, and so-called "modules" were evaluated relative to the expectations of a null model, a disruptive model based on amino acid frequencies at splice junctions, and a formative model defined relative to these. The null model can be excluded for most structural features and is highly improbable when intron sites are grouped by reading frame phase. Phase-dependent correlations with secondary structure and side-chain surface accessibility are particularly strong. However, these phase-dependent correlations are explained largely by the sequence-based disruptive model.     

Characterization of polymorphic microsatellite markers for the green turtle (Chelonia mydas)

We describe primers and polymerase chain reaction conditions to amplify 12 microsatellite loci from the green turtle (Chelonia mydas), including one dinucleotide, four trinucleotide and seven tetranucleotide loci. The primers were tested on 78 individuals from a Pacific population nesting in the Hawaiian Islands. The primer pairs developed in this study yielded an average of 8.33 alleles per locus (range of 3-15 alleles), an average observed heterozygosity of 0.668 (range 0.309-0.910), and an average polymorphic information content of 0.647 (range 0.287-0.894).     

Mitogenome rearrangement in the cold-water scleractinian coral Lophelia pertusa (Cnidaria, Anthozoa) involves a long-term evolving group I intron

Group I introns are genetic insertion elements that invade host genomes in a wide range of organisms. In metazoans, however, group I introns are extremely rare, so far only identified within mitogenomes of hexacorals and some sponges. We sequenced the complete mitogenome of the cold-water scleractinian coral Lophelia pertusa, the dominating deep sea reef-building coral species in the North Atlantic Ocean. The mitogenome (16,150 bp) has the same gene content but organized in a unique gene order compared to that of other known scleractinian corals. A complex group I intron (6460 bp) inserted in the ND5 gene (position 717) was found to host seven essential mitochondrial protein genes and one ribosomal RNA gene. Phylogenetic analysis supports a vertical inheritance pattern of the ND5-717 intron among hexacoral mitogenomes with no examples of intron loss. Structural assessments of the Lophelia intron revealed an unusual organization that lacks the universally conserved ωG at the 3′ end, as well as a highly compact RNA core structure with overlapping ribozyme and protein coding capacities. Based on phylogenetic and structural analyses we reconstructed the evolutionary history of ND5-717, from its ancestral protist origin, through intron loss in some early metazoan lineages, and into a compulsory feature with functional implications in hexacorals.     

A meta-analysis of cancer risk associated with the TP53 intron 3 duplication polymorphism (rs17878362): geographic and tumor-specific effects

We have performed a meta-analysis of cancer risk associated with the rs17878362 polymorphism of the TP53 suppressor gene (PIN3, (polymorphism in intron 3), 16 bp sequence insertion/duplication in intron 3), using a compilation of a total of 25 published studies with 10 786 cases and 11 760 controls. Homozygote carriers of the duplicated allele (A2A2) had a significantly increased cancer risk compared with A1A1 carriers (aggregated odds ratio (OR)=1.45, 95% confidence interval (CI)=1.22–1.74). However, there was no significant effect for the A1A2 heterozygotes (A1A2 versus A1A1 aggregated OR=1.08, 95% CI=0.99–1.18). No significant heterogeneity or publication bias was detected in the data set analysed. When comparing populations groups, increased cancer risk was associated with A2A2 carriage in Indian, Mediterranean and Northern Europe populations but not in the Caucasian population of the United States. Analysis by cancer site showed an increased risk for A2A2 carriers for breast and colorectal, but not for lung cancers. These results support that the A2A2 genotype of rs17878362 is associated with increased cancer risk, with population and tumour-specific effects.