Epigenome-wide profiling of DNA methylation in paired samples of adipose tissue and blood

Many epigenetic association studies have attempted to identify DNA methylation markers in blood that are able to mirror those in target tissues. Although some have suggested potential utility of surrogate epigenetic markers in blood, few studies have collected data to directly compare DNA methylation across tissues from the same individuals. Here, epigenomic data were collected from adipose tissue and blood in 143 subjects using Illumina HumanMethylation450 BeadChip array. The top axis of epigenome-wide variation differentiates adipose tissue from blood, which is confirmed internally using cross-validation and externally with independent data from the two tissues. We identified 1,285 discordant genes and 1,961 concordant genes between blood and adipose tissue. RNA expression data of the two classes of genes show consistent patterns with those observed in DNA methylation. The discordant genes are enriched in biological functions related to immune response, leukocyte activation or differentiation, and blood coagulation. We distinguish the CpG-specific correlation from the within-subject correlation and emphasize that the magnitude of within-subject correlation does not guarantee the utility of surrogate epigenetic markers. The study reinforces the critical role of DNA methylation in regulating gene expression and cellular phenotypes across tissues, and highlights the caveats of using methylation markers in blood to mirror the corresponding profile in the target tissue.     

Genes, Demography, and Life Span: The Contribution of Demographic Data in Genetic Studies on Aging and Longevity

In population studies on aging, the data on genetic markers are often collected for individuals from different age groups. The purpose of such studies is to identify, by comparison of the frequencies of selected genotypes, "longevity" or "frailty" genes in the oldest and in younger groups of individuals. To address questions about more-complicated aspects of genetic influence on longevity, additional information must be used. In this article, we show that the use of demographic information, together with data on genetic markers, allows us to calculate hazard rates, relative risks, and survival functions for respective genes or genotypes. New methods of combining genetic and demographic information are discussed. These methods are tested on simulated data and then are applied to the analysis of data on genetic markers for two haplogroups of human mtDNA. The approaches suggested in this article provide a powerful tool for analyzing the influence of candidate genes on longevity and survival. We also show how factors such as changes in the initial frequencies of candidate genes in subsequent cohorts, or secular trends in cohort mortality, may influence the results of an analysis.     

Comparison of 16S rRNA and protein-coding genes as molecular markers for assessing microbial diversity (Bacteria and Archaea) in ecosystems

PCR amplification of the rRNA gene is the most popular method for assessing microbial diversity. However, this molecular marker is often present in multiple copies in cells presenting, in addition, an intragenomic heterogeneity. In this context, housekeeping genes may be used as taxonomic markers for ecological studies. However, the efficiency of these protein-coding genes compared to 16S rRNA genes has not been tested on environmental data. For this purpose, five protein marker genes for which primer sets are available, were selected (rplB, pyrG, fusA, leuS and rpoB) and compared with 16S rRNA gene results from PCR amplification or metagenomic data from aquatic ecosystems. Analysis of the major groups found in these ecosystems, such as Actinobacteria, Bacteroides, Proteobacteria and Cyanobacteria, showed good agreement between the protein markers and the results given by 16S rRNA genes from metagenomic reads. However, with the markers it was possible to detect minor groups among the microbial assemblages, providing more details compared to 16S rRNA results from PCR amplification. In addition, the use of a set of protein markers made it possible to deduce a mean copy number of rRNA operons. This average estimate is essentially lower than the one estimated in sequenced genomes.     

The value of idiosyncratic markers and changes to conserved tRNA sequences from the mitochondrial genome of hard ticks (Acari: Ixodida: Ixodidae) for phylogenetic inference

Idiosyncratic markers are features of genes and genomes that are so unusual that it is unlikely that they evolved more than once in a lineage of organisms. Here we explore further the potential of idiosyncratic markers and changes to typically conserved tRNA sequences for phylogenetic inference. Hard ticks were chosen as the model group because their phylogeny has been studied extensively. Fifty-eight candidate markers from hard ticks (family Ixodidae) and 22 markers from the subfamily Rhipicephalinae sensu lato were mapped onto phylogenies of these groups. Two of the most interesting markers, features of the secondary structure of two different tRNAs, gave strong support to the hypothesis that species of the Prostriata (Ixodes spp.) are monophyletic. Previous analyses of genes and morphology did not strongly support this relationship, instead suggesting that the Prostriata is paraphyletic with respect to the Metastriata (the rest of the hard ticks). Parallel or convergent evolution was not found in the arrangements of mitochondrial genes in ticks nor were there any reversals to the ancestral arthropod character state. Many of the markers identified were phylogenetically informative, whereas others should be informative with study of additional taxa. Idiosyncratic markers and changes to typically conserved nucleotides in tRNAs that are phylogenetically informative were common in this data set, and thus these types of markers might be found in other organisms.     

Multiple genome alignments facilitate development of NPCL markers: a case study of tetrapod phylogeny focusing on the position of turtles

In recent years, the increasing availability of genomic resources has provided an opportunity to develop phylogenetic markers for phylogenomics. Efficient methods to search for candidate markers from the huge number of genes within genomic data are particularly needed in the era of phylogenomics. Here, rather than using the traditional approach of comparing genomes of two distantly related taxa to develop conserved primers, we take advantage of the multiple genome alignment resources from the the University of California-San Cruz Genome Browser and present a simple and straightforward bioinformatic approach to automatically screen for candidate nuclear protein-coding locus (NPCL) markers. We tested our protocol in tetrapods and successfully obtained 21 new NPCL markers with high success rates of polymerase chain reaction amplification (mostly over 80%) in 16 diverse tetrapod taxa. These 21 newly developed markers together with two reference genes (RAG1 and mitochondrial 12S-16S) are used to infer the higher level relationships of tetrapods, with emphasis on the debated position of turtles. Both maximum likelihood (ML) and Bayesian analyses on the concatenated data combining the 23 markers (21,137 bp) yield the same tree, with ML bootstrap values over 95% and Bayesian posterior probability equaling 1.0 for most nodes. Species tree estimation using the program BEST without data concatenation produces similar results. In all analyses, turtles are robustly recovered as the sister group of Archosauria (birds and crocodilians). The jackknife analysis on the concatenated data showed that the minimum sequence length needed to robustly resolve the position of turtles is 13-14 kb. Based on the large 23-gene data set and the well-resolved tree, we also estimated evolutionary timescales for tetrapods with the popular Bayesian method MultiDivTime. Most of the estimated ages among tetrapods are similar to the average estimates of the previous dating studies summarized by the book The Timetree of Life.     

Search for genetic markers of climatic adaptation in populations of North Eurasia

Genetic diversity of native populations of North Eurasia is investigated using a panel of genetic markers of candidate genes for cold climate adaptation. A high level of within- and between-population variability is detected. Comparative analysis of data on North Eurasian populations combined with data on worldwide populations from the 1000 Genomes and HDGP projects reveals correlations of genetic diversity in candidate genes for cold climate adaptation with key climate parameters, as well as the increase of genetic diversity in markers of this group of genes with the increase of latitude, that is, as modern humans migrated out of Africa. Using the method of searching for extreme empirical values of the coefficient of genetic diversity, signals of directional selection for markers of six genes adaptive to cold (MYOF, LONP2, IFNL4, MKL1, SLC2A12, and CPT1A) are found. The data are discussed in framework of the hypothesis of decanalization of genome–phenome relationships under the pressure of natural selection during human settlement throughout the world.     

Multilocus sequence typing (MLST) for the infra-generic taxonomic classification of entomopathogenic Rickettsiella bacteria

The genus Rickettsiella comprises intracellular bacterial pathogens of a wide range of arthropods that are currently classified in four recognized species and numerous further pathotypes. However, both the delineation of and the synonymization of pathotypes with species are highly problematic. In the sequel of a previous phylogenomic study at the supra-generic level, nine selected genes - the 16S and 23S rRNA genes and the protein-encoding genes dnaG, ftsY, gidA, ksgA, rpoB, rpsA, and sucB - were evaluated for their potential as markers for the generic and infra-generic taxonomic classification of Rickettsiella-like bacteria. A methodological approach combining phylogenetic reconstruction with likelihood-based significance testing was employed on the basis of sequence data from the species Rickettsiella grylli and Rickettsiella popilliae, pathotypes 'Rickettsiella melolonthae' and 'Rickettsiella tipulae'. This study provides the first multilocus sequence typing (MLST) data for the genus Rickettsiella and identifies two new genetic markers, gidA and sucB, for the infra-generic classification within this taxon. In particular, aforesaid genes were found more reliable and informative markers than the corresponding 16S rRNA-encoding sequences that failed to produce strictly significant infra-generic taxonomic assignments. However, gidA- and sucB-based phylogenies were consistent with the currently accepted view of species delineation and species-pathotype synonymization within the genus Rickettsiella.     

Detecting susceptibility genes in case-control studies using set association

Complex diseases are generally caused by intricate interactions of multiple genes and environmental factors. Most available linkage and association methods are developed to identify individual susceptibility genes assuming a simple disease model blind to any possible gene - gene and gene - environmental interactions. We used a set association method that uses single-nucleotide polymorphism markers to locate genetic variation responsible for complex diseases in which multiple genes are involved. Here we extended the set association method from bi-allelic to multiallelic markers. In addition, we studied the type I error rates and power for both approaches using simulations based on the coalescent process. Both bi-allelic set association (BSA) and multiallelic set association (MSA) tests have the correct type I error rates. In addition, BSA and MSA can have more power than individual marker analysis when multiple genes are involved in a complex disease. We applied the MSA approach to the simulated data sets from Genetic Analysis Workshop 13. High cholesterol level was used as the definitive phenotype for a disease. MSA failed to detect markers with significant linkage disequilibrium with genes responsible for cholesterol level. This is due to the wide spacing between the markers and the lack of association between the marker loci and the simulated phenotype.     

Association mapping of complex trait loci with context-dependent effects and unknown context variable

A novel method for Bayesian analysis of genetic heterogeneity and multilocus association in random population samples is presented. The method is valid for quantitative and binary traits as well as for multiallelic markers. In the method, individuals are stochastically assigned into two etiological groups that can have both their own, and possibly different, subsets of trait-associated (disease-predisposing) loci or alleles. The method is favorable especially in situations when etiological models are stratified by the factors that are unknown or went unmeasured, that is, if genetic heterogeneity is due to, for example, unknown genes x environment or genes x gene interactions. Additionally, a heterogeneity structure for the phenotype does not need to follow the structure of the general population; it can have a distinct selection history. The performance of the method is illustrated with simulated example of genes x environment interaction (quantitative trait with loosely linked markers) and compared to the results of single-group analysis in the presence of missing data. Additionally, example analyses with previously analyzed cystic fibrosis and type 2 diabetes data sets (binary traits with closely linked markers) are presented. The implementation (written in WinBUGS) is freely available for research purposes from http://www.rni.helsinki.fi/ approximately mjs/.     

Detailed comparative gene map of rat chromosome 1 with mouse and human genomes and physical mapping of an evolutionary chromosomal breakpoint

We report the localization of 92 new gene-based markers assigned to rat chromosome 1 by linkage or radiation hybrid mapping. The markers were chosen to enrich gene mapping data in a region of the rat chromosome known to contain several of the principal quantitative trait loci in rodent models of human multifactorial disease. The composite map reported here provides map information on a total of 139 known genes, including 80 that have been localized in mouse and 109 that have been localized in human, and integrates the gene-based markers with anonymous microsatellites. The evolutionary breakpoints identifying 16 segments that are homologous regions in the human genome are defined. These data will facilitate genetic and comparative mapping studies and identification of novel candidate genes for the quantitative trait loci that have been localized to the region.     

Meiotic linkage mapping of 52 genes onto the canine map does not identify significant levels of microrearrangement

In an effort to extend our understanding of the evolutionary relationship between the canine and human genomes, we have developed and positioned 52 new gene-associated polymorphic markers on the canine meiotic linkage map. Canine-specific PCR primers were developed from the consensus of published sequences of several mammalian genomes and were designed to span intronic regions, thus optimizing the probability that a polymorphic site was included. The resulting markers were analyzed on a panel of three-generation canine reference families and the data were incorporated into the current meiotic linkage map. The data were compared with those generated by three chromosome paint studies in an effort to understand the distribution and frequency of microrearrangements within the canine genome. Forty-eight of 52 genes map to a chromosomal region predicted to contain genes from the corresponding region of the human genome according to all published reciprocal chromosome paint studies. Meiotic linkage mapping data for three genes can be used to resolve discrepancies between the published reciprocal chromosome paint studies, and for an additional two genes, meiotic mapping data allow evolutionary breakpoints to be more precisely defined. We conclude that microrearrangements of evolutionarily conserved segments between the canine and human genomes are rare, occurring for less than 0.5% of gene data reported to date. In addition, we have found that the placement of genes on the meiotic linkage map is a useful mechanism for resolving discrepancies between existing data sets. Received: 7 February 2001 / Accepted: 9 May 2001     

Genome‐wide survey of nuclear protein‐coding markers for beetle phylogenetics and their application in resolving both deep and shallow‐level divergences

Beetles (Coleoptera) are the most diverse and species‐rich insect group, representing an impressive explosive radiation in the evolutionary history of insects, and their evolutionary relationships are often difficult to resolve. The amount of ‘traditional markers’ (e.g. mitochondrial genes and nuclear rDNAs) for beetle phylogenetics is small, and these markers often lack sufficient signals in resolving relationships for such a rapidly radiating lineage. Here, based on the available genome data of beetles and other related insect species, we performed a genome‐wide survey to search nuclear protein‐coding (NPC) genes suitable for research on beetle phylogenetics. As a result, we identified 1470 candidate loci, which provided a valuable data resource to the beetle evolutionary research community for NPC marker development. We randomly chose 180 candidate loci from the database to design primers and successfully developed 95 NPC markers which can be PCR amplified from standard genomic DNA extracts. These new nuclear markers are universally applicable across Coleoptera, with an average amplification success rate of 90%. To test the phylogenetic utility, we used them to investigate the backbone phylogeny of Coleoptera (18 families sampled) and the family Coccinellidae (39 species sampled). Both phylogenies are well resolved (average bootstrap support >95%), showing that our markers can be used to address phylogenetic questions of various evolutionary depth (from species level to family level). In general, the newly developed nuclear markers are much easier to use and more phylogenetically informative than the ‘traditional markers’, and show great potential to expedite resolution of many parts in the Beetle Tree of Life.     

Design of New Genome- and Gene-Sourced Primers and Identification of QTL for Seed Oil Content in a Specially High-Oil Brassica napus Cultivar

Rapeseed (Brassica napus L.) is one of most important oilseed crops in the world. There are now various rapeseed cultivars in nature that differ in their seed oil content because they vary in oil-content alleles and there are high-oil alleles among the high-oil rapeseed cultivars. For these experiments, we generated doubled haploid (DH) lines derived from the cross between the specially high-oil cultivar zy036 whose seed oil content is approximately 50% and the specially low-oil cultivar 51070 whose seed oil content is approximately 36%. First, to address the deficiency in polymorphic markers, we designed 5944 pairs of newly developed genome-sourced primers and 443 pairs of newly developed primers related to oil-content genes to complement the 2244 pairs of publicly available primers. Second, we constructed a new DH genetic linkage map using 527 molecular markers, consisting of 181 publicly available markers, 298 newly developed genome-sourced markers and 48 newly developed markers related to oil-content genes. The map contained 19 linkage groups, covering a total length of 2,265.54 cM with an average distance between markers of 4.30 cM. Third, we identified quantitative trait loci (QTL) for seed oil content using field data collected at three sites over 3 years, and found a total of 12 QTL. Of the 12 QTL associated with seed oil content identified, 9 were high-oil QTL which derived from the specially high-oil cultivar zy036. Two high-oil QTL on chromosomes A2 and C9 co-localized in two out of three trials. By QTL mapping for seed oil content, we found four candidate genes for seed oil content related to four gene markers: GSNP39, GSSR161, GIFLP106 and GIFLP046. This information will be useful for cloning functional genes correlated with seed oil content in the future.     

Identification of an RFLP interval containing Pch2 on chromosome 7AL in wheat.

The gene Pch2 in 'Cappelle Desprez' is one of two genes found in hexaploid wheat known to confer resistance to eyespot disease. This study was conducted to develop an RFLP linkage map of the distal portion of wheat chromosome 7AL, and to locate and identify markers closely associated with Pch2 for use in marker-assisted selection. Ten loci in addition to Pch2 were mapped on chromosome 7AL, using segregation data from 102 homozygous chromosome 7A recombinant substitution lines derived from 'Chinese Spring' x 'Chinese Spring' ('Cappelle Desprez' 7A). The Pch2 locus was bracketed by two RFLP markers, one 11.0 cM distal to Xcdo347 and the other 18.8 cM proximal to Xwg380. The position of Pch2 on chromosome 7AL is similar to that of Pch1 on chromosome 7DL, suggesting that these resistance genes are homoeoloci. Although no single marker was closely linked to Pch2, simultaneous selection of the flanking RFLP markers Xcdo347 and Xwg380 could be used for selecting Pch2, since double recombination occurred in only 3% of the recombinant population. The use of the flanking RFLP markers to select for Pch2, in combination with previously identified Pch1-linked markers, would facilitate the development of cultivars carrying two genes for resistance to eyespot.     

Chloroplast Genetics of Chlamydomonas. III. Closing the Circle

A novel mapping procedure is presented for organelle genes or any other genetic system exhibiting a measurable frequency of exchanges occurring at a constant rate over a measurable time interval. For a set of markers in a multiply-marked cross, the exchange rates measure relative map distances from a centromere-like attachment point.With this method, we present mapping data and a linear map of genes in the chlcroplast genome of Chlamydomonas. The data are plotted as log (percent remaining heterozygotes) against time and map distances are taken as proportional to slope.A statistical method which is an adaptation of jackknife methodology to a regression problem was developed to estimate slope values. A single line is fitted to pooled data for each marker from several crosses, and then lines are re-fit to a series of pooled data sets in each of which the observations from a single cross have been omitted. From these data sets a final summary slope is computed as well as a statement of its variability. The relative positions of new markers present in single crosses can then be estimated utilizing data from many crosses. The method does not distinguish between one-armed and two-armed linear or circular maps. However, evaluation of this map in conjunction with cosegregation frequency data (Sager and Ramanis 1976b) provides unambiguous evidence of the genetic circularity of the Chlamydomonas chloroplast genome.     

Comparison of Breast Cancer to Healthy Control Tissue Discovers Novel Markers with Potential for Prognosis and Early Detection

This study was initiated to identify biomarkers with potential value for the early detection of poor-outcome breast cancer. Two sets of well-characterized tissues were utilized: one from breast cancer patients with favorable vs. poor outcome and the other from healthy women undergoing reduction mammaplasty. Over 46 differentially expressed genes were identified from a large list of potential targets by a) mining publicly available expression data (identifying 134 genes for quantitative PCR) and b) utilizing a commercial PCR array. Three genes show elevated expression in cancers with poor outcome and low expression in all other tissues, warranting further investigation as potential blood markers for early detection of cancers with poor outcome. Twelve genes showed lower expression in cancers with poor outcome than in cancers with favorable outcome but no differential expression between aggressive cancers and most healthy controls. These genes are more likely to be useful as prognostic tissue markers than as serum markers for early detection of aggressive disease. As a secondary finding was that, when histologically normal breast tissue was removed from a distant site in a breast with cancer, 7 of 38 specimens displayed a cancer-like expression profile, while the remaining 31 were genetically similar to the reduction mammaplasty control group. This finding suggests that some regions of ipsilateral histologically ‘normal’ breast tissue are predisposed to becoming malignant and that normal-appearing tissue with malignant signature might warrant treatment to prevent new primary tumors.     

Molecular mapping of resistance to Leptosphaeria maculans in Australian cultivars of Brassica napus.

Doubled haploid (DH) lines together with a cotyledon bioassay were employed for the molecular analysis of resistance to the blackleg fungus Leptosphaeria maculans in the Australian Brassica napus cultivars Shiralee and Maluka. We used bulked segregant analysis to identify 13 RAPD and two RFLP markers linked to the resistance phenotype and mapped these markers in the segregating DH population. Our data suggest the presence of a single major locus controlling resistance in the cultivar Shiralee, confirming our previous results obtained from Mendelian genetic analyses. In addition, preliminary mapping data for the cultivar Maluka also support a single locus model for resistance and indicate that the resistance genes from 'Shiralee' and 'Maluka' are either linked or possibly identical. The molecular markers identified in this study should be a useful tool for breeding blackleg resistant varieties using marker-assisted selection, and are the essential first step towards the map-based cloning of this resistance gene.     

Expression of a novel receptor tyrosine kinase gene and a paired-like homeobox gene provides evidence of differences in patterning at the oral and aboral ends of hydra

Axial patterning of the aboral end of the hydra body column was examined using expression data from two genes. One, shin guard, is a novel receptor protein-tyrosine kinase gene expressed in the ectoderm of the peduncle, the end of the body column adjacent to the basal disk. The other gene, manacle, is a paired-like homeobox gene expressed in differentiating basal disk ectoderm. During regeneration of the aboral end, expression of manacle precedes that of shin guard. This result is consistent with a requirement for induction of peduncle tissue by basal disk tissue. Our data contrast with data on regeneration of the oral end. During oral end regeneration, markers for tissue of the tentacles, which lie below the extreme oral end (the hypostome), are detected first. Later, markers for the hypostome itself appear at the regenerating tip, with tentacle markers displaced to the region below. Additional evidence that tissue can form basal disk without passing through a stage as peduncle tissue comes from LiCl-induced formation of patches of ectopic basal disk tissue. While manacle is ectopically expressed during formation of basal disk patches, shin guard is not. The genes examined also provide new information on development of the aboral end in buds. Although adult hydra are radially symmetrical, expression of both genes in the bud's aboral end is initially asymmetrical, appearing first on the side of the bud closest to the parent's basal disk. The asymmetry can be explained by differences in positional information in the body column tissue that evaginates to form a bud. As predicted by this hypothesis, grafts reversing the orientation of evaginating body column tissue also reverse the orientation of asymmetrical gene expression.