Evidence for rapid evolution in a grassland biodiversity experiment

In long‐term grassland experiments, positive biodiversity effects on plant productivity commonly increase with time. Subsequent glasshouse experiments showed that these strengthened positive biodiversity effects persist not only in the local environment but also when plants are transferred into a common environment. Thus, we hypothesized that community diversity had acted as a selective agent, resulting in the emergence of plant monoculture and mixture types with differing genetic composition. To test our hypothesis, we grew offspring from plants that were grown for eleven years in monoculture or mixture environments in a biodiversity experiment (Jena Experiment) under controlled glasshouse conditions in monocultures or two‐species mixtures. We used epiGBS, a genotyping‐by‐sequencing approach combined with bisulphite conversion, to provide integrative genetic and epigenetic (i.e., DNA methylation) data. We observed significant divergence in genetic and DNA methylation data according to selection history in three out of five perennial grassland species, namely Galium mollugo, Prunella vulgaris and Veronica chamaedrys, with DNA methylation differences mostly reflecting the genetic differences. In addition, current diversity levels in the glasshouse had weak effects on epigenetic variation. However, given the limited genome coverage of the reference‐free bisulphite method epiGBS, it remains unclear how much of the differences in DNA methylation was independent of underlying genetic differences. Our results thus suggest that selection of genetic variants, and possibly epigenetic variants, caused the rapid emergence of monoculture and mixture types within plant species in the Jena Experiment.     

DNA fingerprinting of Indian isolates of Xanthomonas oryzae pv. oryzae

 A high level of genetic polymorphism was detected among Indian isolates of Xanthomonas oryzae pv. oryzae using hypervariable probes such as a microsatellite oligonucleotide, probe (TG)₁₀, a human minisatellite probe, pV47, an avirulence gene probe, avrXa10 and a repeat clone, pBS101. These DNA probes detected multiple loci in the bacterial genome generating complex DNA fingerprints and differentiated all of the bacterial isolates. Analysis of fingerprints indicated that pV47, (TG)₁₀ and pBS101 have a lower probability of identical match than avrXa10 and therefore are potential probes for DNA fingerprinting and variability analysis of Xanthomonas oryzae pv. oryzae pathogen populations. Cluster analysis based on hybridization patterns using all of the above probes showed five groups at 56% similarity. Studies on the methylation patterns of isolates representing the three important races of X. oryzae pv. oryzae indicated more methylation in the most virulent isolate, suggesting a possible role of methylation in pathogenicity. Received: 8 December 1996 / Accepted: 20 December 1996     

Invasion of diverse habitats by few Japanese knotweed genotypes is correlated with epigenetic differentiation

The expansion of invasive species challenges our understanding of the process of adaptation. Given that the invasion process often entails population bottlenecks, it is surprising that many invasives appear to thrive even with low levels of sequence-based genetic variation. Using Amplified Fragment Length Polymorphism (AFLP) and methylation sensitive-AFLP (MS-AFLP) markers, we tested the hypothesis that differentiation of invasive Japanese knotweed in response to new habitats is more correlated with epigenetic variation than DNA sequence variation. We found that the relatively little genetic variation present was differentiated among species, with less differentiation among sites within species. In contrast, we found a great deal of epigenetic differentiation among sites within each species and evidence that some epigenetic loci may respond to local microhabitat conditions. Our findings indicate that epigenetic effects could contribute to phenotypic variation in genetically depauperate invasive populations. Deciphering whether differences in methylation patterns are the cause or effect of habitat differentiation will require manipulative studies.     

A systematic comparison of quantitative high-resolution DNA methylation analysis and methylation-specific PCR

Assessment of DNA methylation has become a critical factor for the identification, development and application of methylation based biomarkers. Here we describe a systematic comparison of a quantitative high-resolution mass spectrometry-based approach (MassARRAY), pyrosequencing and the broadly used methylation-specific PCR (MSP) technique analyzing clinically relevant epigenetically silenced genes in acute myeloid leukemia (AML). By MassARRAY and pyrosequencing, we identified significant DNA methylation differences at the ID4 gene promoter and in the 5′ region of members of the SFRP gene family in 62 AML patients compared with healthy controls. We found a good correlation between data obtained by MassARRAY and pyrosequencing (correlation coefficient R² = 0.88). MSP-based assessment of the identical samples showed less pronounced differences between AML patients and controls. By direct comparison of MSP-derived and MassARRAY-based methylation data as well as pyrosequencing, we could determine overestimation of DNA methylation data by MSP. We found sequence-context dependent highly variable cut-off values of quantitative DNA methylation values serving as discriminator for the two MSP methylation categories. Moreover, good agreements between quantitative methods and MSP could not be achieved for all investigated loci. Significant correlation of the quantitative assessment but not of MSP-derived methylation data with clinically important characteristics in our patient cohort demonstrated clinical relevance of quantitative DNA methylation assessment. Taken together, while MSP is still the most commonly applied technique for DNA methylation assessment, our data highlight advantages of quantitative approaches for precise characterization and reliable biomarker use of aberrant DNA methylation in primary patient samples, particularly.     

Development of a DNA-labeling system for array-based comparative genomic hybridization.

Chromosomal amplifications and deletions are critical components of tumorigenesis and DNA copy-number variations also correlate with changes in mRNA expression levels. Genome-wide microarray comparative genomic hybridization (CGH) has become an important method for detecting and mapping chromosomal changes in tumors. Thus, the ability to detect twofold differences in fluorescent intensity between samples on microarrays depends on the generation of high-quality labeled probes. To enhance array-based CGH analysis, a random prime genomic DNA labeling method optimized for improved sensitivity, signal-to-noise ratios, and reproducibility has been developed. The labeling system comprises formulated random primers, nucleotide mixtures, and notably a high concentration of the double mutant exo-large fragment of DNA polymerase I (exo-Klenow). Microarray analyses indicate that the genomic DNA-labeled templates yield hybridization signals with higher fluorescent intensities and greater signal-to-noise ratios and detect more positive features than the standard random prime and conventional nick translation methods. Also, templates generated by this system have detected twofold differences in gene copy number between male and female genomic DNA and identified amplification and deletions from the BT474 breast cancer cell line in microarray hybridizations. Moreover, alterations in gene copy number were routinely detected with 0.5 microg of genomic DNA starting sample. The method is flexible and performs efficiently with different fluorescently labeled nucleotides. Application of the optimized CGH labeling system may enhance the resolution and sensitivity of array-based CGH analysis in cancer and medical genetic studies.     

DNA fingerprinting in horses using a simple (TG)n probe and its application to population comparisons

A synthetic polynucleotide (TG)_n was hybridized to equine DNA digested with HinfI and hypervariable hybridization patterns were obtained. Mendelian inheritance of these DNA fingerprinting patterns was confirmed by pedigree analysis. Estimates of the probabilities of identical band patterns in unrelated individuals of different breeds (Swedish Trotters, North Swedish Trotters, Thoroughbreds and Arabians) were in the range 1 times 10-⁴-7 times 10-⁶ The variability derived with the (TG)_n, probe in horses was higher than what we obtained with several other commmonly used probes for DNA fingerprinting. Individuals within breeds tended to be more similar to each other with regard to DNA fingerprint pattern than to individuals of other breeds. Moreover, a parsimony analysis made on the basis of the hybridization patterns gave clustering of individuals within breeds. The possibility of using hypervariable probes for the identification of breed-specific characters is discussed.     

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.     

DNA fingerprint analysis for the detection of induced mutations in mammalian cells in culture

A mutation assay in cultured mammalian cells based on the direct analysis of minisatellite DNA was developed. Band pattern variations reflect DNA alterations ranging from single base changes to complex rearrangements. By DNA fingerprinting a large number of autosomal loci throughout the human genome can be simultaneously checked, therefore minimizing the size of the samples of cell colonies to be scored in the absence of phenotypic selection. For the mutation assay chinese hamster cells (V79) were treated with Nitrosoguanidine and 14 independent colonies were isolated and expanded. DNA fingerprints were obtained after digestion of the DNA extracted from each clone with bothHinfI andHae III, and hybridisation with both 33.15 and 33.6 probes. Twelve colonies from untreated cells were also analysed. Several differences in the band pattern of treated colonies were observed when compared with untreated cells; digestion withHae III and hybridisation with 33.15 probe allowed the detection of the highest frequency of induced variants. The results suggest that minisatellite sequences are hypermutable sites that can be used to monitor the mutagenic potential of chemical agents directly at the DNA level, without phenotypic selection. Moreover, with the method herein decribed, it is possible to distinguish between true mutations and epimutations, such as those caused by changes in DNA methylation.     

DNA extraction procedures meaningfully influence qPCR-based mtDNA copy number determination

Quantitative real time PCR (qPCR) is commonly used to determine cell mitochondrial DNA (mtDNA) copy number. This technique involves obtaining the ratio of an unknown variable (number of copies of an mtDNA gene) to a known parameter (number of copies of a nuclear DNA gene) within a genomic DNA sample. We considered the possibility that mtDNA:nuclear DNA (nDNA) ratio determinations could vary depending on the method of genomic DNA extraction used, and that these differences could substantively impact mtDNA copy number determination via qPCR. To test this we measured mtDNA:nDNA ratios in genomic DNA samples prepared using organic solvent (phenol–chloroform–isoamyl alcohol) extraction and two different silica-based column methods, and found mtDNA:nDNA ratio estimates were not uniform. We further evaluated whether different genomic DNA preparation methods could influence outcomes of experiments that use mtDNA:nDNA ratios as endpoints, and found the method of genomic DNA extraction can indeed alter experimental outcomes. We conclude genomic DNA sample preparation can meaningfully influence mtDNA copy number determination by qPCR.     

DNA fingerprinting based on microsatellite-anchored fragment length polymorphisms,and isolation of sequence-specific PCR markers in lupin (Lupinus angustifolius L.)

We report a method of microsatellite-anchored fragment length polymorphisms for DNA fingerprinting. The method combines the concept of AFLP and the microsatellite-anchor primer technique. Genomic DNA was digested by one restriction enzyme MseI. One AFLP adaptor (MseI adaptor) was ligated onto the restriction fragments. DNA fingerprints were produced by PCR using one microsatellite-anchor primer in combination with one MseI-primer. The method allows co-amplification of over 100 DNA fragments containing microsatellite motifs per PCR. Polymorphisms detected from lupin by this method included those arising from variation in the number of microsatellite repeat units targeted by the microsatellite-anchor primers, from variation on the annealing sites for the SSR-anchor primers, from insertions/deletions outside the SSR region, and from variation in restriction sites. The first three types of polymorphisms were readily converted into sequence-specific PCR markers suitable for marker-assisted breeding.     

Faithful inheritance of cytosine methylation patterns in repeated sequences of the allotetraploid tobacco correlates with the expression of DNA methyltransferase gene families from both parental genomes

The widespread occurrence of epigenetic alterations in allopolyploid species deserves scrutiny that DNA methylation systems may be perturbed by interspecies hybridization and polyploidization. Here we studied the genes involved in DNA methylation in Nicotiana tabacum (tobacco) allotetraploid containing S and T genomes inherited from Nicotiana sylvestris and Nicotiana tomentosiformis progenitors. To determine the inheritance of DNA methyltransferase genes and their expression patterns we examined three major DNA methyltransferase families (MET1, CMT3 and DRM) from tobacco and the progenitor species. Using Southern blot hybridization and PCR-based methods (genomic CAPS), we found that the parental loci of these gene families are retained in tobacco. Homoeologous expression was found in all tissues examined (leaf, root, flower) suggesting that DNA methyltransferase genes were probably not themselves targets of uniparental epigenetic silencing for over thousands of generations of allotetraploid evolution. The level of CG and CHG methylation of selected high-copy repeated sequences was similar and high in tobacco and its diploid progenitors. We speculate that natural selection might favor additive expression of parental DNA methyltransferase genes maintaining high levels of DNA methylation in tobacco, which has a repeat-rich heterochromatic genome. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers AM946602–AM946620 and FM872474–FM872476.     

Familial molar tissues due to mutations in the inflammatory gene, NALP7, have normal postzygotic DNA methylation

An imprinting disorder has been believed to underlie the etiology of familial biparental hydatidiform moles (HMs) based on the abnormal methylation or expression of imprinted genes in molar tissues. However, the extent of the epigenetic defect in these tissues and the developmental stage at which the disorder begins have been poorly defined. In this study, we assessed the extent of abnormal DNA methylation in two HMs caused by mutations in the recently identified 19q13.4 gene, NALP7. We demonstrate normal postzygotic DNA methylation patterns at major repetitive and long interspersed nuclear elements (LINEs), genes on the inactive X-chromosome, three-cancer related genes, and CpG rich regions surrounding the PEG3 differentially methylated region (DMR). Our data provide a comprehensive assessment of DNA methylation in familial molar tissues and indicate that abnormal DNA methylation in these tissues is restricted to imprinted DMRs. The known role of NALP7 in apoptosis and inflammation pinpoints previously unrecognized pathways that could directly or indirectly underlie the abnormal methylation of imprinted genes in molar tissues.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Genome-wide DNA methylation profiling in rheumatoid arthritis identifies disease-associated methylation changes that are distinct to individual T- and B-lymphocyte populations

Changes to the DNA methylome have been described in patients with rheumatoid arthritis (RA). In previous work, we reported genome-wide methylation differences in T-lymphocyte and B-lymphocyte populations from healthy individuals. Now, using HumanMethylation450 BeadChips to interrogate genome-wide DNA methylation, we have determined disease-associated methylation changes in blood-derived T- and B-lymphocyte populations from 12 female patients with seropositive established RA, relative to 12 matched healthy individuals. Array data were analyzed using NIMBL software and bisulfite pyrosequencing was used to validate array candidates. Genome-wide DNA methylation, determined by analysis of LINE-1 sequences, revealed higher methylation in B-lymphocytes compared with T-lymphocytes (P ≤ 0.01), which is consistent with our findings in healthy individuals. Moreover, loci-specific methylation differences that distinguished T-lymphocytes from B-lymphocytes in healthy individuals were also apparent in RA patients. However, disease-associated methylation differences were also identified in RA. In these cases, we identified 509 and 252 CpGs in RA-derived T- and B-lymphocytes, respectively, that showed significant changes in methylation compared with their cognate healthy counterparts. Moreover, this included a restricted set of 32 CpGs in T-lymphocytes and 20 CpGs in B-lymphocytes (representing 15 and 10 genes, respectively, and including two, MGMT and CCS, that were common to both cell types) that displayed more substantial changes in methylation. These changes, apparent as hyper- or hypo-methylation, were independently confirmed by pyrosequencing analysis. Validation by pyrosequencing also revealed additional sites in some candidate genes that also displayed altered methylation in RA. In this first study of genome-wide DNA methylation in individual T- and B-lymphocyte populations in RA patients, we report disease-associated methylation changes that are distinct to each cell type and which support a role for discrete epigenetic regulation in this disease.     

Epigenetic and genetic burden measures are associated with tumor characteristics in invasive breast carcinoma

The development and progression of invasive breast cancer is characterized by alterations to the genome and epigenome. However, the relationship between breast tumor characteristics, disease subtypes, and patient outcomes with the cumulative burden of these molecular alterations are not well characterized. We determined the average departure of tumor DNA methylation from adjacent normal breast DNA methylation using Illumina 450K methylation data from 700 invasive breast tumors and 90 adjacent normal breast tissues in The Cancer Genome Atlas. From this we generated a novel summary measure of altered DNA methylation, the DNA methylation dysregulation index (MDI), and examined the relation of MDI with tumor characteristics and summary measures that quantify cumulative burden of genetic mutation and copy number alterations. Our analysis revealed that MDI was significantly associated with tumor stage (P = 0.017). Across invasive breast tumor subtypes we observed significant differences in genome-wide DNA MDIs (P = 4.9E–09) and in a fraction of the genome with copy number alterations (FGA) (P = 4.6E–03). Results from a linear regression adjusted for subject age, tumor stage, and estimated tumor purity indicated a positive significant association of MDI with both MCB and FGA (P = 0.036 and P < 2.2E–16). A recursively partitioned mixture model of all 3 somatic alteration burden measures resulted in classes of tumors whose epigenetic and genetic burden profile were associated with the PAM50 subtype and mutations in TP53, PIK3CA, and CDH1. Together, our work presents a novel framework for characterizing the epigenetic burden and adds to the understanding of the aggregate impact of epigenetic and genetic alterations in breast cancer.     

Parental alleles of an imprinted mouse transgene replicate synchronously

Molecular features of imprinted genes include differences in expression, methylation, and the timing of DNA replication between parental alleles. Whereas methylation differences always seem to be associated with differences in expression, differences in the timing of replication between parental homologs are not always seen at imprinted loci. These observations raise the possibility that differences in replication timing may not be an essential feature underlying genomic imprinting. In this study, we examined the timing of replication of the two alleles of the imprinted RSVIgmyc transgene in individual embryonic cells using fluorescence in situ hybridization (FISH). The cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. Thus, allele-specific differences in replication, if they indeed govern RSVIgmyc imprinting, should be found in RSVIgmyc sequences. We found that the parental alleles of RSVIgmyc, which exhibit differences in methylation, replicated at the same time. Synchronous replication was also seen in embryonic cells containing a modified version of RSVIgmyc that exhibited parental allele differences in both methylation and expression. These findings indicate that maintenance of expression and methylation differences between alleles does not require a difference in replication timing. The differences in replication timing of endogenous imprinted alleles detected by FISH might therefore reflect structural differences between the two alleles that could be a consequence of imprinting or, alternatively, could be unrelated to imprinting. Dev. Genet. 23:275–284, 1998. © 1998 Wiley-Liss, Inc.     

DNA content, interphase AgNOR-area, number of 3 HrDNA hybridization signals and the methylation level in coding rDNA sequence in different organs of Lupinus luteus L.

Nuclear DNA and AgNOR-area quantity have been measured cytophotometrically, the number of silver grains afterin situ ³H rDNA/DNA hybridization has been counted, and the level of rDNA methylation has been estimated in root meristems, differentiated root zones, hypocotyls, cotyledons, and leaves of 7-day-old seedlings ofLupinus luteus. DNA content increases by endoreplication, reaching the average DNA C-value of 4.2 in the meristem, 6.6C in hypocotyl, 15.9C in cotyledon, and 3.3C in leaf. The quantity of AgNOR-area, the highest in root meristem, does not follow DNA C-value. Number of grains afterin situ ³H rDNA/DNA hybridization corresponds to DNA amount, but their distribution differs, showing in many cases perinucleolar localization in organs displaying low AgNOR-area quantity. Southern hybridization has not demonstrated detectable changes in level rDNA methylation. We suggest that the low level of rDNA expression in non-meristematic tissues could be related to rDNA inactivation caused by its translocation into nucleolus-associated heterochromatin.     

Evaluating Neanderthal Genetics and Phylogeny

The retrieval of Neanderthal (Homo neanderthalsensis) mitochondrial DNA is thought to be among the most significant ancient DNA contributions to date, allowing conflicting hypotheses on modern human (Homo sapiens) evolution to be tested directly. Recently, however, both the authenticity of the Neanderthal sequences and their phylogenetic position outside contemporary human diversity have been questioned. Using Bayesian inference and the largest dataset to date, we find strong support for a monophyletic Neanderthal clade outside the diversity of contemporary humans, in agreement with the expectations of the Out-of-Africa replacement model of modern human origin. From average pairwise sequence differences, we obtain support for claims that the first published Neanderthal sequence may include errors due to postmortem damage in the template molecules for PCR. In contrast, we find that recent results implying that the Neanderthal sequences are products of PCR artifacts are not well supported, suffering from inadequate experimental design and a presumably high percentage (>68%) of chimeric sequences due to “jumping PCR” events. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. [Reviewing Editor: Dr. Martin Kreitman]     

Dot-blot-SNP analysis for practical plant breeding and cultivar identification in rice

We report dot-blot hybridization with allele-specific oligonucleotides for single nucleotide polymorphisms (SNPs) analysis to be applicable for practical plant breeding and cultivar identification. Competitive hybridization of a digoxigenin-labeled oligonucleotide having the sequence of a mutant allele (or a wild-type allele) together with an unlabeled oligonucleotide having the sequence of a wild-type allele (or a mutant allele) was highly effective to reduce background signals in dot-blot hybridization. All 100 tested genes (200 alleles) in rice having SNPs or insertions/deletions were detected in an allele-specific manner. Genotypes of 43 rice cultivars were identified by this technique, and eight SNP markers were found to be sufficient for distinguishing all the cultivars from each other. Dot-blot analysis was also applied to genotyping of Wx and Sd1 of F₄ plants in a conventional breeding program. Since dot-blot analysis with competitive hybridization provides a highly reliable, simple, and cost-effective technique for SNP analysis of a large number of samples, this technique is expected to realize the practical use of a novel breeding method, in which plants or breeding lines are selected by SNP analyses of many genes in a laboratory.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Field performance evaluation and genetic integrity assessment of cryopreserved papaya clones

This paper is the first report of field performance and evaluation of morphological traits following cryopreservation in four genotypes of Carica papaya (Z6, 97, TS2 and 35). It also describes the successful establishment of in vitro plantlets following vitrification-based cryopreservation of shoot tips and their acclimatisation through to field establishment. Cloned plants resulting from untreated controls, as well as controls taken at three other stages of the cryopreservation process (dissection, pre-treatment, plant vitrification solution 2 (PVS2) treatment) and cryopreserved plants were established to ensure a rigorous appraisal of any variation. Results indicate no differences between any of the control plants or cryopreserved plants for either growth performance or morphology. In addition, both randomly amplified DNA fingerprinting and amplified DNA methylation polymorphism markers were used to assess any genomic or methylation changes in genotype 97 at four different developmental stages post cryopreservation (in vitro, acclimatisation and field). Only small genomic DNA modifications (0–8.3%) were detected in field stage plants and methylation modifications (0–4.3%) were detected at both the in vitro and field stages for samples treated with PVS2 or cryopreservation.