RECONSTRUCTING RETICULATION HISTORY IN A PHYLOGENETIC FRAMEWORK AND THE POTENTIAL OF ALLOPATRIC SPECIATION DRIVEN BY POLYPLOIDY IN AN AGAMIC COMPLEX IN CRATAEGUS (ROSACEAE)

Polyploidy plays a prominent role in the speciation process in plants. Many species are known to be part of agamic complexes comprising sexual diploids and more or less exclusively asexual polyploids. However, polyploid formation has been studied in very few cases, primarily because of the challenges in examining these cases phylogenetically. In this study, we demonstrate the use of a variety of phylogenetic approaches to unravel origins and infer reticulation history in a diploid–polyploid complex of black‐fruited Crataegus. The tree approaches are shown to be useful in testing alternative hypotheses and in revealing genealogies of nuclear genes, particularly in polyploid organisms that may contain multiple copies. Compared to trees, network approaches provide a better indication of reticulate relationships among recently diverged taxa. Taken together, our data point to both the autopolyploid and allopolyploid origins of triploids in natural populations of Crataegus suksdorfii, whereas tetraploids are formed via a triploid bridge, involving the backcross of allotriploid offspring with their diploid C. suksdorfii parent, followed by gene introgression from sympatric C. douglasii. Our findings provide empirical evidence for different pathways of polyploid formation that are all likely to occur within natural populations and the allopatric establishment of neopolyploids subsequent to their formation.     

Insertion of core CpG island element into human CMV promoter for enhancing recombinant protein expression stability in CHO cells

The human cytomegalovirus promoter (hCMV) is susceptible to gene silencing in CHO cells, most likely due to epigenetic events, such as DNA methylation and histone modifications. The core CpG island element (IE) from the hamster adenine phosphoribosyltransferase gene has been shown to prevent DNA methylation. A set of modified hCMV promoters was developed by inserting one or two copies of IE in either forward or reverse orientations either upstream of the hCMV enhancer, between the enhancer and core promoter (CP), or downstream of the CP. The modified hCMV with one copy of IE inserted between the enhancer and core promoter in reverse orientation (MR1) was most effective at enhancing expression stability without compromising expression level when compared with the wild‐type (WT) hCMV. A third of 18 EGFP expressing clones generated using MR1 retained 70% of their starting expression level after 8 weeks of culture in the absence of selection pressure, while none of 18 WT hCMV generated clones had expression above 50%. MR1 also improved antibody expression stability of methotrexate (MTX) amplified CHO cell lines. Stably transfected pools generated using MR1 maintained 62% of their original monoclonal antibody titer after 8 weeks of culture in the absence of MTX, compared to only 37% for WT hCMV pools. Low levels of CpG methylation within both WT hCMV and MR1 were observed in all the analyzed cell lines and the methylation levels did not correlate to the expression stability, suggesting IE enhances expression stability by other mechanisms other than preventing methylation. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:523–534, 2014     

Recent and recurrent polyploidy in Tragopogon (Asteraceae): cytogenetic, genomic and genetic comparisons

Tragopogon mirus Ownbey and T. miscellus Ownbey are allopolyploids that formed repeatedly during the past 80 years following the introduction of three diploids (T. dubius Scop., T. pratensis L. and T. porrifolius L.) from Europe to western North America. These polyploid species of known parentage are useful for studying the consequences of recent and recurrent polyploidization. We summarize recent analyses of the cytogenetic, genomic and genetic consequences of polyploidy in Tragopogon. Analyses of rDNA ITS (internal transcribed spacer) + ETS (external transcribed spacer) sequence data indicate that the parental diploids are phylogenetically well separated within Tragopogon (a genus of perhaps 150 species), in agreement with isozymic and cpDNA data. Using Southern blot and cloning experiments on tissue from early herbarium collections of T. mirus and T. miscellus (from 1949) to represent the rDNA repeat condition closer to the time of polyploidization than samples collected today, we have demonstrated concerted evolution of rDNA. Concerted evolution is ongoing, but has not proceeded to completion in any polyploid population examined; rDNA repeats of the diploid T. dubius are typically lost or converted in both allopolyploids, including populations of independent origin. Molecular cytogenetic studies employing rDNA probes, as well as centromeric and subtelomeric repeats isolated from Tragopogon, distinguished all chromosomes among the diploid progenitors (2n = 12). The diploid chromosome complements are additive in both allopolyploids (2n = 24); there is no evidence of major chromosomal rearrangements in populations of either T. mirus or T. miscellus. cDNA‐AFLP display revealed differences in gene expression between T. miscellus and its diploid parents, as well as between populations of T. miscellus of reciprocal origin. Approximately 5% of the genes examined in the allopolyploid populations have been silenced, and an additional 4% exhibit novel gene expression relative to their diploid parents. Some of the differences in gene expression represent maternal or paternal effects. Multiple origins of a polyploid species not only affect patterns of genetic variation in natural populations, but also contribute to differential patterns of gene expression and may therefore play a major role in the long‐term evolution of polyploids. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82 , 485–501.     

Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize

An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25–30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.     

Plant defense gene promoter enhances the reliability of<Emphasis Type="Italic"> shiva-1</Emphasis> gene-induced resistance to soft rot disease in potato

PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding. A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter. The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent. As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease. In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant. More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies. The highly resistant CaMV 35S recombinant also was present as a single copy. The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.     

Transgene copy number can be positively or negatively associated with transgene expression

Two different types of T-DNA insert were found in tobacco plants transformed with Agrobacterium tumefaciens. High-expressing (H) types had one copy of the T-DNA at a locus and produced high expression of the transgene uidA, as measured by uidA RNA levels and -glucuronidase activity; low-expressing (L) types had inverted repeats of the T-DNA at a locus and produced low uidA expression. H-types from different transformants acted additively, and cross-fertilization between two different homozygous transformants with H-type inserts produced F₁ plants with GUS activity that equalled the parents and individual F₂ plants with 50%, 100%, 150% and 200% of parental values. However, the L-type inserts worked in trans to suppress uidA expression from H-type inserts when both were present in the same genome. Hence when a transformant homozygous for the L-type insert was crossed to one homozygous for the H-type, all plants in the F₁ and F₂ generations with both types of insert had low GUS activity while F₂ segregants that only had the H-type inserts had high GUS activity again. Suppression of the H-type gene was associated with increased methylation of the insert. Particle acceleration was used to introduce further copies of uidA into tissues of the transformants. Regardless of the promoter used, those plants with endogenous L-type inserts showed none of the distinct loci of GUS activity readily visible in material with no inserts, showing that L-type inserts could suppress not only the uidA expression of genomic homologues, but also of copies added in vitro.     

Aberrant CpG methylation of the TFAP2A gene constitutes a mechanism for loss of TFAP2A expression in human metastatic melanoma

Metastatic melanoma is a deadly treatment-resistant form of skin cancer whose global incidence is on the rise. During melanocyte transformation and melanoma progression the expression profile of many genes changes. Among these, a gene implicated in several steps of melanocyte development, TFAP2A, is frequently silenced; however, the molecular mechanism of TFAP2A silencing in human melanoma remains unknown. In this study, we measured TFAP2A mRNA expression in primary human melanocytes compared to 11 human melanoma samples by quantitative real-time RT-PCR. In addition, we assessed CpG DNA methylation of the TFAP2A promoter in these samples using bisulfite sequencing. Compared to primary melanocytes, which showed high TFAP2A mRNA expression and no promoter methylation, human melanoma samples showed decreased TFAP2A mRNA expression and increased promoter methylation. We further show that increased CpG methylation correlates with decreased TFAP2A mRNA expression. Using The Cancer Genome Atlas, we further identified TFAP2A as a gene displaying among the most decreased expression in stage 4 melanomas vs. non-stage 4 melanomas, and whose CpG methylation was frequently associated with lack of mRNA expression. Based on our data, we conclude that TFAP2A expression in human melanomas can be silenced by aberrant CpG methylation of the TFAP2A promoter. We have identified aberrant CpG DNA methylation as an epigenetic mark associated with TFAP2A silencing in human melanoma that could have significant implications for the therapy of human melanoma using epigenetic modifying drugs.     

Transcriptional silencing and developmental reactivation of cry1Ab gene in transgenic rice

One transgenic rice line lacking Cry1Ab expression product was screened in the progenies of Agrobacterium-transformed transgenic rice variety Zhong 8215 with a cry1Ab gene under field releasing conditions by using GUS histochemical assay and Western blot. Molecular hybridization results revealed that the cry1Ab gene was silenced in the transgenic rice variety Zhong 8215 and two copies of ubiquitin promoter were integrated into the rice genome. The silencing of cry1Ab gene in transgenic rice was found to be due to the methylation of the ubiquitin promoter as revealed by methylation analysis. Meanwhile, different concentrations of demethylation reagent 5-azacytidine combining with different treatment time were employed to treat the silenced transgenic rice seeds. The results indicated that 5-azacytidine could reactivate 8%–30% of the silenced transgenic rice plants and the expression level of the reactivated cry1Ab transgene could reach as high as 0.147% of the total soluble protein. Treatment with low concentration of 5-azacytidine (45 mg/L for 1 d and 2 d) could lead to the highest reactivation ratio and the highest expression level of the cry1Ab gene.     

Introduction and constitutive expression of a rice chitinase gene in bread wheat using biolistic bombardment and the bar gene as a selectable marker

 Our long-term goal is to control wheat diseases through the enhancement of host plant resistance. The constitutive expression of plant defense genes to control fungal diseases can be engineered by genetic transformation. Our experimental strategy was to biolistically transform wheat with a vector DNA containing a rice chitinase gene under the control of the CaMV 35 S promoter and the bar gene under control of the ubiquitin promoter as a selectable marker. Immature embryos of wheat cv ‘Bobwhite’ were bombarded with plasmid pAHG11 containing the rice chitinase gene chi11 and the bar gene. The embryos were subcultured on MS2 medium containing the herbicide bialaphos. Calli were then transferred to a regeneration medium, also containing bialaphos. Seventeen herbicide-resistant putative transformants (T₀) were selected after spraying with 0.2% Liberty, of which 16 showed bar gene expression as determined by the phosphinothricin acetyltransferase (PAT) assay. Of the 17 plants, 12 showed the expected 35-kDa rice chitinase as revealed by Western blot analysis. The majority of transgenic plants were morphologically normal and self-fertile. The integration, inheritance and expression of the chi11 and bar genes were confirmed by Southern hybridization, PAT and Western blot analysis of T₀ and T₁ transgenic plants. Mendelian segregation of herbicide resistance was observed in some T₁ progenies. Interestingly, a majority of the T₁ progeny had very little or no chitinase expression even though the chitinase transgene was intact. Because PAT gene expression under control of the ubiquitin promoter was unaffected, we conclude that the CaMV 35 S promoter is selectively inactivated in T₁ transgenic wheat plants. Received: 12 May 1998 / Accepted: 15 May 1998     

Can polyploidy confer invasive plants with a wider climatic tolerance? A test using Solidago canadensis

Polyploidy can cause variation in plant functional traits and thereby generate individuals that can adapt to fluctuating environments and exploit new environments. However, few empirical studies have tested for an association between ploidy level and climatic tolerance of invasive cytotypes relative to conspecific native‐range cytotypes. Here, we used an invasive plant Solidago canadensis to test whether invasive populations had a higher proportion of polyploids, greater height and stem‐base diameter, and occupied a wider range of climatic conditions than conspecific native‐range populations. We also tested whether the invasive populations had overcome genetic founder effects. We sampled a total of 80 populations in parts of the invaded range in China and native range in North America for in situ measurements of plant height and stem‐base diameter in the field and for population genetic and cytotype analyses. To examine climatic correlates, we augmented our field‐sampled data with occurrence records obtained from Global Biodiversity Information Facility. All, except one, of the populations that we sampled in China occurred in a humid subtropical climate. In contrast, the North American populations occurred in humid continental, humid subtropical, and semi‐arid climatic zones. All populations of S. canadensis in China were purely hexaploid, while the North American populations were diploid, tetraploid, and hexaploid. The invasive hexaploids were significantly taller and had a larger stem‐base diameter than native hexaploids. Native hexaploids were significantly taller and had larger stem‐base diameter than native diploids. Climatic correlate assessment found that invasive and native populations occupied different climatic envelopes, with invasive populations occurring in warmer and less seasonal climates than native populations. However, there was no significant correlation between ploidy level and climatic envelope of S. canadensis. Molecular phylogeography data suggest reduced genetic founder effects in the invaded range. Overall, these results suggest that polyploidy does not influence S. canadensis climatic tolerance.     

A different methylation profile of circadian genes promoter in breast cancer patients according to clinicopathological features

ABSTRACT

One of the supposed mechanisms that may lead to breast cancer (BC) is an alteration of circadian gene expression and DNA methylation. We undertook an integrated approach to identify methylation pattern of core circadian promoter regions in BC patients with regard to clinical features. We performed a quantitative methylation-specific real-time PCR analysis of a promoter methylation profile in 107 breast tumor and matched non-tumor tissues. A panel of circadian genes CLOCK, BMAL1, PERIOD (PER1, 2, 3), CRYPTOCHROME (CRY1, 2) and TIMELESS as well as their association with clinicopathological characteristics were included in the analysis. Three out of the eight analyzed genes exhibited marked hypermethylation (PER1, 2, 3), whereas CLOCK, BMAL1, CRY2 showed significantly lower promoter CpG methylation in the BC tissues when compared to the non-tumor tissues. Among variously methylated genes we found an association between the elevated methylation level of PERs promoter region and molecular subtypes, histological subtypes and tumor grading of BC. Methylation status may be associated with a gene expression level of circadian genes in BC patients. An aberrant methylation pattern in circadian genes in BC may provide information that could be used as novel biomarkers in clinics and molecular epidemiology as well as play an important role in BC etiology.     

Origins of polyploids: an example from peonies (Paeonia) and a model for angiosperms

The majority of tetraploid peonies are allopolyploids derived from crosses between phylogenetically distinct diploid lineages. Tetraploid Paeonia obovata was previously considered to be an autopolyploid because it is morphologically indistinguishable from the diploid of the same species. The presence of the Adh2 gene in tetraploid P. obovata but the inability to amplify the Adh2 gene from Chinese diploids of P. obovata, however, suggests that the tetraploid was not an autotetraploid derivative of the geographically adjacent diploid populations in China. The Adh gene phylogenies rather suggest that the tetraploid originated from crosses between two geographical races of diploid P. obovata distributed in China and Japan. The intermediate status of tetraploid P. obovata between auto‐ and allopolyploidy highlights the need for population genetic analyses of polyploid origins along the continuous range of genomic divergence. Here we present a model that describes the probabilities of polyploid formation and establishment as a function of genomic divergence between diploid progenitors. The probability of polyploid formation (P_f) is obtained from the multiplication of the probability of production of unreduced gametes (P_g) and the probability of ‘hybridization’ (P_h). P_f stays relatively stable when the genomic divergence is low, and then decreases progressively rapidly with the increase of genomic divergence between diploid progenitors. The probability of polyploid establishment (P_e), which depends on the rate of appearance of stable beneficial gene combinations and the rate of fertility restoration, is positively correlated with the genomic divergence of diploid parents. Multiplication of P_f and P_e gives an overall probability of polyploid origins (P_o) that varies continuously along the genomic divergence between diploid progenitors. © 2004 The Linnean Society of London, Biological Journal of the Linnean Society, 2004, 82 , 561–571.     

Polyploidy and DNA methylation: new tools available

Most plant species are recent or ancient polyploids (displaying at least one round of genome duplication in their history). Cultivated species (e.g. wheat, cotton, canola, sugarcane, coffee) and invasive species are often relatively recent polyploids, and frequently of hybrid origin (i.e. allopolyploids). Despite the genetic bottleneck occurring during the allopolyploid speciation process, the formation of such species from two divergent lineages leads to fixed heterozygosity decisive to their success. New phenotypes and new niche occupation are usually associated with this mode of speciation, as a result of both genomic rearrangements and gene expression changes of different magnitudes depending on the different polyploid species investigated. These gene expression changes affecting newly formed polyploid species may result from various, interconnected mechanisms, including (i) functional interactions between the homoeologous copies and between their products, that are reunited in the same nucleus and cell; (ii) the fate of duplicated copies, selective pressure on one of the parental copy being released which could lead to gene loss, pseudogenization, or alternatively, to subfunctionalization or neofunctionalization; and (iii) epigenetic landscape changes that in turn affect gene expression. As one of the interrelated processes leading to epigenetic regulation of gene expression, the DNA methylation status of newly formed species appears to be consistently affected following both hybridization and genome doubling. In this issue, Verhoeven et al. have investigated the fate of DNA methylation patterns that could affect naturally occurring new asexual triploid lineages of dandelions. As a result of such a ploidy level change, the authors demonstrate stably transmitted DNA methylation changes leading to unique DNA methylation patterns in each newly formed lineage. Most studies published to date on plant DNA methylation polymorphism were performed using restriction enzymes sensitive to methylation. Recently, new high‐throughput methods were made available, thanks to the development of ‘next‐generation sequencing’ techniques. The combination of these methods offers powerful and promising tools to investigate epigenetic variation in both model and non‐model systems.     

Divergent DNA methylation contributes to duplicated gene evolution and chilling response in tea plants

The tea plant (Camellia sinensis) is a thermophilic cash crop and contains a highly duplicated and repeat-rich genome. It is still unclear how DNA methylation regulates the evolution of duplicated genes and chilling stress in tea plants. We therefore generated a single-base-resolution DNA methylation map of tea plants under chilling stress. We found that, compared with other plants, the tea plant genome is highly methylated in all three sequence contexts, including CG, CHG and CHH (where H = A, T, or C), which is further proven to be correlated with its repeat content and genome size. We show that DNA methylation in the gene body negatively regulates the gene expression of tea plants, whereas non-CG methylation in the flanking region enables a positive regulation of gene expression. We demonstrate that transposable element-mediated methylation dynamics significantly drives the expression divergence of duplicated genes in tea plants. The DNA methylation and expression divergence of duplicated genes in the tea plant increases with evolutionary age and selective pressure. Moreover, we detect thousands of differentially methylated genes, some of which are functionally associated with chilling stress. We also experimentally reveal that DNA methyltransferase genes of tea plants are significantly downregulated, whereas demethylase genes are upregulated at the initial stage of chilling stress, which is in line with the significant loss of DNA methylation of three well-known cold-responsive genes at their promoter and gene body regions. Overall, our findings underscore the importance of DNA methylation regulation and offer new insights into duplicated gene evolution and chilling tolerance in tea plants.