Methylation of ribosomal cistrons in diploid and tetraploid Odontophrynus americanus (Amphibia,Anura)

Odontophrynus americanus (Amphibia, Anura) genomic DNA from diploid and tetraploid specimens was treated with restriction enzymes sensitive to cytosine and adenine methylation (5 meC and 6 meA). In both diploids and tetraploids a high proportion of the total DNA was not cleaved by 5 meC-sensitive enzymes as observed on agarose gels stained with ethidium bromide. The DNAs were transferred to nitrocellulose filters and hybridized with cloned fragments containing sequences of Xenopus laevis 28S and 18S ribosomal DNA (rDNA). A high level of methylation of the ribosomal repeat units was revealed by 5 meC-sensitive enzymes in blood, liver, kidney and testis tissues. Adenine was methylated to a lesser degree and similarly in the rDNA from both germinative and somatic tissues. Comparison of the results obtained with DNA of diploids and tetraploids showed that methylation of ribosomal genes was increased in tetraploid genomes of adult frogs, but exact quantitative determinations could not be performed by this methodology. Cloning of the 28S region of the rDNA repeat unit was performed in the gtWESC vector. Restriction patterns obtained with methylation-sensitive enzymes using diploid and tetraploid derived clones confirmed the high level of methylation of the corresponding region of the ribosomal repeat unit in genomic DNAs. The implications of these results in the regulation of expression of the ribosomal genes in diploids and tetraploids are discussed.     

Habitat differentiation, hybridization and gene flow patterns in mixed populations of diploid and autotetraploid Dactylorhiza maculata s.l. (Orchidaceae)

Detailed ecological, morphological and molecular analyses were performed in mixed populations of diploid and autotetraploid Dactylorhiza maculata s.l. in Scandinavia. Comparisons were made with pure populations of either diploid ssp. fuchsii or tetraploid ssp. maculata. It was shown that mixed populations are the result of secondary contact between ssp. fuchsii and ssp. maculata. No patterns of recent and local autopolyploidization were found. Morphology and nuclear DNA markers (internal transcribed spacers of nuclear ribosomal DNA) showed that diploids and tetraploids from mixed populations have similar levels of differentiation to diploids and tetraploids from pure populations. Vegetation analyses, as well as analyses of environmental variables, revealed that diploid and tetraploid individuals in mixed populations are ecologically well differentiated on a microhabitat level. Diploids and tetraploids in pure populations have wider ecological amplitudes than they do in mixed populations. Triploid hybrids grew in intermediate microhabitats between diploids and tetraploids in the mixed populations. Plastid DNA markers indicated that both diploids and tetraploids may act as the maternal parent. Based on morphology and nuclear markers triploids are more similar to tetraploids than to diploids. There were indications of introgressive gene flow between ploidy levels. Plastid markers indicated that gene flow from diploid to tetraploid level is most common, but nuclear markers suggested that gene flow in opposite direction also may occur. Similar patterns of differentiation and gene flow appeared in localities that represented contrasting biogeographic regions. Disturbance and topography may explain why hybridization was slightly more common and the differentiation patterns somewhat less clear in the Scandinavian mountains than in the coastal lowland. An erratum to this article can be found at     

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.     

Other tetraploid species and conspecific diploids as sources of genetic variation for an autotetraploid

? Premise of the study: Most plants are polyploid and have more than two copies of the genome. The evolutionary success of polyploids is often attributed to their potential to harbor increased genetic variation, but it is poorly understood how polyploids can attain such variation. Because of their formation bottleneck, newly formed tetraploids start out with little variation. Tetraploids may attain genetic variation through a combination of new mutations, recurrent formation, and gene exchange with diploid ancestors or related tetraploid species. We explore the role of gene exchange and introgression in autotetraploid Rorippa amphibia, a species that harbors more genetic variation than its diploid ancestors. ? Methods: We crossed autotetraploid R. amphibia to diploid conspecifics and tetraploid R. sylvestris and backcrossed resulting F(1) hybrids. We used flow cytometry to determine the ploidy of all progeny. ? Key results: Tetraploids of R. amphibia and R. sylvestris were interfertile; F(1) hybrids were fertile and could backcross. Crosses between diploids and tetraploids yielded a small number of viable, often tetraploid progeny. This indicates that unreduced gametes can facilitate gene flow from diploids to tetraploids. We detected a frequency of unreduced gametes of around 2.7 per 1000, which was comparable between diploids and tetraploids. ? Conclusions: Introgression from tetraploid R. sylvestris provides a realistic source of variation in autotetraploid R. amphibia. Only in a scenario where other compatible partners are absent, for example immediately after tetraploidization, gene flow through unreduced gametes from diploids could be an important source of genetic variation for tetraploids.     

Climate‐dependent variation in cold tolerance of weedy rice and rice mediated by OsICE1 promoter methylation

The mechanisms by which weedy rice (Oryza sativa f. spontanea) has adapted to endure low‐temperature stress in northern latitudes remain unresolved. In this study, we assessed cold tolerance of 100 rice varieties and 100 co‐occurring weedy rice populations, which were sampled across a broad range of climates in China. A parallel pattern of latitude‐dependent variation in cold tolerance was detected in cultivated rice and weedy rice. At the molecular level, differential cold tolerance was strongly correlated with relative expression levels of CBF cold response pathway genes and with methylation levels in the promoter region of OsICE1, a regulator of this pathway. Among all methylated cytosine sites of the OsICE1 promoter, levels of CHG and CHH methylation were found to be significantly correlated with cold tolerance among accessions. Furthermore, within many of the collection locales, weedy rice shared identical or near‐identical OsICE1 methylation patterns with co‐occurring cultivated rice. These findings provide new insights on the possible roles that methylation variation in the OsICE1 promoter may play in cold tolerance, and they suggest that weedy rice can rapidly acquire cold tolerance via methylation patterns that are shared with co‐occurring rice cultivars.     

铜胁迫对不同抗性种群海州香薷酸性转化酶基因启动子甲基化的影响

采用亚硫酸氢盐测序技术,检测了Cu胁迫对海州香薷（Elsholtzia haichowensis Sun） Cu抗性和非抗性种群酸性转化酶基因启动子甲基化的影响。结果表明,海州香薷液泡转化酶基因（vINV）和细胞壁转化酶基因（cwINV）的启动子在CG位点分别表现出超甲基化和低甲基化的现象。酸性转化酶基因启动子CHG和CHH位点的甲基化状态受Cu胁迫影响较大。Cu胁迫下,vINV和cwINV启动子分别有1个CHG和6个CHH位点的甲基化状态在Cu抗性种群和非抗性种群之间表现出较大差异。抗Cu种群中这些甲基化差异位点对Cu胁迫不敏感,但在非抗性种群中这些位点的甲基化水平在Cu胁迫后出现大幅上升或下降。有些甲基化差异位点位于或者临近预测的启动子顺式作用元件区域,可能参与Cu胁迫下酸性转化酶基因的表达调控。     

Pollen competition as a unilateral reproductive barrier between sympatric diploid and tetraploid Chamerion angustifolium

Speciation requires the evolution of barriers to gene exchange between descendant and progenitor populations. Cryptic reproductive barriers in plants arise after pollination but before fertilization as a result of pollen competition and interactions between male gametophytes and female reproductive tissues. We tested for such gametic isolation between the polyploid Chamerion angustifolium and its diploid progenitor by conducting single (diploid or tetraploid) and mixed ploidy (1 : 1 diploid and tetraploid) pollinations on both cytotypes and inferring siring success from paternity analysis and pollen-tube counts. In mixed pollinations, polyploids sired most (79%) of their own seeds as well as those of diploids (61%) (correcting for triploid block, siring success was 70% and 83%, respectively). In single donor pollinations, pollen tubes from tetraploids were more numerous than those from diploids at four different positions in each style and for both diploid and tetraploid pollen recipients. The lack of a pollen donor x recipient interaction indicates that the tetraploid siring advantage is a result of pollen competition rather than pollen-pistil interactions. Such unilateral pollen precedence results in an asymmetrical pattern of isolation, with tetraploids experiencing less gene flow than diploids. It also enhances tetraploid establishment in sympatric populations, by maximizing tetraploid success and simultaneously diminishing that of diploids through the production of inviable triploid offspring.     

植物表观遗传修饰与病原菌胁迫应答研究进展

真核生物基因表达受到染色质结构的调控,组蛋白与DNA的共价修饰构成表观遗传标签,并在植物胁迫应答如防御病原菌侵染过程中起重要作用.病原菌侵染可引起基因组整体DNA甲基化模式变化及胁迫应答基因的位点特异性去甲基化,导致植物抗性基因表达上调或下调,并进一步调控植物对病原菌的胁迫应答;组蛋白去乙酰化酶HDAC通过茉莉酸途径增强植物对病原菌的胁迫应答;此外,染色质重塑复合物Swr1复合体通过识别DNA基元和组蛋白乙酰化修饰状态靶向基因启动子,负调控SA敏感基因.该文从DNA甲基化、组蛋白乙酰化、甲基化修饰,染色质重塑等方面着重阐述植物与病原菌互作过程中发生的主要事件的分子基础及其研究进展.     

Relationships among genetic distance,forage yield and heterozygosity in isogenic diploid and tetraploid alfalfa populations

Isogenic diploid and tetraploid alfalfa (Medicago sativa L.) was studied with molecular markers to help understand why diploid performance and breeding behavior does not always predict that of tetraploids. In a previous study of partially heterozygous alfalfa genotypes, we detected a low correlation between yields of isogenic diploid (2x) and tetraploid (4x) single-cross progenies, and genetic distances were more highly correlated with yields of tetraploids than diploids. These differences may be related to the level of RFLP heterozygosity expected among progenies derived from heterozygous parents at the two ploidy levels. The objectives of this study were to determine the relationships among genetic distance, forage yield and heterozygosity in isogenic 2 x and 4 x alfalfa populations. Four diploid genotypes were chromosome doubled to produce corresponding isogenic autotetraploids, and these genotypes were mated in 4 × 4 diallels to produce 6 single-cross families at each ploidy level for field evaluation. Allele compositions of parents were determined at 33 RFLP loci by monitoring segregation of homologous restriction fragments among individuals within progenies, and these were used to estimate RFLP heterozygosity levels for all single-cross progenies at both ploidy levels. RFLP heterozygosity rankings were identical between progenies of isogenic diploid and tetraploid parents; but significant associations (P < 0.05) between estimated heterozygosity levels and forage yield were detected only at the tetraploid level. Since tetraploid families were nearly 25% more heterozygous than the corresponding diploid families, inconsistencies in the association between molecular marker diversity and forage yields of isogenic 2 x and 4 x single crosses may be due to recessive alleles that are expressed in diploids but masked in tetraploids. The gene action involved in heterosis may be the same at both ploidy levels; however, tetraploids benefit from greater complementary gene interactions than are possible for equivalent diploids. Present address: AgResearch Grasslands, New Zealand Pastoral Agriculture Research Institute, Palmerston North, New Zealand     

Frequencies and variation in cytosine methylation patterns in diploid and tetraploid cytotypes of Paspalum notatum

Paspalum notatum Flügge is a grass species organized as an agamic complex. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in diploid and tetraploid genotypes, and to determine the occurrence of methylation changes associated with tetraploidization by using methylation-sensitive amplification polymorphism (MSAP) markers. No differences were found in the average proportions of methylated CCGG sites between cytotypes, but methylation patterns were significantly more variable in tetraploids. In both groups of plants, epigenetic and non-epigenetic variation correlated significantly when compared by Mantel tests. The evaluation of 159 common MSAP markers showed that 18.86 % of them differed in their methylation status in the different ploidies. Dendrogram analysis, reflecting epigenetic distances, showed that the four diploids and one experimentally-obtained sexually-reproducing tetraploid, grouped together. MSAP analysis performed on a diploid plant and its autotetraploid derivative showed that new epialleles emerged after tetraploidization. Sequencing of several MASP markers showed homologies with low copy genes, non-coding sequences and transposon/retrotransposon elements.     

Methyl jasmonate regulated diploid and tetraploid black locust (<Emphasis Type="Italic">Robinia pseudoacacia</Emphasis> L.) tolerance to salt stress

Methyl jasmonate (MeJA) is an essential and promising plant growth regulation factor that can improve plant development and growth. Here, we explored the mechanism by which MeJA regulates the tolerance of black locust (Robinia pseudoacacia L.) to salt stress. In this study, diploid and tetraploid R. pseudoacacia were subjected to three treatments: 500 mM NaCl; 100 μM MeJA; and 500 mM NaCl and 100 μM MeJA, and the changes in plant growth, endogenous MeJA levels and the anti-oxidative metabolism of leaves were investigated. The results showed that salt stress significantly inhibited plant growth and induced the accumulation of Na⁺ and Cl^? ions, malondialdehyde (MDA) content and reactive oxygen species. However, these adverse effects could be alleviated by applying MeJA, which was followed by a marked increase in the activities of antioxidant enzymes. In addition, some genes encoding several antioxidant enzymes were also up-regulated. Simultaneously, the endogenous MeJA content in MeJA-treated plants was lower than in salt-treated plants. It is noteworthy that tetraploids always possessed higher salt tolerance and obtained greater positive effects from MeJA than diploids. These results suggested that MeJA might play a protective role in defense responses, enabling diploid and tetraploid black locust, especially tetraploid, to better tolerate the adverse effects of salt stress.     

A CYTOGENETIC ANALYSIS OF CERTAIN POLYPLOIDS IN GRINDELIA (COMPOSITAE)

Two diploid taxa, Grindelia procera and G. camporum, and 3 tetraploid ones, G. camporum, G. hirsutula, and G. stricta, have been studied to ascertain their interrelationships. Meiosis in diploid parental strains was regular, the common chromosome configuration being 5 rod bivalents and 1 ring bivalent. The average chiasmata frequency per chromosome was 0.60. Pollen fertility was about 90% in all strains examined. Diploid interspecific hybrids had normal meiosis with an average chiasmata frequency of 0.56 per chromosome. No heterozygosity for inversions or interchanges was detected, and pollen fertility was above 85%. Meiosis in parental tetraploid strains was characterized by the presence of quadrivalents in addition to a complementary number of bivalents. The average chiasmata frequency per chromosome was 0.59 and pollen fertility was generally about 80%. Tetraploid interspecific hybrids also had quadrivalents, normal meiosis, and high pollen fertility. Close genetic relationships between the diploids and between the tetraploids are indicated, and geographical, ecological, and seasonal barriers to gene exchange exist. Attempts to obtain hybrids between diploids and tetraploids were successful in a few cases. The hybrids were tetraploid and had normal meiosis and fertility similar to parental and F₁ tetraploids. Their origin was by the union of unreduced gametes of the diploid female parent and normal pollen from the tetraploid parent. On the basis of chromosome homology, normal meiosis, plus high fertility exhibited in the diploid, tetraploid, and diploid X tetraploid interspecific hybrids, these species of Grindelia are considered to be a part of an autopolyploid complex. Gene exchange between diploids and diploids, tetraploids and tetraploids, and diploids and tetraploids is possible. Tetraploid G. camporum may have originated by hybridization between G. procera and diploid G. camporum with subsequent doubling of chromosomes and selection for the combined characteristics of the diploids.     

Bisulphite sequencing reveals dynamic DNA methylation under desiccation and salinity stresses in rice cultivars

DNA methylation governs gene regulation in plants in response to environmental conditions. Here, we analyzed role of DNA methylation under desiccation and salinity stresses in three (IR64, stress-sensitive; Nagina 22, drought-tolerant and Pokkali, salinity-tolerant) rice cultivars via bisulphite sequencing. Methylation in CG context within gene body and methylation in CHH context in distal promoter regions were positively correlated with gene expression. Hypomethylation in Nagina 22 and hypermethylation in Pokkali in response to desiccation and salinity stresses, respectively, were correlated with higher expression of few abiotic stress response related genes. Most of the differentially methylated and differentially expressed genes (DMR-DEGs) were cultivar-specific, suggesting an important role of DNA methylation in abiotic stress responses in rice in cultivar-specific manner. DMR-DEGs harboring differentially methylated cytosines due to DNA polymorphisms between the sensitive and tolerant cultivars in their promoter regions and/or coding regions were identified, suggesting the role of epialleles in abiotic stress responses.     

DNA methylation of retrotransposons,DNA transposons and genes in sugar beet (Beta vulgaris L.)

The methylation of cytosines shapes the epigenetic landscape of plant genomes, coordinates transgenerational epigenetic inheritance, represses the activity of transposable elements (TEs), affects gene expression and, hence, can influence the phenotype. Sugar beet (Beta vulgaris ssp. vulgaris), an important crop that accounts for 30% of worldwide sugar needs, has a relatively small genome size (758 Mbp) consisting of approximately 485 Mbp repetitive DNA (64%), in particular satellite DNA, retrotransposons and DNA transposons. Genome‐wide cytosine methylation in the sugar beet genome was studied in leaves and leaf‐derived callus with a focus on repetitive sequences, including retrotransposons and DNA transposons, the major groups of repetitive DNA sequences, and compared with gene methylation. Genes showed a specific methylation pattern for CG, CHG (H = A, C, and T) and CHH sites, whereas the TE pattern differed, depending on the TE class (class 1, retrotransposons and class 2, DNA transposons). Along genes and TEs, CG and CHG methylation was higher than that of adjacent genomic regions. In contrast to the relatively low CHH methylation in retrotransposons and genes, the level of CHH methylation in DNA transposons was strongly increased, pointing to a functional role of asymmetric methylation in DNA transposon silencing. Comparison of genome‐wide DNA methylation between sugar beet leaves and callus revealed a differential methylation upon tissue culture. Potential epialleles were hypomethylated (lower methylation) at CG and CHG sites in retrotransposons and genes and hypermethylated (higher methylation) at CHH sites in DNA transposons of callus when compared with leaves.     

Isoenzyme polymorphism of the sorbitol dehydrogenase and the NADP-dependent isocitrate dehydrogenases in the fish family Cyprinidae

Among members of the fish family Cyprinidae , the existence of a diploid-tetraploid relationship is well established. The analysis of individual gene loci, using isoenzyme polymorphism as genetic markers, does not always confirm the expected gene duplication in the tetraploids. Of the markers used in this study, only the M-form of the NADP-dependent isocitrate dehydrogenase follows this expectation; the data suggest the existence of a single gene locus for the enzyme in diploids, while the observations on tetraploids were consistent with control by two distinct loci. For two other enzymes, the S-form of the NADP-dependent isocitrate dehydrogenase and sorbitol dehydrogenase, no difference seems to exist in the number of gene loci between diploids and tetraploids. A comparison between Cyprinid fish (order Ostariophysi ) and members of the order Isospondyli in which another diploid-tetraploid relationship was established, reveals that gene duplications are more frequently demonstrable within tetraploid Isospondyli than in tetraploid Cyprinidae. From this, it is concluded that polyploidization occurred earlier in evolution of Cyprinidae than of Isospondyli.     

Influence of ploidy level on morphology,growth and drought susceptibility in <Emphasis Type="Italic">Spathiphyllum wallisii</Emphasis>

In this study, we analysed morphological, anatomical and physiological effects of polyploidisation in Spathiphyllum wallisii in order to evaluate possible interesting advantages of polyploids for ornamental breeding. Stomatal density was negatively correlated with increased ploidy level. Stomatal size increased in polyploids. Tetraploid Spathiphyllum plants had more ovate and thicker leaves. The inflorescence of tetraploids had a more ovate and thicker spathum, a more cylindrical spadix and a thicker but shorter flower stalk. Biomass production of the tetraploids was reduced, as expressed by lower total dry weights, and tetraploids produced fewer shoots and leaves compared with their diploid progenitors. Furthermore, tetraploid Spathiphyllum plants were more resistant to drought stress compared with diploid plants. After 15 days of drought stress, diploids showed symptoms of wilting, while the tetraploids showed almost no symptoms. Further, measurements of stomatal resistance, leaf water potential, relative water content and proline content indicated that the tetraploid genotypes were more resistant to drought stress compared with the diploids.