Comparative fecundity of morphs in a mixed monoclinous-andromonoecious population ofWurmbea dioica subsp.alba (Liliaceae) in Western Australia

A population ofWurmbea dioica subsp.alba in Western Australia contained monoclinous and andromonoecious individuals in roughly equal proportions. The average number of flowers per inflorescence for the former was 2.6 and for the latter 2.9, with its terminal flower staminate. Ovule number and seed production per perfect flower of both morphs decreased progressively from lower to upper flowers in the inflorescence. Two-flowered monoclinous individuals had a greater percentage of ovules maturing to seed than did threeflowered ones, but seed production of the latter exceeded that of the former. No differences in percent seed set or in seed production were noted for similar individuals of andromonoecious individuals. Although monoclinous and andromonoecious plants apparently contributed equally to the pollen pool, seed production of the former exceeded that of the latter. Thus, the energetic costs of monocliny exceed those of andromonoecy in this population.     

DEVELOPMENTAL PLASTICITY,GENETIC VARIATION,AND THE EVOLUTION OF ANDROMONOECY IN SOLANUM HIRTUM (SOLANACEAE)

Studies of andromonoecious species have shown that sex expression (proportions of hermaphrodite and staminate flowers) is quite variable. It is not known, however, whether this variation is due to variation among individuals for genetically fixed patterns of allocation to staminate and hermaphrodite flowers (population level variation) and/or to developmental plasticity of individuals in a heterogeneous environment (organismal level variation). Distinguishing between these two levels of variation is important for understanding the evolution of andromonoecy. This study investigates levels of variation in sex expression in the andromonoecious Solanum hirlum. Sex expression in this species is shown to be plastic among individuals of the same genotype (organismal level variation) and determined, in part, by the resource status of the individual. Among the genotypes examined there is also genetic variation for developmental plasticity. Thus, developmental plasticity can potentially respond to selection, and the evolution of this developmental system may have been instrumental in the establishment and maintenance of andromonoecy in S. hirtum.     

FUNCTIONAL DIOECY AND ANDROMONOECY IN SOLANUM

Field and laboratory studies of 19 diclinous species endemic to Australia help to clarify the nature and evolution of andromonoecy, androdioecy, and dioecy in the genus Solanum. Ten species are andromonoecious; typically these species bear inflorescences with a single, large basal hermaphroditic flower and 12–60 distal, smaller staminate flowers. We suggest that the andromonoecious condition was derived from hermaphroditic-flowered ancestors in part by hemisterilization of flowers but largely by addition of staminate flowers. The resultant larger inflorescences are hypothesized to serve both to attract and to entrain pollinators, yielding more or higher-quality seed set in hermaphroditic flowers and/or greater dispersion of pollen from staminate flowers. We suggest that andromonoecy may also serve to reduce selling. Nine other species are morphologically androdioecious but functionally dioecious. In these species, staminate flowers, like those of the andromonoecious species, bear anthers with copious tricolporate pollen and a highly reduced gynoecium. The morphologically hermaphroditic flowers are functionally pistillate and borne singly in inflorescences, and they bear anthers with inaperturate pollen. The inaperturate pollen, although viable, never germinates and is hypothesized to be retained in pistillate flowers as a reward to pollinators in the nectarless Solanum flowers. All other species of Solanum studied with pollen dimorphism in which one pollen morph is inaperturate are also best treated as functionally dioecious. We conclude that there is no evidence for androdioecy in Solanum. A review of other families suggests that there is little support for this unusual breeding system in any other angiosperm group either. Preliminary analyses suggest that andromonoecy and dioecy are polyphyletic in Solanum. Furthermore, dioecy is as likely to have arisen from hermaphroditic as from andromonoecious ancestors.     

Divergent methylation pattern in adult stage between two forms of Tetranychus urticae (Acari: Tetranychidae)

The two‐spotted spider mite, Tetranychus urticae Koch has two forms: green form and red form. Understanding the molecular basis of how these two forms established without divergent genetic background is an intriguing area. As a well‐known epigenetic process, DNA methylation has particularly important roles in gene regulation and developmental variation across diverse organisms that do not alter genetic background. Here, to investigate whether DNA methylation could be associated with different phenotypic consequences in the two forms of T. urticae, we surveyed the genome‐wide cytosine methylation status and expression level of DNA methyltransferase 3 (Tudnmt3) throughout their entire life cycle. Methylation‐sensitive amplification polymorphism (MSAP) analyses of 585 loci revealed variable methylation patterns in the different developmental stages. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between female adults of the two forms. The gene expression of Tudnmt3 was detected in all examined developmental stages, which was significantly different in the adult stage of the two forms. Together, our results reveal the epigenetic distance between the two forms of T. urticae, suggesting that DNA methylation might be implicated in different developmental demands, and contribute to different phenotypes in the adult stage of these two forms.     

Newly developed MSAP analysis reveals the different polymorphism patterns in transgenic tobacco plants with the dsRNA MET1 gene

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we isolated NtMET1 from Nicotiana tabacum cv. Havana (SR1) and obtain transgenic plants that reduced MET1 expression level with the double-strand RNA (dsRNA) MET1 gene. Transgenic tobacco plants showed dwarf and abnormal flower development when compared with the wild type. Using methylation-sensitive amplified polymorphism (MSAP) analysis, the patterns of cytosine methylation in transformed plants and the wild type were compared. MseI/HpaII selection primers showed an interesting polymorphism, and 153 DNA bands of interest were detected. Among these, 30 selective fragments were sequenced and analyzed with a BLAST search by successful MSAP modifications. The homology search showed that the transposons and tandem repeated sequences were related to the phenotypes. These results suggested that the decreased degree of methylation by dsRNA strategy caused abnormal growth and development in N. tabacum.     

Andromonoecy in Solanum lycocarpum A. St. -Hil. (Solanaceae): Floral attributes,visitors and variation in sexual expression over time

Sexual expression in andromonoecious species—those in which a single individual can bear both staminate and hermaphroditic flowers—may vary among reproductive events in the same plant, among individuals and across populations. This variation influences, in turn, the individual contribution of hermaphroditic plants via male and female fitness functions (i.e., Lloydʼs phenotypic gender). However, temporal variation in sexual expression in andromonoecious species and its relationship with seasonal changes in climatic conditions remain poorly understood. Here we analyze floral attributes, visitors and variation in sexual expression in three populations of Solanum lycocarpum A. St. -Hil. Seasonality in the production of floral types, the mating system and floral visitors were also investigated. Hermaphroditic flowers produced more pollen grains, but the pollen of staminate flowers had higher viability. Only hermaphroditic flowers produced fruits, and ovules in staminate flowers were sterile. Solanum lycocarpum is mainly pollinated by large bees with the ability to vibrate flowers. Phenotypic gender varied throughout the year, and the seasonal production of staminate flowers is associated with the local climate. We suggest that the higher and seasonally variable relative abundance of staminate flowers compared to the low and uniform production of hermaphroditic flowers may be explained by (a) the very high energetic costs incurred in producing large fruits, which in turn make hermaphroditic flower production very costly, and (b) the potentially lower energy expenditure of the smaller staminate flowers with reduced pistils and non-viable ovules that allow them to rapidly respond to climate variability.     

Diversification of andromonoecy in Solanum section Lasiocarpa (Solanaceae): the roles of phenotypic plasticity and architecture

Quantitative analyses of sexual expression show extensive interspecific variation in the strength of andromonoecy (proportions of hermaphroditic and staminate flowers) among Solanum species in the monophyletic section Lasiocarpa. The roles of phenotypic plasticity and inter- and intra-inflorescence architecture in the diversification of andromonoecy within this small clade were analyzed. Four species that represent a range of expression of andromonoecy were examined. Staminate flowers produced within inflorescences ranged from 3% (S. candidum) to 7% (S. ferox) in weakly andromonoecious species and from 39% (S. pseudolulo) to 60% (S. quitoense) in more strongly andromonoecious species. Manipulation of fruit set on clonal replicates of multiple genotypes demonstrated variation among species for phenotypic plasticity. The strongly andromonoecious species, S. pseudolulo and S. quitoense, were not plastic and produced a large proportion of staminate flowers regardless of fruiting treatment, whereas S. candidum and S. ferox were phenotypically plastic and produced significantly more staminate flowers in the presence of developing fruit. Staminate flower production of all four species varied both within and among inflorescences. A greater proportion of staminate flowers were produced in distal (later produced) inflorescences. Within inflorescences, hermaphroditic flowers occurred in basal positions, whereas staminate flowers, when produced, occurred more distally. This pattern of staminate flower production is qualitatively the same in all species investigated; however, quantitative variation in the transition from hermaphroditic to staminate flower production within and among inflorescences is associated with variation in the strength of andromonoecy. At least three factors have contributed to the diversification of andromonoecy in section Lasiocarpa including the presence or absence of phenotypic plasticity in response to fruit set, quantitative variation in intra- and inter-inflorescence architectural effects, and total flower production.     

Analysis of DNA methylation patterns of PLBs derived from <Emphasis Type="Italic">Cymbidium hybridium</Emphasis> based on MSAP

The best known and most thoroughly studied epigenetic phenomenon is DNA methylation, which plays an important role in regulating gene expression during plant regeneration and development. In this study, the methylation-sensitive amplified polymorphism (MSAP) technique was carried out to determine differences in methylation profiles between two forms of protocorm-like bodies (PLBs), continuously proliferating PLBs (cPLBs) and spontaneously-differenting PLBs (sdPLBs), derived from cultures of Cymbidium hybridium. A total of 72 selective primer combinations were used to assess the status of cytosine methylation of DNA in these tissues. Of 4,440 fragments obtained 911 fragments, each representing a recognition site cleaved by one or both of the isoschizomers (Hpa II and Msp I), were amplified and were significantly different between the two forms of PLBs. Frequency of total and full-methylation of cPLBs and sdPLBs were 26.7/12.2%, 24.1/11.1%, respectively. In addition, 14 types of MSAP patterns detected in the two forms of PLBs belonged to two classes, type I and II. Sequencing of 14 differentially methylated fragments and their subsequent blast search revealed that cytosine methylated 5′-CCGG-3′ sequences were equally distributed in the coding and non-coding regions. Southern blotting was conducted to verify the methylation polymorphism.     

MSAP markers and global cytosine methylation in plants: a literature survey and comparative analysis for a wild‐growing species

Methylation of DNA cytosines affects whether transposons are silenced and genes are expressed, and is a major epigenetic mechanism whereby plants respond to environmental change. Analyses of methylation‐sensitive amplification polymorphism (MS‐AFLP or MSAP) have been often used to assess methyl‐cytosine changes in response to stress treatments and, more recently, in ecological studies of wild plant populations. MSAP technique does not require a sequenced reference genome and provides many anonymous loci randomly distributed over the genome for which the methylation status can be ascertained. Scoring of MSAP data, however, is not straightforward, and efforts are still required to standardize this step to make use of the potential to distinguish between methylation at different nucleotide contexts. Furthermore, it is not known how accurately MSAP infers genome‐wide cytosine methylation levels in plants. Here, we analyse the relationship between MSAP results and the percentage of global cytosine methylation in genomic DNA obtained by HPLC analysis. A screening of literature revealed that methylation of cytosines at cleavage sites assayed by MSAP was greater than genome‐wide estimates obtained by HPLC, and percentages of methylation at different nucleotide contexts varied within and across species. Concurrent HPLC and MSAP analyses of DNA from 200 individuals of the perennial herb Helleborus foetidus confirmed that methyl‐cytosine was more frequent in CCGG contexts than in the genome as a whole. In this species, global methylation was unrelated to methylation at the inner CG site. We suggest that global HPLC and context‐specific MSAP methylation estimates provide complementary information whose combination can improve our current understanding of methylation‐based epigenetic processes in nonmodel plants.     

Flower-like heads from flower-like meristems: pseudanthium development in <Emphasis Type="Italic">Davidia involucrata</Emphasis> (Nyssaceae)

Flower-like inflorescences (pseudanthia) have fascinated botanists for a long time. They are explained as condensed inflorescences implying that the pseudanthium develops from an inflorescence meristem (IM). However, recent developmental studies identified a new form of reproductive meristem, the floral unit meristem (FUM). It differs from IMs by lacking acropetal growth and shares fractionation, expansion and autonomous space filling with flower meristems (FM). The similarity among FUMs and FMs raises the question how far flower-like heads originate from flower-like meristems. In the present paper, pseudanthium development in Davidia involucrata is investigated using scanning electron microscopy. D. involucrata has pincushion-shaped heads composed of densely aggregated, perianthless flowers and associated with two large showy bracts. Early developmental stages show a huge naked FUM. The FMs appear almost simultaneously and lack subtending bracts. With ongoing FUM expansion new space is generated which is immediately used by further FM fractionation. The heads have only staminate flowers or are andromonoecious with staminate and a single perfect flower in oblique position. All FMs lack perianth structures and fractionate a variable number of stamen primordia. The perfect FM is much larger than the staminate FMs and forms a syncarpous gynoecium with inferior ovary. Pseudanthium development in D. involucrata confirms the morphogenetic similarity to FMs as to acropetal growth limitation, meristem expansion and fractionation. It thus should not be interpreted as a condensed inflorescence, but as a flower equivalent. Furthermore as the FUM develops inside a bud, its development is considered to be influenced by mechanical pressure. The oblique position of the perfect flower, the developmental delay of the proximal flowers, and the variable number of stamens which were observed in the pseudanthium development, can be caused by mechanical pressure. Next to the Asteraceae, D. involucrata offers a further example of a pseudanthium originating from a FUM. More knowledge on FUMs is still needed to understand diversification and evolution of flower-like inflorescences.     

Tissue culture-induced locus-specific alteration in DNA methylation and its correlation with genetic variation in <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> Benth. et Hook. f

We have reported recently that tissue culture induced a high level of genetic variation at the primary nucleotide sequence in regenerants of medicinal plant Codonopsis lanceolata. It is not known, however, whether epigenetic variation in the form of alteration in DNA methylation also occurred in these plants. Here, we investigated possible alterations in level and pattern of cytosine methylation at the CCGG sites in the same set of regenerants relative to the donor plant, by the MSAP method employing a pair of isoschizomers, HpaII and MspI, which recognize the same restriction site but are differentially sensitive to cytosine methylation at the CCGG sites. A total of 1,674 MSAP profiles were resolved using 39 primer combinations. Of these, 177 (10.5%) profiles were polymorphic among the regenerants and/or between the regenerant(s) and the donor plant, in EcoRI + HpaII or EcoRI + MspI digest but not in both, indicating alteration in cytosine methylation patterns of specific loci, though their estimated total level of methylation remained more or less the same as the donor plant. Gel blot analysis validated most of the variant MSAP profiles as bona fide alteration in methylation patterns. Correlation analysis between the MSAP data and the previously reported ISSR and RAPD data revealed significant correlations, suggesting their possible intrinsic interrelatedness. Thirty-seven typical variant MSAP profiles were isolated and sequenced, of which 5 showed significant homology to known-function genes, 2 to chloroplast sequences, whilst the rest 30 did not find a match in the database. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. W. L. Guo and R. Wu contributed equally to this work.     

Differential DNA methylation between two wing phenotypes adults of Sogatella furcifera

Sogatella furcifera is a major rice pest with wing dimorphism . DNA methylation is an important epigenetic modification that plays a role in gene regulation and phenotype variation in most organisms. The objective of the current research was to survey the frequencies and variation of cytosine methylation at CCGG sequences in macropterous female adults (MFA) and brachypterous female adults (BFA) of S. furcifera, and to determine the occurrence of methylation changes associated with wing phenotypes by using methylation‐sensitive amplification polymorphism (MSAP). No differences were found in the average proportions of methylated CCGG sites between MFA and BFA, but there were significant differences for methylation patterns between MFA and BFA. The fully methylated ratio was 5.81% in BFA, much higher than 2.40% in MFA; while the hemi‐methylated ratio was 4.35% in BFA, much lower than 8.35% in MFA. These results provide circumstantial evidence that DNA methylation might be related to wing phenotype variation in S. furcifera. We also cloned and got 14 satisfactory sequences, which displayed variable cytosine methylation patterns between MFA and BFA. All these data will facilitate the researches on the epigenetic mechanisms of insect wing polymorphism. genesis 51:819–826. © 2013 Wiley Periodicals, Inc.     

Detection of epigenetic variation in tissue-culture-derived plants of <Emphasis Type="Italic">Doritaenopsis</Emphasis> by methylation-sensitive amplification polymorphism (MSAP) analysis

Somaclones exhibiting variations with flower characteristics were recovered from the tissue-culture-derived plants of Doritaenopsis. Two molecular techniques, random amplified polymorphic DNA (RAPD) and methylation-sensitive amplification polymorphism (MSAP) analyses, were used to characterize the somaclones. RAPD analysis, using 100 randomly selected primers, failed to differentiate variants and normal plants, even though some primers (six out of 100 primers) exhibited 6–10 distinct banding patterns. However, MSAP analysis revealed the differences in the DNA methylation patterns in the normal and variant plants which were correlated with phenotypic variation. In all, 311, 337, 366, and 343 fragments were obtained with normal and V1, V2, and V3 variant plants, respectively; each representing recognition site cleaved by either or both of the isoshizomers were amplified using 12 combination of primers. A total of 36 (11.6%), 77 (22.9%), 73 (19.9%), and 47 (13.7%) sites were found to be methylated at cytosine in the genomes of normal and V1, V2, and V3 variant Doritaenopsis plants. This study demonstrates usefulness of MSAP to detect DNA methylation events in tissue cultured Doritaenopsis plants.     

The expression of andromonoecy in Solanum hirtum (Solanaceae): phenotypic plasticity and ontogenetic contingency

Sex expression (the proportions of staminate and hermaphrodite flowers produced) in andromonoecious Solarium hirtum is phenotypically plastic, and there is genetic variation for sex expression plasticity. Changes in sex expression phenotype are inherently the result of altered development. However, the underlying developmental components of sex expression plasticity and of differences in plasticity among genotypes are unknown. This study takes an explicitly genetic and developmental approach to the study of phenotypic plasticity and examines changes in sex expression of ten clonally replicated genotypes at three levels of organization: among inflorescences, within inflorescences, and at the level of developing floral meristems. Changes in sex expression of individuals and differences among individuals are the result of a predictable interplay of resource, architectural, and floral level response within the hierarchical construction of the shoot system. Phenotypic plasticity of whole plant sex expression is ultimately due to sexual lability of individual developing flowers: floral sex is not determined until a primordium size of 9–10 mm. Until that time, sex expression remains labile and developing floral primordia can respond to changes in plant resource status. This flower level developmental lability, however, is expressed within the constraints set by the architecture and ontogenetic history of the organism. Only those floral primordia produced in distal portions of each inflorescence are labile, capable of developing into either a staminate or hermaphrodite flower, whereas those primordia in basal positions invariably develop as hermaphrodite flowers. The genotypes differ with respect to the architectural components of phenotypic plasticity and it is this architectural variation that results in differences in plasticity among genotypes. The phenomenon, in which the developmental fate of a primordium depends upon where and when it is produced within the architecture of an organism and what events have preceded it during ontogeny, can be termed “ontogenetic contingency.”     

Changes in DNA-methylation during zygotic embryogenesis in interspecific hybrids of beans (<Emphasis Type="Italic">Phaseolus</Emphasis> ssp.)

Hybrid embryos resulting from crosses between Phaseolus species often fail to reach maturity and some combinations frequently abort at early developmental stages. The genetic or molecular basis for these consistent developmental defects is at present not clear. However, an extremely complex genetic system, thought to be caused by major epigenetic changes associated with gene expression changes, has been shown to be active in plant species. We have investigated DNA methylation in two interspecific hybrids, Phaseolus vulgaris × Phaseolus coccineus and its reciprocal crosses, using methylation sensitive amplification polymorphism (MSAP). The potential use of MSAP for detecting methylation variation during embryogenesis in interspecific hybrids is discussed. Significant differences in the DNA methylation patterns were observed in abortive (interspecific hybrids) and non abortive (parental) genotypes. Taken together, our results strongly suggest that generalized alterations in DNA methylation profiles could play a causative role in early interspecific embryo abortion in vivo. A considerable change in the methylation pattern during embryogenesis could be involved in the disruption of the regulation or maintenance of the embryogenesis process of Phaseolus interspecific hybrids. The results also support the earlier hypothesis that DNA methylation is critical for the regulation of plant embryogenesis and gene expression.     

Utility of the methylation-sensitive amplified polymorphism (MSAP) marker for detection of DNA methylation polymorphism and epigenetic population structure in a wild barley species (<Emphasis Type="Italic">Hordeum brevisubulatum</Emphasis>)

We report here that by using a modified scoring criterion, the methylation-sensitive amplified polymorphism or MSAP marker can be used effectively to detect polymorphism in DNA methylation patterns within and among populations of a perennial wild barley species, Hordeum brevisubulatum. Twenty-four selected individual genotypes representing four natural populations of H. brevisubulatum distributed in the Songnen Prairie in northeastern China were studied. The utility of MSAP was evidenced by its detection of high levels of polymorphism in DNA methylation patterns between individuals within a given population, and the clear inter-population differentiation in methylation patterns (methylation-based epigenetic population structure) revealed among the four populations. The resolving power of MSAP to detect DNA methylation polymorphism was found to be comparable with that of a retrotransposon-based sequence-specific amplified polymorphism marker, or SSAP, to detect genetic polymorphism in the same set of plants, suggesting that MSAP with a modified scoring criterion can be used efficiently to detect DNA methylation polymorphism and assess epigenetic population structure in natural plant populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.