Hypomethylation of the aquatic invasive plant,Ludwigia grandiflora subsp. hexapetala mimics the adaptive transition into the terrestrial morphotype

Ongoing global changes affect ecosystems and open up new opportunities for biological invasion. The ability of invasive species to rapidly adapt to new environments represents a relevant model for studying short-term adaptation mechanisms. The aquatic invasive plant, Ludwigia grandiflora subsp. hexapetala, is classified as harmful in European rivers. In French wet meadows, this species has shown a rapid transition from aquatic to terrestrial environments with emergence of two distinct morphotypes in 5 years. To understand the heritable mechanisms involved in adjustment to such a new environment, we investigate both genetic and epigenetic as possible sources of flexibility involved in this fast terrestrial transition. We found a low overall genetic differentiation between the two morphotypes arguing against the possibility that terrestrial morphotype emerged from a new adaptive genetic capacity. Artificial hypomethylation was induced on both morphotypes to assess the epigenetic hypothesis. We analyzed global DNA methylation, morphological changes, phytohormones and metabolite profiles of both morphotype responses in both aquatic and terrestrial conditions in shoot and root tissues. Hypomethylation significantly affected morphological variables, phytohormone levels and the amount of some metabolites. The effects of hypomethylation depended on morphotypes, conditions and plant tissues, which highlighted differences among the morphotypes and their plasticity. Using a correlative integrative approach, we showed that hypomethylation of the aquatic morphotype mimicked the characteristics of the terrestrial morphotype. Our data suggest that DNA methylation rather than a new adaptive genetic capacity is playing a key role in L. grandiflora subsp. hexapetala plasticity during its rapid aquatic to terrestrial transition.     

Congruence between morphological and molecular markers inferred from the analysis of the intra-morphotype genetic diversity and the spatial structure of Oxalis tuberosa Mol.

Oxalis tuberosa is an important crop cultivated in the highest Andean zones. A germplasm collection is maintained ex situ by CIP, which has developed a morphological markers system to classify the accessions into morphotypes, i.e. groups of morphologically identical accessions. However, their genetic uniformity is currently unknown. The ISSR technique was used in two experiments to determine the relationships between both morphological and molecular markers systems. The intra-morphotype genetic diversity, the spatial structures of the diversity and the congruence between both markers systems were determined. In the first experience, 44 accessions representing five morphotypes, clearly distinct from each other, were analyzed. At the molecular level, the accessions exactly clustered according to their morphotypes. However, a genetic variability was observed inside each morphotype. In the second experiment, 34 accessions gradually differing from each other on morphological base were analyzed. The morphological clustering showed no geographical structure. On the opposite, the molecular analysis showed that the genetic structure was slightly related to the collection site. The correlation between both markers systems was weak but significant. The lack of perfect congruence between morphological and molecular data suggests that the morphological system may be useful for the morphotypes management but is not appropriate to study the genetic structure of the oca. The spatial structure of the genetic diversity can be related to the evolution of the species and the discordance between the morphological and molecular structures may result from similar selection pressures at different places leading to similar forms with a different genetic background.     

Construction of genetic linkage map of the medicinal and ornamental plant <Emphasis Type="Italic">Catharanthus roseus</Emphasis>

An integrated genetic linkage map of the medicinal and ornamental plant Catharanthus roseus, based on different types of molecular and morphological markers was constructed, using a F₂ population of 144 plants. The map defines 14 linkage groups (LGs) and consists of 131 marker loci, including 125 molecular DNA markers (76 RAPD, 3 RAPD combinations; 7 ISSR; 2 EST-SSR from Medicago truncatula and 37 other PCR based DNA markers), selected from a total of 472 primers or primer pairs, and six morphological markers (stem pigmentation, leaf lamina pigmentation and shape, leaf petiole and pod size, and petal colour). The total map length is 1131.9 cM (centiMorgans), giving an average map length and distance between two markers equal to 80.9 cM and 8.6 cM, respectively. The morphological markers/genes were found linked with nearest molecular or morphological markers at distances varying from 0.7 to 11.4 cM. Linkage was observed between the morphological markers concerned with lamina shape and petiole size of leaf on LG1 and leaf, stem and petiole pigmentation and pod size on LG8. This is the first genetic linkage map of C. roseus.     

The many faces of Peridinium cinctum (Peridiniaceae,Peridiniales): morphological and molecular variability in a common dinophyte

Peridinium cinctum is a common freshwater dinophyte with a long history of research. Erich Lindemann was the first to assess intraspecific variability in this species focusing on plate pattern variation. Since then, this issue has been neglected but with the application of DNA sequence diagnostics, a combination of morphological and molecular characters may enable taxonomic delimitations. Our aim was to identify distinct morphotypes using plate pattern as the main characteristic and then compare them to the geographic occurrence of particular ribotypes (as inferred from sequences of the Internal Transcribed Spacer: ITS) in samples from Central Europe. Approximately 200 observations were carried out under the inverse light microscope for each of a total of 15 strains. We observed two main variations from the abundant plate pattern in P. cinctum, namely an unusual position of the 2a plate and the irregular shape of the 1a plate. In 88 (predominantly clonal) strains, we identified five different ribotypes (submitted as 71 new GenBank entries) which had no clear correlation to the defined morphotypes and/or spatial occurrences. In four cases, we detected two distinct ribotypes at the same locality. However, samples collected south of the Danube River presented a different predominant morphotype from the rest of the samples, thus implying a potential biogeographic signal as inferred from morphology. In general, there is morphological and molecular variability in P. cinctum, which is under-studied and which may uncover geographic or ecological correlations or even the existence of cryptic species.     

Insight into Date Palm Diversity: Genetic and Morphological Investigations

Date palm (Phoenix dactylifera) is an important crop plant both from nutritional and economic points of view. The assessment of genetic diversity and population differentiation of date palms are evaluative for its dynamic conservation and sustainable utilization of its genetic diversity. Estimates of genetic diversity based on molecular markers and fruit characteristics were performed in samples of 23 date palms growing in Ahvaz city (Khuzestan province of Iran). Clustering based on fruit morphological features separated the cultivars in different groups. These cultivars differed significantly in morphological features (P =?0.001). Start codon targeted (SCoT) polymorphism markers revealed a good level of genetic variability (10.17 to 45.76%) in these cultivars. Moreover, STRUCTURE analysis revealed the presence of within-population genetic variability. Analysis of molecular variance revealed a significant genetic difference among date palms, while it showed a higher degree of within-cultivar genetic variability compared with that of among-population diversity. Some degree of common shared alleles occurred between date palm cultivars. Gst versus Nm analysis showed that some of the SCoT markers have a high discrimination power and may have a potential local adaptive value. The Mantel test showed a significant association (r =?0.40, P =?0.001) between morphological and genetic distances. Therefore, both morphological and SCoT molecular data can be used in genetic screening of date palms in the available germplasm.

     

A multi‐marker DNA barcoding approach to save time and resources in vegetation surveys

Vegetation surveys have a long tradition in ecological studies, but several limitations in the morphological identification of species have been recognized. The objective of this study was to evaluate the effectiveness of DNA barcoding in plant species identification to save field technicians time and resources. Vegetation surveys were performed in four plots of semi‐dry grassland in the Italian subalpine region of Lombardy. Two identification approaches were employed: a conventional morphological identification and a molecular multi‐marker DNA barcoding method. Results showed that morphological identification of 49 species collected from the study area (five field inspections) required a substantial amount of time to complete relative to the molecular method. The same 49 samples were analysed using the following DNA multi‐marker barcodes: rbcL, matK and trnH‐psbA. rbcL showed 100% amplification success with standard primers, but low interspecific genetic variability. matK demonstrated some amplification problems with standard primers; however, consistent genetic diversity was observed. Finally, the trnH‐psbA spacer region exhibited reliable amplification success and the highest molecular variability. In a comparison with publicly available databases, trnH‐psbA and matK returned the highest proportion of identified samples, whereas rbcL returned several misidentifications. The DNA barcoding approach is a powerful tool in vegetation surveys and may significantly reduce the time and cost spent for species identification. However, to effectively apply DNA barcoding in vegetation surveys, exhaustive local or regional molecular databases must be defined. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169 , 518–529.     

Chloroplast DNA diversity in the genusRubus (Rosaceae) revealed by Southern hybridization

The variability in chloroplast DNA type of 20Rubus genotypes was examined by Southern hybridization. DNA extracted from theRubus accessions was digested with two restriction enzymes (EcoRI and EcoRV) and heterologous chloroplast DNA sequences from barley and pea were used as probes to detectRubus chloroplast DNA sequences on Southern blots ofRubus total DNA. Restriction fragment length polymorphism was detected and a total of 92 restriction fragments were generated by the probe/enzyme combinations examined. Cladistic principles based on the parsimony assumption were used to assemble a phylogenetic tree based on chloroplast restriction fragment length data. The phylogenetic tree grouped the taxonomically defined species and is in general agreement with information based on morphological criteria. However, the Japanese red raspberryR. illecebrosus was shown to have diverged considerably in terms of evolutionary time from other species in subg.Idaeobatus. Furthermore, the molecular approach provides a quantitative estimate of the relationship between species that is difficult to obtain from morphological data. In order to complement the chloroplast DNA information a ribosomal DNA probe was also included in the analysis and provided further information on the phylogenetic relationships withinRubus.     

The influence of genetic background on resistance to the cabbage aphid (Brevicoryne brassicae) in Kale (Brassica oleracea var. acephala)

Resistance to Brevicoryne brassicae has been identified in the progeny of two selected kale (B. oleracea var. acephala) plants, one from the F1 hybrid cultivar ‘Arsis RS’ and one from the landrace ‘Butzo’. These plants were crossed with susceptible B. oleracea morphotypes that have different periods to flowering. The type of susceptible plant line used had an effect on the resistance phenotypc of the progeny. Tested F2 populations derived from these crosses show that resistance is not under simple genetic control. This, in addition to variation in aphid numbers within accessions, suggests that separation of genetic components of control from environmental ‘noise’ for any accession may only be possible by the production of double haploid plant lines.     

Identification of quantitative trait loci controlling developmental characteristics of Brassica oleracea L

A segregating population of F₁-derived doubled haploid (DH) lines of Brassica oleracea was used to detect and locate QTLs controlling 27 morphological and developmental traits, including leaf, flowering, axillary bud and stem characters. The population resulted from a cross between two very different B. oleracea crop types, an annual cauliflower and a biennial Brussels sprout. A principal component analysis (PCA), based on line means, allowed all the traits to be grouped into distinct categories according to the first five Principal Components. These were: leaf traits (PC1), flowering traits (PC2), axillary bud traits (PC3 and 5) and stem traits (PC4). Between zero and four putative QTL were located per trait, which individually explained between 6% and 43% of the additive genetic variation, using the multiple-marker regression approach to QTL mapping. For lamina width, bare petiole length and stem length two QTL with opposite effects were detected on the same linkage groups. Intra- and inter-specific comparative mapping using RFLP markers identified a QTL on linkage group O8 accounting for variation in vernalisation, which is probably synonymous with a QTL detected on linkage group N19 of Brassica napus. In addition, a QTL for petiole length detected on O3 of this study appeared to be homologous to a QTL detected on another B. oleracea genetic map (Camargo et al. 1995). Received: 28 March 2001 / Accepted: 25 June 2001     

Inheritance of isozyme and RFLP markers in Brassica campestris and comparison with B. oleracea

Summary Using primarily cDNA restriction fragment length polymorphism markers (RFLPs) previously located to Brassica oleracea (cabbage, 2n=18) chromosomes, we initiated a comparative RFLP map in an F₂ population of B. campestris (turnip x mock pak-choi, 2n=20). As with B. oleracea, the genome of B. campestris showed extensive gene duplication, and the majority of detected duplicated loci were unlinked. Only 6 of the 49 identified loci were represented as a single copy, and 3 of these 6 were clustered on a single linkage group showing a distorted segregation ratio. Comparison with B. Oleracea indicates this synteny is conserved between species. Two other linkage groups also appeared syntenic between B. oleracea and B. campestris. One single copy locus appears to have changed synteny between B. oleracea and B. campestris. These observations suggest that B. oleracea and B. campestris share a common ancestor, but that chromosome repatterning has occurred during or after speciation. Within B. campestris, 5 loci appeared duplicated in one parent or the other, and 2 of these were linked. Differentiation through subspecies-specific duplication or deletion events is suggested as one mechansim for the evolution of numerous morphotypes within each of these species.     

Ecological differentiation,speciation, and rarity: How do they match in Tephroseris longifolia agg. (Asteraceae)?

Tephroseris longifolia agg. is a complex group of outcrossing perennials distributed throughout Central Europe. Recent morphological study revealed six morphotypes corresponding to five previously distinguished subspecies, together with Alpine and Pannonian morphotypes of T. longifolia subsp. longifolia. The delimited morphotypes differ in relative DNA content, geographical range, and rarity. We compared ecological niches of the six morphotypes in order to assess the impact of ecological differentiation on the speciation processes within the T. longifolia agg. Further, we examined whether morphotypes with small range are more ecologically specialized than their widespread relatives. The distribution area of the aggregate includes the Alps, Apennines, Carpathians, and the Pannonian Basin. Ecological variables linked to climate, topography, soil, and vegetation were gathered from 135 circular plots recorded in 35 localities. Related variables were grouped to describe the partial ecological niches: climatic, topographic, pedological, biotic, and coenotic (based either on vascular plants or on bryophytes), each of them visualized as an envelope in the two‐dimensional nonmetric multidimensional scaling ordination space. Each partial ecological niche for a given morphotype was characterized by its position (location of the envelope centroid), breadth (surface of the envelope), and overlaps with envelopes of the other morphotypes. Mantel statistics based on Spearman correlation coefficients were used to quantify differentiation of morphotypes in ecological parameters represented by the partial ecological niches. The significant niche differentiation was confirmed for climatic, topographic, pedological, and vascular plant‐based coenotic niches. Ecological niche differentiation corresponded well to morphological and partially also to karyological differentiation. Narrowly distributed morphotypes occupied more specific habitats and had narrower ecological niches than their widespread relatives. Ecological differentiation could be considered an important driver in allopatric speciation within the T. longifolia agg. Our results demonstrate that quantification of ecological divergence is helpful in assessing evolutionary history of closely related taxa.     

A new broccoli × broccoli immortal mapping population and framework genetic map: tools for breeders and complex trait analysis

A unique broccoli × broccoli doubled haploid (DH) population has been created from the F₁ of a cross between two DH broccoli lines derived from cultivars Green Duke and Marathon. We genotyped 154 individuals from this population with simple sequence repeat and amplified fragment length polymorphism markers to create a B. oleracea L. var. italica ‘intra-crop’ specific framework linkage map. The map is composed of nine linkage groups with a total length of 946.7 cM. Previous published B. oleracea maps have been constructed using diverse crosses between morphotypes of B. oleracea; this map therefore represents a useful breeding resource for the dissection of broccoli specific traits. Phenotype data have been collected from the population over five growing seasons; the framework linkage map has been used to locate quantitative trait loci for agronomically important broccoli traits including head weight (saleable yield), head diameter, stalk diameter, weight loss and relative weight loss during storage, as well as traits for broccoli leaf architecture. This population and associated linkage map will aid breeders to directly map agronomically important traits for the improvement of elite broccoli cultivars.     

Brassica taxonomy based on nuclear restriction fragment length polymorphisms (RFLPs)

Summary RFLPs were used to study genome evolution and phylogeny in Brassica and related genera. Thirtyeight accessions, including 10 accessions of B. rapa (syn. campestris), 9 cultivated types of B. oleracea, 13 nine-chromosome wild brassicas related to B. oleracea, and 6 other species in Brassica and allied genera, were examined with more then 30 random genomic DNA probes, which identified RFLPs mapping to nine different linkage groups of the B. rapa genome. Based on the RFLP data, phylogenetic trees were constructed using the PAUP microcomputer program. Within B. rapa, accessions of pak choi, narinosa, and Chinese cabbage from East Asia constituted a group distinct from turnip and wild European populations, consistent with the hypothesis that B. rapa had two centers of domestication. A wild B. rapa accession from India was positioned in the tree between European types and East Asian types, suggesting an evolutionary pathway from Europe to India, then to South China. Cultivated B. oleracea morphotypes showed monophyletic origin with wild B. oleracea or B. alboglabra as possible ancestors. Various kales constitute a highly diverse group, and represent the primitive morphotypes of cultivated B. oleracea from which cabbage, broccoli, cauliflower, etc. probably have evolved. Cauliflower was found to be closely related to broccoli, whereas cabbage was closely related to leafy kales. A great diversity existed among the 13 collections of nine-chromosome wild brassicas related to B. oleracea, representing various taxonomic states from subspecies to species. Results from these studies suggested that two basic evolutionary pathways exist for the diploid species examined. One pathway gave rise to B. fruticulosa, B. nigra, and Sinapis arvensis, with B. adpressa or a close relative as the initial ancestor. Another pathway gave rise to B. oleracea and B. rapa, with Diplotaxis erucoides or a close relative as the initial ancestor. Raphanus sativus and Eruca sativus represented intermediate types between the two lineages, and might have been derived from introgression or hybridization between species belonging to different lineages. Molecular evidence for an ascending order of chromosome numbers in the evolution of Brassica and allied genera was obtained on the basis of RFLP data and phylogenetic analysis.     

Analysis of organelle genomes in a somatic hybrid derived from cytoplasmic male-sterile Brassica oleracea and atrazine-resistant B. campestris

Summary An atrazine-resistant, male-fertile Brassica napus plant was synthesized by fusion of protoplasts from the diploid species B. oleracea and B. campestris. Leaf protoplasts from B. oleracea var. italica carrying the Ogura male-sterile cytoplasm derived from Raphanus sativus were fused with etiolated hypocotyl protoplasts of atrazine-resistant B. campestris. The selection procedure was based on the inability of B. campestris protoplasts to regenerate in the media used, and the reduction of light-induced growth of B. oleracea tissue by atrazine. A somatic hybrid plant that differed in morphology from both B. oleracea and B. campestris was regenerated on medium containing 50 M atrazine. Its chromosome number was 36–38, approximately that of B. napus. Furthermore, nuclear ribosomal DNA from this hybrid was a mixture of both parental rDNAs. Southern blot analyses of chloroplast DNA and an assay involving tetrazolium blue indicated that the hybrid contained atrazine-resistant B. campestris chloroplasts. The hybrid's mitochondrial genome was recombinant, containing fragments unique to each parent, as well as novel fragments carrying putative crossover points. Although the plant was female-sterile, it was successfully used to pollinate B. napus.     

Diploid Brassica napus somatic hybrids: characterization of nuclear and organellar DNA

Summary Five somatic hybrids between Brassica campestris and B. oleracea were obtained. Molecular, morphological and cytological information all suggest that the resynthesized B. napus plants were hybrids. All five plants were diploid (2n=38) and had mainly bivalents at meiosis. Seedset was low after selfing but normal after crossing with B. napus. Molecular proof of the hybrid nature of these plants was obtained by hybridization of a rDNA repeat to total DNA. Analysis of chloroplast DNA restriction patterns revealed that all hybrids had chloroplasts identical to the B. oleracea parent. The analysis of mitochondrial DNA indicated that three hybrids had restriction patterns identical to those of B. campestris, and the other two had restriction patterns similar to those of B. oleracea. The 11.3 kb plasmid present in mitochondria of the B. campestris parent was also found in mitochondria of all five hybrids. This suggests that the plasmid from a B. campestris type of mitochondria was transferred into mitochondria of a B. oleracea type.     

Postembryonal morphological variation of <Emphasis Type="Italic">Daphnia galeata</Emphasis> in water bodies of different types

Despite many ecological studies the population morphological variability within the freshwater crustacean genus Daphnia is poorly investigated, especially during postembryonic development. Unusual phenotypic plasticity of some Daphnia species results in tremendous difficulties in morphological species delineation. The ontogenetic morphological variation of this species was studied, revealing the general trends in body shape variation in different populations. The morphotypes of size and age groups of D. galeata turned out to be more variable at the mouth of the Kargat River (Lake Chany basin) than in Lake Todzha (Bol’ shoi Yenisei River basin); however, the growth of characters such as the helmet and tail spine was described by an allometric function and their absolute sizes decreased with age in Daphnia from both water basins. It is shown that the first mature size-and-age group of D. galeata is most suitable for investigating population morphological variation.     

Phloem‐specific resistance in Brassica oleracea against the whitefly Aleyrodes proletella

The cabbage whitefly [Aleyrodes proletella L. (Hemiptera: Aleyrodidae)] is becoming a serious pest in Brassica oleracea L. (Brassicaceae) crops. However, almost nothing is known about the interaction of this insect with its host plants. Previous studies have shown differences in the natural occurrence of adults, eggs, and nymphs on the closely related B. oleracea cultivars Christmas Drumhead and Riviera grown in the field. In this study, we aimed to identify the nature of these differences and to gain insight into the resistance mechanisms against A. proletella. We used no‐choice experiments on field‐ and greenhouse‐grown plants to show that the differences between the two cultivars are mainly based on antibiosis (traits that reduce herbivore performance) and not on antixenosis (traits that deter herbivory). This was further supported by laboratory choice experiments that indicated little or no discrimination between the two cultivars based on plant volatiles. We showed that resistance is dependent on plant age, that is, resistance increased during plant development, and is mainly independent of environmental factors. Analysis of probing behaviour revealed that the resistance trait affects A. proletella at the phloem level and that morphological differences between the two cultivars are most likely not involved. We suggest that compounds present in the phloem reduce sap ingestion by the whitefly and that this explains the observed resistance.     

Nucleotide based validation of Ocimum species by evaluating three candidate barcodes of the chloroplast region

The genus Ocimum comprises of several medicinally important species which frequently fall prey to adulteration due to misidentification. A proficient method is hence required to solve the problems that exist in differentiating its various morphotypes. In plants, candidate DNA barcodes of the chloroplast and nuclear regions have proved to be a great success in the validation of several plant families. Hence, this study involves the use of the molecular‐based DNA barcoding method to identify some of the most common and useful species of the genus Ocimum (Tulsi). Here, DNA amplification of three candidate barcodes of the chloroplast genome viz. matK, rbcL and psbA‐trnH was performed, to access their ability to produce high sequence variability. The discrimination among species was performed using the Kimura 2‐parameter and maximum composite likelihood methods. On analysing the sequence data, the psbA‐trnH region proved to be the most suitable candidate barcode and gave an overall variation of 7.3% at the interspecies level. A clear differentiation was found at the species level, showing a maximum distance of 0.264 between dissimilar species. Also, phylogenetic analysis led to the successful identification of hybrids, while it failed to do so at the variety level. Hence, it can be inferred that DNA barcoding is ideal for species‐level identification of the genus Ocimum.     

The patterns of population differentiation in a <Emphasis Type="Italic">Brassica rapa</Emphasis> core collection

With the recent advances in high throughput profiling techniques the amount of genetic and phenotypic data available has increased dramatically. Although many genetic diversity studies combine morphological and genetic data, metabolite profiling has yet to be integrated into these studies. For our study we selected 168 accessions representing the different morphotypes and geographic origins of Brassica rapa. Metabolite profiling was performed on all plants of this collection in the youngest expanded leaves, 5 weeks after transplanting and the same material was used for molecular marker profiling. During the same season a year later, 26 morphological characteristics were measured on plants that had been vernalized in the seedling stage. The number of groups and composition following a hierarchical clustering with molecular markers was highly correlated to the groups based on morphological traits (r = 0.420) and metabolic profiles (r = 0.476). To reveal the admixture levels in B. rapa, comparison with the results of the programme STRUCTURE was needed to obtain information on population substructure. To analyze 5546 metabolite (LC–MS) signals the groups identified with STRUCTURE were used for random forests classification. When comparing the random forests and STRUCTURE membership probabilities 86% of the accessions were allocated into the same subgroup. Our findings indicate that if extensive phenotypic data (metabolites) are available, classification based on this type of data is very comparable to genetic classification. These multivariate types of data and methodological approaches are valuable for the selection of accessions to study the genetics of selected traits and for genetic improvement programs, and additionally provide information on the evolution of the different morphotypes in B. rapa.