Clonal fingerprinting in the genus <Emphasis Type="Italic">Populus</Emphasis> L. by nuclear microsatellite loci regarding differences between sections,species and hybrids

Reliable methods for clone identification are desired to characterise and distinguish breeding products within the genus Populus L. (Salicaceae). Ten nuclear microsatellite loci (PMGC14, PMGC456, PMGC2163, PTR2, PTR7, WPMS05, WPMS09, WPMS14, WPMS15 and WPMS20) were applied on a clone collection with several species and hybrids belonging to the sections Tacamahaca (balsam poplars), Aigeiros (black poplars, cottonwoods) and Populus (white poplars and aspens) and intersectional hybrids between black and balsam poplars. The members of the different sections and species do not always share their allelic ladders. Some shifts of one or two nucleotides in allele length were observed for several loci. This could be explained by nucleotide sequence differences in the flanking regions of loci in diverse taxonomic groups. Such shifts of allelic ladders result in irregular patterns in hybrid genotypes. The set of loci should have a sufficient amount of variation for a differentiation between clones, even if they are full siblings originating from crossing experiments.     

The effects of spacing on growth, morphology and biomass production and allocation in two hybrid poplar clones growing in the boreal region of Canada

Intra-clonal competition was studied in young hybrid poplar plantations to assess the effects of spacing on growth, biomass production and allocation, and morphological characteristics of above- and below-ground tree parts. Three spacings were used as whole-plots (1 × 1 m, 3 × 3 m and 5 × 5 m), with two hybrid poplar clones as subplots (BT747, Populus balsamifera L. × P. trichocarpa Torr. & Gray; MB915, P. maximowiczii A. Henry × P. balsamifera L.) in a split-plot design. After six growing seasons, diameter at breast height (dbh) increased by about 120% from the 1 × 1 m to the 5 × 5 m spacing for clone MB915, while there was no significant change in dbh for the other clone. The effect of spacing on height growth was opposite for the clones; it increased by about 175% from the narrowest to the widest spacing for clone MB915, while it decreased by about 27% for clone BT747. Estimates of above-ground biomass production after six growing seasons were significantly reduced with increasing spacing, with 29.6, 4.9 and 3.2 MgDM ha⁻¹ on average from the narrowest to the widest spacing. Branch traits and the vertical distribution of leaf area were the most affected by spacing for both clones, while live crown ratio and percentage of syllepsis did not change. Spacing also affected proportions of biomass allocated to stem, leaves, and branches, but allocation to roots did not change.     

Stoichiometric differences in food quality: impacts on genetic diversity and the coexistence of aquatic herbivores in a <Emphasis Type="Italic">Daphnia</Emphasis> hybrid complex

The maintenance of genetic and species diversity in an assemblage of genotypes (clones) in the Daphnia pulex species complex (Cladocera: Anomopoda) in response to variation in the carbon:phosphorus ratio (quantity and quality) of the green alga, Scenedesmus acutus, was examined in a 90-day microcosm competition experiment. Results indicated that mixed assemblages of seven distinct genotypes (representing clonal lineages of D. pulex, D. pulicaria and interspecific hybrids) showed rapid loss of genetic diversity in all treatments (2 × 2 factorial design, high vs. low quantity, and high vs. low quality). However, the erosion of diversity (measured as the effective number of clones) was slowest under the poorest food conditions (i.e., low quantity, low quality) and by the conclusion of the experiment (90 days) had resulted in the (low, low) treatment having significantly greater genetic diversity than the other three treatments. In addition, significant genotype (clone) × (food) environment interactions were observed, with a different predominant species/clone found under low food quality versus high food quality (no significant differences were detected for the two food quantities). A clone of D. pulex displaced the other clones under low food quality conditions, while a clone of D. pulicaria displaced the other clones in the high food quality treatments. Subsequent life-history experiments were not sufficient to predict the outcome of competitive interactions among members of this clonal assemblage. Our results suggest that genetic diversity among herbivore species such as Daphnia may be impacted not only by differences in food quantity but also by those in food quality and could be important in the overall maintenance of genetic diversity in natural populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

In vitro rooting of genetically related Eucalyptus urophylla × Eucalyptus grandis clones in relation to the time spent in culture

In vitro responsiveness of microshoots derived from three genetically related and different age Eucalyptus urophylla × Eucalyptus grandis clones kept cultivated by monthly subcultures was assessed on two rooting media in relation to the time spent in culture. Significant differences of rooting capacity were found between the two 22-year-old half sibling genotypes 147 and 149 according to the concentration of BA added to the media and also to light intensity. The contrast was even more salient with the 1-year-old clone S, which displayed the poorest rooting ability despite being full sibling with clone 147 and much younger. The various criteria observed, i.e. rooting rate, number of roots produced, root length, microshoot height and shoot tip necrosis varied greatly and differently for the three origins according to the successive time replicates of the same rooting protocols, with rooting rates of more than 80% for clone 147 at certain dates. These results were discussed, suggesting the influence of endogenous rhythms as the most rationale cause of the time-related fluctuations of responses observed in steady in vitro environment, notwithstanding possible interferences of non-optimal tissue culture conditions. Such unexpected variations of rootability between closely related genotypes, and the notable interactions pointed out between the time spent in culture and the rooting conditions deserve to be taken into consideration before stating definitive judgment on the rooting capacities of a given genotype for arborescent species.     

A fast and simple genetic survey reveals the spread of poplar hybrids at a natural Elbe river site

It is known that various poplar species and cultivated poplar hybrids have the potential to interbreed and produce fertile offspring. Conservation strategies for the genetic resources of the endangered Eurasian black poplar (Populus nigra L.) thus rely on a monitoring which enables the identification and verification of the pure species status. At the same time, the risk of hybrid dispersal and introgressive gene flow has to be estimated. In the present study a combination of two molecular markers, one from chloroplast DNA and the other from nuclear DNA, was applied to evaluate a large P. nigra population on the Elbe River. Hybrid clones of P. × canadensis are scattered within this population and also occur as plantations in the surrounding landscape. By means of the DNA markers the taxonomic status of 208 adult trees in the population and 140 young poplars along the riverbank was monitored. From the analysed young poplars, almost 20 percent were found to exhibit at least one of the two P. deltoides or P. × canadensis diagnostic alleles or genotypes, respectively. Possible vegetative spreads of F1 hybrids and precedent mating scenarios are discussed. Most interestingly we found clear evidence for a small number of backcross hybrids where P.  × canadensis acted as pollen donor. This case had long been debated and thought to be less probable, so far.     

Clonal identification by microsatellite loci in sporadic flowering of a dwarf bamboo species, <Emphasis Type="Italic">Sasa cernua</Emphasis>

Dwarf bamboo species are monocarpic. They flower simultaneously and die after several decades. The type of flowering in the genus Sasa ranges from sporadic to gregarious. In order to determine whether or not the sporadic flowering of dwarf bamboo is fixed genetically, we investigated the distribution of clones using eight microsatellite (SSR) loci in a sporadic flowering patch of Sasa cernua Makino, a major dwarf bamboo species found in central Hokkaido. In May 2006, flowering occurred on 60.5% of living culms in a 1600 m² patch. We established a 50 × 10 m study plot in this patch and noted 1267 clumps consisting of 2529 living culms. We investigated all 1267 clumps and identified six multilocus genotypes as clones using five variable SSR loci. All flowering clumps belonged to the same clone. On the other hand, non-flowering vegetative clumps were also discovered to be of the same clone. These data suggest that all flowering culms originated from a single clone of a sporadic flowering patch of S. cernua. Clonal analysis for investigation of sporadic flowering of S. cernua revealed that only a portion of a clone flowers and dies instead of the whole clone.     

Genetic relationships and clonal identity in a collection of commercially relevant poplar cultivars assessed by AFLP and SSR

A collection of 66 poplar commercial clones widely cultivated in Italy, China and in other countries of southern Europe and belonging to various poplar species and hybrids, have been fingerprinted using both amplified fragment length polymorphism (AFLP) and simple sequence repeats (SSR) techniques. Three AFLP primer combinations and six SSRs unambiguously genotyped the analysed poplar collection, with the exception of three groups of six, four and two individuals, which turned out to be indistinguishable even if they met the standards currently applied for distinctness, uniformity and stability (DUS) testing when registered. High levels of variation were detected with both molecular techniques; a total of 201 AFLP bands were amplified of which 96% turned out to be polymorphic and up to 15 SSR alleles were identified at a single locus, with a mean of 9.3 alleles per locus in the case of Populus × canadensis. The probability of matching fortuitously any two genotypes at all the SSR loci in the case of P. × canadensis was less then 7.5×10^–9. The AFLP-derived dendrogram and principal coordinate analysis (PCOORDA) clustered the clones with respect to their taxonomic classification, and allowed their genetic interrelationships to be established. Correct identification of poplar varieties is essential for ensuring the effective correspondence between the real and the declared identity of a clone, to avoid commercial frauds, and to establish breeding programmes. Molecular markers may play a major role to satisfy all these needs.     

The potential for arms race and Red Queen coevolution in a protist host–parasite system

The dynamics and consequences of host–parasite coevolution depend on the nature of host genotype‐by‐parasite genotype interactions (G × G) for host and parasite fitness. G × G with crossing reaction norms can yield cyclic dynamics of allele frequencies (“Red Queen” dynamics) while G × G where the variance among host genotypes differs between parasite genotypes results in selective sweeps (“arms race” dynamics). Here, we investigate the relative potential for arms race and Red Queen coevolution in a protist host–parasite system, the dinoflagellate Alexandrium minutum and its parasite Parvilucifera sinerae. We challenged nine different clones of A. minutum with 10 clones of P. sinerae in a fully factorial design and measured infection success and host and parasite fitness. Each host genotype was successfully infected by four to ten of the parasite genotypes. There were strong G × Gs for infection success, as well as both host and parasite fitness. About three quarters of the G × G variance components for host and parasite fitness were due to crossing reaction norms. There were no general costs of resistance or infectivity. We conclude that there is high potential for Red Queen dynamics in this host–parasite system.     

A reliable multiplexed microsatellite set for genotyping <Emphasis Type="Italic">Fragaria</Emphasis> and its use in a survey of 60 <Emphasis Type="Italic">F</Emphasis>. × <Emphasis Type="Italic">ananassa</Emphasis> cultivars

We have identified a reliable set of multiplexed microsatellite (SSR) markers for the genotyping of strawberry cultivars and their octoploid progenitors. Over 100 SSRs were screened in two F. × ananassa genotypes and from these, 32 that showed promise for genotyping were selected for further analysis. These SSRs were used to screen a set of 16 strawberry cultivars and a set of fingerprints were produced. Those SSRs that produced reliable, reproducible and easy to interpret fingerprints, that could also distinguish readily between the 16 strawberry cultivars screened, and which could be conveniently included in three multiplex reactions, were selected to form the genotyping set. The genotyping set, consisting of 10 previously-reported SSRs was used to fingerprint a total of 56 cultivated strawberry, and four octoploid Fragaria species accessions. The SSRs used could reliably distinguish between all 60 genotypes surveyed, including sibling cultivars derived from the same parental lines. The primers could be combined for multiplex PCR and represent a useful and convenient genotyping set for Fragaria that will permit fingerprinting data to be shared between laboratories.     

Successful hybridisation of normally incompatible hybrid aspen (Populus tremula?��?P. tremuloides) and eastern cottonwood (P. deltoides)

Hybrid aspen (Populus tremula × P. tremuloides) belong to the section Populus. Eastern cottonwood (P. deltoides) is a member of the section Aigeiros within the genus Populus. These poplar sections are generally considered to be incompatible. Here, we describe successful hybridisation between these parents, producing an offspring family with 27 individuals. The hybrid character of individuals was proven by genotypes at 16 nuclear microsatellite loci. One individual was suspected to have more than the diploid chromosome number of 2n = 38 due to the observation of more than two alleles at several loci. This individual is a triploid, ascertained by flow cytometry. Two distinct growth classes of tall and dwarf plants were observed in the progeny, reflecting different degrees of postzygotic incompatibility. Two loci linked to the tested microsatellites have an effect on height growth. Some fast-growing individuals were micropropagated to test them for biomass performance together with other clones in field trials.     

<Emphasis Type="Italic">Dasytricha</Emphasis> Dominance in Surti Buffalo Rumen Revealed by 18S rRNA Sequences and Real-Time PCR Assay

The genetic diversity of protozoa in Surti buffalo rumen was studied by amplified ribosomal DNA restriction analysis, 18S rDNA sequence homology and phylogenetic and Real-time PCR analysis methods. Three animals were fed diet comprised green fodder Napier bajra 21 (Pennisetum purpureum), mature pasture grass (Dicanthium annulatum) and concentrate mixture (20% crude protein, 65% total digestible nutrients). A protozoa-specific primer (P-SSU-342f) and a eukarya-specific primer (Medlin B) were used to amplify a 1,360 bp fragment of DNA encoding protozoal small subunit (SSU) ribosomal RNA from rumen fluid. A total of 91 clones were examined and identified 14 different 18S RNA sequences based on PCR–RFLP pattern. These 14 phylotypes were distributed into four genera-based 18S rDNA database sequences and identified as Dasytricha (57 clones), Isotricha (14 clones), Ostracodinium (11 clones) and Polyplastron (9 clones). Phylogenetic analyses were also used to infer the makeup of protozoa communities in the rumen of Surti buffalo. Out of 14 sequences, 8 sequences (69 clones) clustered with the Dasytricha ruminantium-like clone and 4 sequences (13 clones) were also phylogenetically placed with the Isotricha prostoma-like clone. Moreover, 2 phylotypes (9 clones) were related to Polyplastron multivesiculatum-like clone. In addition, the number of 18S rDNA gene copies of Dasytricha ruminantium (0.05% to ciliate protozoa) was higher than Entodinium sp. (2.0 × 10⁵ vs. 1.3 × 10⁴) in per ml ruminal fluid.     

<Emphasis Type="Italic">In vitro</Emphasis> rooting of two <Emphasis Type="Italic">Eucalyptus urophylla X Eucalyptus grandis</Emphasis> mature clones

Abstract   The rooting capacity of microshoots derived from two mature Eucalyptus urophylla X Eucalyptus grandis half-sib clones kept for 3 y under intensive micropropagation was assessed in different in vitro conditions. A first set of experiments established that clone 147 microshoots rooted earlier and in greater proportions, while producing more adventitious roots overall than their homologs from clone 149. Modifying the composition of the basal 1/2-MS-derived rooting medium by 1/4-MS or Knop macronutrients, or reducing sucrose concentration to 10 g l⁻¹ did not enhance the rooting rates. However, together with the growth regulators added, they had a significant effect on the number of adventitious roots formed. With rooting rates reaching 81%, the higher rootability of clone 147 over clone 149 was further confirmed by the second set of experiments with significant effects of the various auxins tested and strong clone × auxin interactions on the proportions of rooted microshoots and on the number of adventitious roots. The best rooting scores were given by 5 μM indole-3-butyric acid (IBA) and 12.5 μM 1-naphthaleneacetic acid (NAA), whereas the microshoots exposed to 5 or 12.5 μM indole-3-acetic acid (IAA) were less responsive. Lower light intensities did not improve significantly root capacities, although differences might exist according to the genotype. Overall, root and shoot elongation was stimulated by light. At the end of the experiment, the rooted microshoots were markedly taller than the non-rooted ones, with significant influences of auxins and light intensity, and to a lesser extent, of the genotypes.     

Novel Chloroplast Microsatellite (cpSSR) Markers for Genetic Diversity Assessment of Cultivated and Wild Hevea Rubber

Understanding the genetic diversity and population structure of Hevea is of great importance for managing its conservation and for utilization of rubber genetic resources. In this study, we investigated the genetic diversity and population structure of eight populations of Hevea rubber genotypes from Malaysia (MY), India (IN), Sri Lanka (LK), Indonesia (ID), France (FR), Thailand (TH), Brazil (BR), and China (CN), in addition to individual primary clones, using 10 nuclear and 11 polymorphic novel chloroplast microsatellite markers (nSSRs and cpSSRs, respectively). The BR population exhibited the greatest genetic diversity (H _e ) for both nSSR (0.841) and haploid genetic diversity (cpSSR; 0.207) markers. Bayesian analysis was applied to infer genetic structure based on nSSR data using STRUCTURE software, and a neighbor-joining tree was used to construct an unrooted phylogram based on Nei’s genetic distance that clustered these Hevea genotypes into three groups. Six polymorphic cpSSR markers produced 13 alleles and eight chlorotypes. Seven chlorotypes, A, B, C, D, E, F, and H were detected among Brazilian populations from Acre (AC), Rondônia (RO), and Mato Grosso (MT) locations. The G and H chlorotypes were found in Asiatic genotypes or Wickham clones and only one genotype, 15 AC-F-7 38-62 from AC location. These results provide valuable data for in situ or ex situ conservation and utilization of germplasm collections for breeding programs.

     

Genotypic variability and stability of poplars and willows grown onnitrate-contaminated soils

Abstract

Phyto-recurrent selection is an established method for selecting tree genotypes for phytoremediation. To identify promising Populus (poplar) and Salix (willow) genotypes for phytotechnologies, our objectives were to (1) evaluate the genotypic variability in survival, height, and diameter of poplar and willow clones established on soils heavily contaminated with nitrates; and (2) assess the genotypic stability in survival and diameter of selected poplar clones after one and eleven growing seasons. We tested 27 poplar and 10 willow clones planted as unrooted cuttings, along with 15 poplar genotypes planted as rooted cuttings. The trees were tested at an agricultural production facility in the Midwestern, United States. After 11 growing seasons, using phyto-recurrent selection, we surveyed survival and measured the diameter of 27 poplar clones (14 unrooted, 13 rooted) that were selected based on superior survival and growth throughout plantation development. Overall, willow exhibited the greatest survival, while poplar had the greatest height and diameter. At 11 years after planting, superior clones were identified that exhibited above-average diameter growth at the establishment- and rotation-age, most of which had stable genotypic performance over time. Selection of specific clones was favorable to genomic groups, based on the geographic location and soil conditions of the site.     

Entamoeba Clone‐Recognition Experiments: Morphometrics,Aggregative Behavior,and Cell‐Signaling Characterization

Studies on clone‐ and kin‐discrimination in protists have proliferated during the past decade. We report clone‐recognition experiments in seven Entamoeba lineages (E. invadens IP‐1, E. invadens VK‐1:NS, E. terrapinae, E. moshkovskii Laredo, E. moshkovskii Snake, E. histolytica HM‐1:IMSS and E. dispar). First, we characterized morphometrically each clone (length, width, and cell‐surface area) and documented how they differed statistically from one another (as per single‐variable or canonical‐discriminant analyses). Second, we demonstrated that amebas themselves could discriminate self (clone) from different (themselves vs. other clones). In mix‐cell‐line cultures between closely‐related (E. invadens IP‐1 vs. E. invadens VK‐1:NS) or distant‐phylogenetic clones (E. terrapinae vs. E. moshkovskii Laredo), amebas consistently aggregated with same‐clone members. Third, we identified six putative cell‐signals secreted by the amebas (RasGap/Ankyrin, coronin‐WD40, actin, protein kinases, heat shock 70, and ubiquitin) and which known functions in Entamoeba spp. included: cell proliferation, cell adhesion, cell movement, and stress‐induced encystation. To our knowledge, this is the first multi‐clone characterization of Entamoeba spp. morphometrics, aggregative behavior, and cell‐signaling secretion in the context of clone‐recognition. Protists allow us to study cell–cell recognition from ecological and evolutionary perspectives. Modern protistan lineages can be central to studies about the origins and evolution of multicellularity.     

Bark morphological and chemical features are differentially correlated with disease resistance and yield in hybrid poplar taxa

In the southeastern United States, the establishment of short-rotation intensively cultured plantations of hybrid poplar has been hindered by its susceptibility to stem cankers. We evaluated the tradeoffs between biomass yield and disease tolerance in hybrid poplar genotypes belonging to P. deltoides × P. maximowiczii (DM), P. deltoides × P. nigra (DN), P. trichocarpa × P. maximowiczii (TM), and P. deltoides × P. deltoides (DD) taxa. We hypothesized that canker resistant genotypes will have thicker bark but bark thickness and biomass yield will be negatively correlated. After two growing seasons, the DD genotypes developed thicker bark compared to the genotypes of other taxa and bark thickness was not correlated with biomass yield in the DD genotypes (R² = 0.002). However, in the TM, DM, and DN genotypes, bark thickness was negatively correlated with biomass yield (R² = 0.33–0.77). Disease incidence studies revealed that the DM genotypes were most susceptible to canker whereas no disease was detected in DD genotypes. Furthermore, bark analysis conducted by Fourier transform infrared spectroscopy coupled with multivariate analysis showed that that DD genotypes to be chemically separate from the three hybrid genotypes and that bark chemistry was correlated with canker disease incidence. Taken together, these results reveal that it is possible to generate hybrid poplar genotypes with thicker bark, disease resistance, and higher biomass yields. This insight should guide further efforts to develop genetically improved hybrid poplar genotypes, both in terms of biomass yield and disease tolerance, for cultivation in the southeastern United States. Hybrid poplar cultivation in southeastern United States is hindered by its susceptibility to stem cankers. We evaluated tradeoffs between yield and canker disease resistance in various hybrid poplar genotypes. After two growing seasons, the DD genotypes showed disease resistance and developed thicker bark that was chemically distinct from the other genotypes. Bark thickness was not correlated with yield in the DD genotypes but was negatively correlated with yield in the other genotypes. These results will guide the development of hybrid poplar genotypes that are both disease resistant and high yielding for cultivation in the southeastern United States.     

Somatic hybridization between the diploids of <Emphasis Type="Italic">S.</Emphasis> × <Emphasis Type="Italic">michoacanum</Emphasis> and <Emphasis Type="Italic">S. tuberosum</Emphasis>

Interspecific somatic hybrids between a diploid potato clone DG 81-68 susceptible to Phytophthora infestans (Mont.) de Bary and a resistant diploid tuber-bearing species Solanum × michoacanum were generated and analyzed. About 30 regenerants displaying an intermediate morphology were obtained as a result of three separate PEG-mediated fusion experiments. The RAPD analysis confirmed the hybridity of all the regenerants. About 50% of the hybrid plants exhibited vigorous growth and were stable in culture, while the rest of them rooted poorly and grew slowly in vitro. Most of the hybrid clones were at the tetraploid level (70%), while 30% of the clones examined were at the hexaploid level. The S. × michoacanum (+) DG 81-68 hybrids with growth anomalies were aneuploid. The variation in late blight resistance of the hybrid clones was found in detached leaflet tests, with enhanced resistance characteristic for three tetraploid hybrids.     

Analysis of <Emphasis Type="Italic">Malus</Emphasis><Emphasis Type="Italic">S</Emphasis>-RNase gene diversity based on a comparative study of old and modern apple cultivars and European wild apple

Malus S-RNase genetic diversity was analyzed in Malus × domestica cultivars and compared to European wild apple (Malus sylvestris). Using PCR-based approaches, the S-RNase genotype of 140 M. × domestica cultivars, 196 M. sylvestris trees and 27 M. sylvestris—M. × domestica hybrids was determined. S-RNase allelic richness in M. sylvestris was much higher than in M. × domestica, indicating the negative influence of domestication on S-RNase diversity. Heterogeneity of the S-RNase allelic distribution is much higher in cultivated apple than in wild apple, which shows that breeding leads to strong departure from the expected homogeneity of genes under negative frequency-dependent selection. The majority of the M. × domestica S-alleles has been found in M. sylvestris as well, which points to strong conservation of the S-locus gene structure. Based on the sequence of all different SCAR-fragments, which comprise both the hypervariable PS1 region and the single intron, S-RNase genetic diversity was further explored. It provided some clues to the occurrence of new S-alleles among the multitude of novel S-RNase sequences that have been identified, which were mostly unique for the group of M. sylvestris individuals. The determination of the S-RNase genotypes of old cultivars and M. sylvestris will enable their introduction into new breeding strategies. As M. sylvestris has become an endangered species in Belgium, the knowledge gathered in this study will be an important tool for selecting useful genotypes for a core collection.