Increasing the Efficiency of Microsatellite Discovery from Poorly Enriched Libraries in Coniferous Forest Species

Microsatellites are PCR based markers widely applicable in many plant species. Although the discovery and development of new microsatellites is a costly and technically demanding process, they are widely used in many plant systems. In coniferous tree species, the large genome size and repetitive DNA regions further aggravate the process, often resulting in libraries with very low enrichment efficiencies. Such species have proven intractable to efficient microsatellite discovery. For example, enriched microsatellite libraries produced for seven angiosperm species had enrichment efficiencies up to 50%, whereas libraries for three conifers, generated concurrently, under identical conditions, all had enrichment efficiencies below 10%. This report describes a simple strategy for recovering useful microsatellites from two of these conifer libraries, Pinus elliottii and Araucaria cuninghamii. Enrichment efficiencies increased from 0% to 100% and 1.5% to 98%, respectively. The strategy utilised DIG labelled oligo probes to identify clones containing microsatellite inserts. The technique is cost effective and rapid, utilising basic techniques and equipment that are widely available.     

Bahiagrass,Corn, Cotton Rotations,and Pesticides for Managing Nematodes,Diseases, and Insects on Peanut

Florunner peanut was grown after 1 and 2 years of Tifton 9 bahiagrass, corn, cotton, and continuous peanut as whole-plots. Pesticide treatments aldicarb (3.4 kg a.i./ha), flutolanil (1.7 kg a.i./ha), aldicarb + flutolanil, and untreated (control) were sub-plots. Numbers of Meloidogyne arenaria second-stage juveniles in the soil and root-gall indices of peanut at harvest were consistently lower in plots treated with aldicarb and aldicarb + flutolanil than in flutolanil-treated and untreated plots. Percentages of peanut leaflets damaged by thrips and leafhoppers were consistently greater in flutolaniltreated and untreated plots than in plots treated with aldicarb or aldicarb + flutolanil but not affected by cropping sequences. Incidence of southern stem rot was moderate to high for all chemical treatments except those that included flutolanil. Stem rot loci were low in peanut following 2 years of bahiagrass, intermediate following 2 years of corn or cotton, and highest in continuous peanut. Rhizoctonia limb rot was more severe in the peanut monoculture than in peanut following 2 years of bahiagrass, corn, or cotton. Flutolanil alone or combined with aldicarb suppressed limb rot compared with aldicarb-treated and untreated plots. Peanut pod yields were 4,186 kg/ha from aldicarb + flutolanil-treated plots, 3,627 kg/ha from aldicarb-treated plots, 3,426 kg/ha from flutolanil-treated plots, and 3,056 kg/ha from untreated plots. Yields of peanut following 2 years of bahiagrass, corn, and cotton were 29% to 33% higher than yield of monocultured peanut.     

The Rooting of the Universal Tree of Life Is Not Reliable

Several composite universal trees connected by an ancestral gene duplication have been used to root the universal tree of life. In all cases, this root turned out to be in the eubacterial branch. However, the validity of results obtained from comparative sequence analysis has recently been questioned, in particular, in the case of ancient phylogenies. For example, it has been shown that several eukaryotic groups are misplaced in ribosomal RNA or elongation factor trees because of unequal rates of evolution and mutational saturation. Furthermore, the addition of new sequences to data sets has often turned apparently reasonable phylogenies into confused ones. We have thus revisited all composite protein trees that have been used to root the universal tree of life up to now (elongation factors, ATPases, tRNA synthetases, carbamoyl phosphate synthetases, signal recognition particle proteins) with updated data sets. In general, the two prokaryotic domains were not monophyletic with several aberrant groupings at different levels of the tree. Furthermore, the respective phylogenies contradicted each others, so that various ad hoc scenarios (paralogy or lateral gene transfer) must be proposed in order to obtain the traditional Archaebacteria–Eukaryota sisterhood. More importantly, all of the markers are heavily saturated with respect to amino acid substitutions. As phylogenies inferred from saturated data sets are extremely sensitive to differences in evolutionary rates, present phylogenies used to root the universal tree of life could be biased by the phenomenon of long branch attraction. Since the eubacterial branch was always the longest one, the eubacterial rooting could be explained by an attraction between this branch and the long branch of the outgroup. Finally, we suggested that an eukaryotic rooting could be a more fruitful working hypothesis, as it provides, for example, a simple explanation to the high genetic similarity of Archaebacteria and Eubacteria inferred from complete genome analysis.     

Real-time analyses of chloroplast and mitochondrial division and differences in the behavior of their dividing rings during contraction

The time courses of chloroplast and mitochondrial division and the morphological changes in the plastid-dividing ring (PD ring) and mitochondrion-dividing ring (MD ring) during chloroplast and mitochondrial division were studied in Cyanidioschyzon merolae De Luca, Taddei and Varano. To accomplish this, chloroplast and cell division of living cells were continuously video-recorded under light microscopy, and the morphological changes in the PD and MD rings were analyzed quantitatively and three-dimensionally by transmission electron microscopy (TEM). Under the light microscope, the diameters of the chloroplast and the cell decreased at uniform velocities, the speed depending on the temperature. To study in detail the sequential morphological change of the mitochondrion in M phase and the contractile mechanism in the divisional planes of the chloroplast and the mitochondrion, we observed the PD and MD rings, which are believed to promote contraction, under TEM, using the diameter of the chloroplast as an index of the time. Three PD rings (an outer PD ring on the cytoplasmic face of the outer envelope, a middle PD ring in the intermembrane space, and an inner PD ring on the stromal face of the inner envelope) were clearly observed, but only the outer MD ring could be observed. The PD ring started to contract soon after it formed, while the contraction of the MD ring did not occur immediately after formation, but was delayed until the contraction of the PD ring was almost complete. Once the MD ring began to contract, the rate of decrease of its circumference was 4 times as high as that of the PD ring. As the outer PD and MD rings contracted, they grew thicker and maintained a constant volume, while the thickness of the inner PD ring did not change and its volume decreased at a constant rate with contraction. In the early stage of contraction, the widths of the three PD rings increased in order, from the outer to the inner ring. With contraction, their widths changed at different rates until they came to have much the same width. In cross-section, the MD ring was wider where it was next to the chloroplast than at the opposite side, adjacent to the nucleus in the early stage of contraction. By the late stage, the widths of the two sides became equal. In our observations, the microbody elongated along the outer MD ring and touched the outer PD ring during contraction of the PD and MD rings. These results clearly revealed differences between the mode of contraction of the outer, middle, and inner PD rings, and between the PD and the MD rings. They also revealed the coordinated widening of the three PD rings, and suggested that the microbody plays a role in the contraction of the PD and MD rings. Received: 1 July 1998 / Accepted: 1 September 1998     

Hydrogen and oxygen isotope ratios of tree-ring cellulose for riparian trees grown long-term under hydroponically controlled environments

Saplings of three riparian tree species (alder, birch and cottonwood) were grown for over 5 months in a hydroponics system that maintained the isotopic composition of source water in six treatments, ranging from –120 to +180‰δD and –15 to +10‰δ¹⁸O. The trees were grown in two greenhouses maintained at 25°C and at either 40 or 75% relative humidity, creating differences in transpiration rates and leaf water isotopic evaporative enrichment. The cellulose produced in the annual growth ring was linearly related to source water with differences in both slope and offset associated with greenhouse humidity. The slope of the isotopic composition of source water versus tree-ring cellulose was less than 1 for both δD and δ¹⁸O indicating incomplete isotopic exchange of carbohydrate substrate with xylem water during cellulose synthesis. Tests using the outer portion of the tree-ring and new roots were similar and showed that the tree-ring values were representative of the cellulose laid down under the imposed environmental conditions. The fraction of H and O in carbohydrate substrate that isotopically exchange with medium water was calculated to be 0.36 and 0.42 respectively, and biochemical mechanisms for these observed fractions are discussed. A mechanistic model of the biochemical fractionation events for both δD and δ¹⁸O leading to cellulose synthesis was robust over the wide range of cellulose stable isotope ratios. The experimental results indicate that both water source and humidity information are indeed recorded in tree-ring cellulose. These results help to resolve some of the disparate observations regarding the interpretation of stable isotope ratios in tree-rings found in the literature. Received: 4 January 1999 / Accepted: 12 August 1999     

The development of symmetry,rigidity and anchorage in the structural root system of conifers

The stability of shallowly rooted trees can be strongly influenced by the symmetry of the ‘structural’ system of woody roots. Root systems of forest trees are often markedly asymmetric, and many of the factors affecting symmetry, including root initiation and the growth of primary and woody roots, are poorly understood. The internal and environmental factors that control the development, with respect to symmetry and rigidity, of shallow structural root systems are reviewed and discussed with particular reference to Sitka spruce (Picea sitchensis Bong. Carr.). Areas where there is insufficient knowledge are highlighted. A scheme is proposed that represents the root system as a set of spokes that are variable in number, size and radial distribution. Rigidity can vary between and along each of the spokes. The root system is presented as a zone of competition for assimilates, where allocation to individual roots depends upon their position and local variations in conditions. Factors considered include the production of root primordia of different sizes, effects of soil conditions such as the supply of mineral nutrients and water on growth of primary and woody roots, and the effect of forces caused by wind action on growth of the cambium, giving rise to roots which, in cross section, resemble I- or T-beams, and efficiently resist bending. This revised version was published online in June 2006 with corrections to the Cover Date.     

Structural root architecture of 5-year-old Pinus pinaster measured by 3D digitising and analysed with AMAPmod

Pinus pinaster (Ait.) is a high yielding forest tree, producing nearly a fourth of French marketed timber essentially from intensively managed stands located in southwestern France, in the Landes Forest. This species has generally a poor stem straightness, especially when it grows in poor sandy podzol of the Landes Forest, affected by summer droughts and winter floods. Above- and below-ground architecture and biomass as well as stem straightness were measured on twenty-nine 5-year-old planted trees uprooted by pulling with a lumbering crane. A very precise numeric representation of the geometry and topology of structural root architecture was gained using a low-magnetic-field digitising device (Danjon et al., 1998; Sinoquet and Rivet, 1997). Data were analysed with AMAPmod, a database software designed to analyse plant topological structures (Godin et al., 1997). Several characteristics of root architecture were extracted by queries including root number, length, diameter, volume, spatial position, ramification order, branching angle and inter-laterals length. Differences between root systems originated from their dimensions, but also from the proportion of deep roots and the taproot size, which represented 8% of the total root volume. The proportion of root volume in the zone of rapid taper was negatively correlated with the proportion of root volume in the taproot indicating a compensation between taproot and main lateral root volume. Among all studied root characteristics the maximal rooting depth, the proportion of deep roots and the root partitioning coefficient were correlated with stem straightness. This revised version was published online in June 2006 with corrections to the Cover Date.     

Low-molecular-weight aliphatic carboxylic acids in soil solutions under different vegetations determined by capillary zone electrophoresis

Concentrations of low-molecular-weight aliphatic carboxylic acids in soil solution were determined by a newly developed capillary zone electrophoresis method. Soil solution samples were collected by centrifugation of soil from the A horizon of a Danish, homogeneous, nutrient-rich Hapludalf in adjacent forested and arable plots. The forested plots of 0.5 ha were 33-year old stands of beech (Fagus sylvatica L.), oak (Quercus robur L.), grand fir (Abies grandis Lindl.), and Norway spruce (Picea abies (L.) Karst.), while sugar beet (Beta vulgaris L.) and winter wheat (Triticum aestivum L.) were the agricultural crops this year. High variability in soil solution concentrations of metal cations (Al, Ca, K, Mg, Na), monocarboxylic acids (formic, acetic, lactic, and valeric acids), and di- and tricarboxylic acids (oxalic, malic, succinic, and citric acids) were found within each plot. Despite this short-range within-plot variability, higher concentrations of di- and tricarboxylic acids were found in the forested soils than in the arable soils. The vegetation seemed to favour some monocarboxylic acids, but the total monocarboxylic acid concentrations showed little relation to the vegetation. Probably due to much less soil water in the Norway spruce plot, the low-molecular-weight aliphatic carboxylic acid concentrations in the samples from that plot were much higher than those found in samples from the other plots. Carbon in low-molecular-weight aliphatic carboxylic acids only accounts for a few percent of dissolved organic carbon, and no general relation was found between carbon in low-molecular-weight aliphatic carboxylic acids and dissolved organic carbon, although the correlation between carbon in di- and tricarboxylic acids and dissolved organic carbon was significant. This revised version was published online in June 2006 with corrections to the Cover Date.     

Genetic diversity of Couratari multiflora and Couratari guianensis (Lecythidaceae): consequences of two types of rarity in central Amazonia

We quantified the within-population genetic variation of Couratari multiflora and C. guianensis, two tree species found in terra firme forests of central Amazonia. Both species have some ecological features in common, but they differ in population abundance across their geographic ranges. While C. multiflora has been found only in low-density populations in all sites studied to date, C. guianensis is relatively common in some sites and very scarce in others. In a 400-ha plot, we found 41 and 29 adults of C. multiflora and C. guianensis, respectively. Twenty-two saplings of C. guianensis and 103 seedlings of C. multiflora were also examined. The mean expected heterozygosities (H_em) of seedlings and adults of C. multiflora were 0.431 and 0.436, and the mean fixation indices (F_m), 0.114 and 0.176, respectively. For C. guianensis, saplings and adults presented H_em equal to 0.425 and 0.429, and the F_m were 0.393 and 0.527, respectively. These low-density populations of two congeneric species did not differ in terms of genetic diversity, but rather they differed in terms of mean observed heterozygosity (H_om), and therefore F_m. The species with variable population density had lower H_om and greater F_m relative to the species that is always found in low-density.