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DNA sequence variation and selection of tag single-nucleotide polymorphisms at candidate genes for drought-stress response in Pinus taeda L.

Genetic association studies are rapidly becoming the experimental approach of choice to dissect complex traits, including tolerance to drought stress, which is the most common cause of mortality and yield losses in forest trees. Optimization of association mapping requires knowledge of the patterns of nucleotide diversity and linkage disequilibrium and the selection of suitable polymorphisms for genotyping. Moreover, standard neutrality tests applied to DNA sequence variation data can be used to select candidate genes or amino acid sites that are putatively under selection for association mapping. In this article, we study the pattern of polymorphism of 18 candidate genes for drought-stress response in Pinus taeda L., an important tree crop. Data analyses based on a set of 21 putatively neutral nuclear microsatellites did not show population genetic structure or genomewide departures from neutrality. Candidate genes had moderate average nucleotide diversity at silent sites (pi(sil) = 0.00853), varying 100-fold among single genes. The level of within-gene LD was low, with an average pairwise r2 of 0.30, decaying rapidly from approximately 0.50 to approximately 0.20 at 800 bp. No apparent LD among genes was found. A selective sweep may have occurred at the early-response-to-drought-3 (erd3) gene, although population expansion can also explain our results and evidence for selection was not conclusive. One other gene, ccoaomt-1, a methylating enzyme involved in lignification, showed dimorphism (i.e., two highly divergent haplotype lineages at equal frequency), which is commonly associated with the long-term action of balancing selection. Finally, a set of haplotype-tagging SNPs (htSNPs) was selected. Using htSNPs, a reduction of genotyping effort of approximately 30-40%, while sampling most common allelic variants, can be gained in our ongoing association studies for drought tolerance in pine.

Base Sequence↗

Facilitation by Pinus flexilis during succession: a hierarchy of mechanisms benefits other plant species.

Studies of facilitation have primarily been limited to single mechanisms, species, or environments. We examined interacting mechanisms governing the facilitative effects of Pinus flexilis on two later successional understory species, Pseudotsuga menziesii and Ribes cereum, in different microhabitats and seasons at the ecotone between the Rocky Mountain forests and Great Plains grasslands in Montana, USA. In field surveys, 69% of Pseudotsuga and 91% of Ribes were located beneath P. flexilis even though P. flexilis subcrowns accounted for a small proportion of available habitat. For three years, we monitored the survival of Pseudotsuga and Ribes seedlings experimentally planted beneath P. flexilis and in the open at a windward and a leeward site. Survival of both species was highest beneath P. flexilis at a site topographically protected from strong unidirectional winds (38% for Pseudotsuga and 63% for Ribes), and lowest at a windward site and in the open where tree crowns did not provide shelter from winds (2% and 6%, respectively). These results suggest that wind amelioration contributed to the facilitative effect of P. flexilis. However, even at the leeward site, where wind speed was low, survival of Pseudotsuga and Ribes was higher beneath P. flexilis, suggesting the importance of shade. To explore the relative importance of different mechanisms, we designed an experiment with six treatments: "shade," "shade + wind," "shade + drift," "wind," "drift," and a "control." After two years, we found shade to be of overwhelming importance for the survival of Pseudotsuga and Ribes. Without shade, no other treatments were significant, but once shade was provided, wind amelioration and snow pack accumulation increased survival of Pseudotsuga, suggesting that these different facilitative mechanisms functioned in a nested hierarchical manner: some mechanisms were important only when others were already functioning. Many studies have demonstrated multiple interacting mechanisms in the way that plants interact, but to our knowledge hierarchical interactive processes have not been previously documented. If the effects of positive or competitive mechanisms are often hierarchical, then studies of isolated mechanisms may not accurately assess their importance in nature.

Ecosystem↗

Modeling seed dispersal distances: implications for transgenic Pinus taeda.

Predicting forest-tree seed dispersal across a landscape is useful for estimating gene flow from genetically engineered (GE) or transgenic trees. The question of biocontainment has yet to be resolved, although field-trial permits for transgenic forest trees are on the rise. Most current field trials in the United States occur in the Southeast where Pinus taeda L., an indigenous species, is the major timber commodity. Seed dispersal distances were simulated using a model where the major determinants were: (1) forest canopy height at seed release, (2) terminal velocity of the seeds, (3) absolute seed release, and (4) turbulent-flow statistics, all of which were measured or determined within a P. taeda plantation established from seeds collected from wild forest-tree stands at the Duke Forest near Durham, North Carolina, USA. In plantations aged 16 and 25 years our model results showed that most of the seeds fell within local-neighborhood dispersal distances, with estimates ranging from 0.05 to 0.14 km from the source. A fraction of seeds was uplifted above the forest canopy and moved via the long-distance dispersal (LDD) process as far as 11.9-33.7 km. Out of 10(5) seeds produced per hectare per year, roughly 440 seeds were predicted to be uplifted by vertical eddies above the forest canopy and transported via LDD. Of these, 70 seeds/ha traveled distances in excess of 1 km from the source, a distance too great to serve as a biocontainment zone. The probability of LDD occurrence of transgenic conifer seeds at distances exceeding 1 km approached 100%.

Ecology↗

Abortifacient effects of a unique class of vasoactive lipids from Pinus ponderosa needles.

Pinus ponderosa needle (PN) ingestion by late pregnant cows results in decreased uterine blood flow, premature parturition, and retained placentae. Further, plasma from PN-fed cows increases caruncular arterial tone (i.e., induces prolonged contraction) in an isolated perfused bovine placentome. A novel class of vasoactive lipids was isolated and identified using a bovine placentome assay-guided fractionation of CH2Cl2 extracts of PN. Placentome perfusion tests indicated that 1-12-dodecanedioyl-dimyristate (14-12-14) was the most potent of the PN lipids for increasing caruncular arterial tone. Late pregnant guinea pigs (GP) were used to evaluate the abortifacient activity of these vasoactive lipids. In Study 1, on d 50 of gestation, part of the control diet was replaced with chopped PN (Diet A) or chopped PN subjected to sequential extraction with diethyl ether (Et2O; Diet B); Et2O and CH2Cl2 (Diet C); and Et2O, CH2Cl2, and methanol (Diet D). The GP on Diets A and B exhibited shorter (P<.01) gestation lengths and reduced (P<.01) pig birth weights than GP on the control diet or Diets C and D. Further, only GP on Diets A and B exhibited retained placentae. In Study 2, on d 50 of gestation, part of the control diet was replaced with chopped PN that had been subjected to exhaustive CH2Cl2 extraction and then infiltrated with either CH2Cl2 alone (Diet E), CH2Cl2 containing 14-12-14 (Diet F), or CH2Cl2 containing isocupressic acid (Diet G); then solvents were evaporated. The GP consuming Diet F had shorter (P<.05) gestation lengths and reduced (P<.05) pig birth weights than did GP consuming Diets E or G. The GP consuming Diet F also exhibited a high incidence of retained placentae. These data provide evidence that a unique class of vasoactive lipids in PN exhibit abortifacient activity in guinea pigs.

Abortifacient Agents↗

Effects of Pinus massoniana bark extract on cell proliferation and apoptosis of human hepatoma BEL-7402 cells.

AIM: To study the effects of Pinus massoniana bark extract (PMBE) on cell proliferation and apoptosis of human hepatoma BEL-7402 cells and to elucidate its molecular mechanism. METHODS: BEL-7402 cells were incubated with various concentrations (20-200 microg/mL) of PMBE for different periods of time. After 48 h, cell proliferation was determined by 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide (MTT) assay. Apoptosis was evaluated by morphological observation, agarose gel electrophoresis, and flow cytometry analysis. Possible molecular mechanisms were primarily explored through immunohistochemical staining. RESULTS: PMBE (20-200 microg/mL) significantly suppressed BEL-7402 cell proliferation in a time- and dose-dependent manner. After treatment of BEL-7402 cells with 160 microg/mL PMBE for 24, 48, or 72 h, a typical apoptotic "DNA ladder" was observed using agarose gel electrophoresis. Nuclear condensation and boundary aggregation or split, apoptotic bodies were seen by fluorescence and electron microscopy. Sub-G1 curves were displayed by flow cytometry analysis. PMBE decreased the expression levels of Bcl-2 protein in a time-dependent manner after treatment of cells with 160 microg/mL PMBE. CONCLUSION: PMBE suppresses proliferation of BEL-7402 cells in a time- and dose-dependent manner and induces cell apoptosis by possibly downregulating the expression of the bcl-2 gene.

Apoptosis↗

Low-copy microsatellite markers for Pinus taeda L.

Eighteen low-copy and genomic microsatellite markers were tested for Mendelian inheritance and then assayed in 41 Pinus taeda L. samples drawn from five regions in the southern United States. The PCR products had multiple alleles, high levels of polymorphism, and little non-specific priming. Fifteen of the 18 markers were informative for a P. taeda three-generation RFLP (restriction fragment length polymorphism) pedigree, and a P. taeda population survey revealed three to 28 alleles per locus. The highest allele numbers and polymorphic information content (PIC) values were associated with complex repeat sequences and (or) with sequences consisting of the longer strings of perfect repeats. The abundance of low- to rare-frequency alleles also accounted for high PIC values in both types of markers. Low-copy microsatellites are useful for the large, complex pine genome, especially in the absence of entire gene sequences in public databases and with the low levels of polymorphism in markers developed from expressed sequence tags (ESTs).

Alleles↗

Aging in Pinus sylvestris L. seeds: changes in viability and lipids.

Aging of Scots pine seeds (Pinus sylvestris L.) leads to changes in seed quality, such as loss of germinability, delayed growth and abnormality in developing seedlings. The knowledge of biochemical changes responsible for these aging processes is plentiful in some seeds, which are of world-wide interest, but for pine seeds these studies are rare. The aim of the present study, was to analyse pine seeds of varying ages in order to identify biochemical changes occurring in aged pine seeds, and to see if a correlation existed between these results and traditionally used seed-quality parameters, such as germinability and electrolyte leakage.

Cycadopsida↗

[Genetic diversity of isoenzymes in mountain pine (Pinus mugo Turra) in natural populations in the Ukrainian Carpathian mountains].

Electrophoretic spectra of GOT, GDH, DIA, MDH, SOD, FDH, ADH, ACP, IDH enzymes in the megagametophytes of seeds of 69 mountain pine (Pinus mugo Turra) trees from natural populations of the Ukrainian Carpathian mountains have been described. 19 loci products had efficient electrophoretic separation. The analysis of alleles segregation of the heterozygous trees on the whole confirms monogenic inheritance of the discovered variants.

Alleles↗

[Individual biomass of natural Pinus densiflora].

The aboveground biomass of individuals with different growth potentials in natural Pinus densiflora forest with different stand densities was measured in Yanbian, Jilin Province. The variation of individual biomass affected by densities was in order of dominant tree < intermediate tree < suppressed tree, while the distribution proportion of biomass in different organs affected by densities was: in order of trunk > branch > needle > bark. The biomass components of P. densifliora with different growth potentials varied markedly with the approaching of density class III, and the change of intermediate trees was similar to the whole stand. The vertical distributions of biomass of different trees were different from each other, but all showed that the biomass of trunks and barks was mainly distributed below 6 m high from ground, that of branches was within 6-10 m high, that of needles was uniform in the upper, middle and lower layers, and that of branches and needles in upper layer was least affected by density.

Biomass↗

[Variation of dormancy characteristic of different Pinus bungeana seed sources].

Studies on the dormancy characteristics of Pinus bungeana seeds from 7 representative seed sources show that the difference of water permeability of seed capsule was very significant among seed sources. The seeds from northern part of distribution area had a much higher water permeability than from the south. According to water permeability, the seven seed sources were divided into 2 geographical groups, with Qinling Mountain as the division line. The difference of seed ventilation was caused firstly by outer seed capsule, and secondly by inner one. The variation of permeability had a similar trend with the ventilation. The inhibition ability of seed extracts was significantly different among different seed sources, which was increased with the concentration of seed extracts. Based on the inhibition ability of extracts from seed capsule and endosperm, the seven seed sources were divided into 2 and 3 geographical groups, respectively. The germination capacity decreased with increasing latitude, and seeds with poor germination capacity took relatively more time for germination. All population groups based on different division criteria showed such a character, i.e., the difference of seed characteristics was more significant among groups than in same group, and the distance of seed source was longer among groups than in a group. The difference of dormancy characteristics of different P. bungeana seed sources was the result of interaction between climatic and geographical factors.

Germination↗

[Genetic variation of geographical provenance of Pinus massoniana--review and analysis].

Pinus massoniana is a significant tree species constituting the subtropical forests in China. Based on morphological, physio-ecological, chromosome, and molecular levels, the genetic variation of geographical provenance of P. massoniana and its distribution were reviewed, and the methodologies on genetic diversity and the genetic variation patterns of geographical provenance of P. massoniana were synthetically analyzed. The Key problems on molecular ecology of P. massoniana were discussed.

Ecology↗

[Ecophysiological response of Pinus koraiensis seedlings to elevated CO2].

Studies on the ecophysiological response of Pinus koraiensis seedlings were conducted in open-top chambers which were controlled with CO2 concentration of about 500 and 700 mumol.mol-1. The result shows that there were different responses in net photosynthetic rate and stomatal conductance at high CO2(500 and 700 mumol.mol-1) and ambient CO2 concentrations. Under 500 mumol.mol-1 CO2, the carboxylase activity of RuBP was the highest, the photosynthesis showed "up-ward", the average daily net CO2 assimilation was the greatest, and the contents of chlorophyll and soluble sugars were the highest. While under 700 mumol.mol-1 CO2, the photosynthesis showed "down-ward" and was lower than that of the control, and the enzyme activity and the contents of substances were the lowest.

Carbohydrate Metabolism↗

[Genetic control of the isoenzymes in Cembra pine (Pinus cembra L.) in the Ukrainian Carpathian Mountains].

Genetic control of GOT, GDH, DIA, MDH, SOD, FDH, ADH, ACP, and LAP enzymes was studied in the seed megagametophytes of cembra pine (Pinus cembra L.) from the natural population of the Ukrainian Carpa-thian mountains. Efficient electrophoretic separation was obtained for 21 loci products. The analysis of allele segregation in heterozygous trees confirms monogenic inheritance of the revealed variants.

Alleles↗

Evaluation of Platanus occidentalis and Pinus sylvestris as bioindicators for lead and cadmium by slurry sampling-electrothermal atomic absorption spectrometry.

Two higher plants (Platanus occidentalis and Pinus sylvestris) were used as bioindicators for assessment of contamination by Pb and Cd in an urban and industrialized area. Both toxic metals in plant tissue were determined by transverse-heated electrothermal atomic absorption spectrometry-longitudinal Zeeman background correction combined with automated ultrasonic slurry sampling. Twenty sampling points were established to observe a relationship between metal concentration in plant and traffic density. Screening optimization of variables influencing slurry preparations (sonication time, sonication power, and surfactant concentration) followed a fractional factorial design at 2 levels. No significant effects were observed in the interval of variables studied. Limits of detection of Pb and Cd in the plants were 0.28 and 0.025 microg/g (dry weight), respectively. Metal concentrations in the plants were in the range of 5-51 and 0.05-0.7 microg/g for Pb and Cd, respectively. Factor analysis to data of metal concentration in both plants, extractable metal content in soil, distance to traffic, and traffic density, showed a significant association between features such as concentration of Pb and Cd in leaves of P. occidentalis and traffic density, which accounted for the largest variance. Other features such as extractable metal contents in soil accounted for lesser variance, meaning that soils in the area investigated were less suitable than plants for monitoring metal pollution caused by vehicular traffic.

Cadmium↗

[Genetic variation and differentiation of peat-bog and dry-meadow populations of the dwarf mountain pine Pinus mugo Turra in the highlands of the Ukrainian Carpathians].

Based on electrophoretic analysis of 21 isozyme loci controlling 10 enzyme systems, the intra- and inter-population variation was studied in two peat-bog and three dry-meadow populations of the dwarf mountain pine Pinus mugo Turra from the highlands of the Ukrainian Carpathians. In the studied samples (a total of 164 trees), on average 62% of the studied genes were polymorphic; the mean heterozygosity was 21.3%. The dry-meadow populations differed from the peat-bog populations by allele and genotype diversity and by heterozygosity although the indices characterizing population heterogeneity (Fst and Gst) were small (0.027 and 0.032, respectively). Nei's genetic distances between the populations ranged of 0.011 to 0.032 with the mean of 0.018.

Genetic Variation↗

[Isolation and structure identification of ligan glycosides from pine needles of Pinus massoniana lamb].

AIM: To study the chemical constituents of the pine needles of Pinus massoniana lamb.. METHODS: Various chromatographic techniques were used to separate and purify. Their physico-chemical properties and spectral data (UV, IR, MS, 1H-1 H COSY, HMQC, DEPT, HMBC and ORD ect.) were measured for structure elucication. RESULTS: Three compounds were isolated from the n-BuOH fraction of water-extracts. Their structures were identified as massonianoside A (4), massonianoside A: (7S, 8R)-3, 4, 9'-trihydroxyl-3-methyoxyl-7, 8-dihydrobenzofunan-1'-propanolneoligan-9-O-alpha-L-rhamnopyranoside, massonianoside C (5), (7S, 8R)-9,9'-dihydroxyl-3,3'-dimethyoxyl-7,8-dihydrobenzofunan-1'- propanolneoligan-4-O-alpha-L-rhamnopyranoside and cedrusin-4-O-beta-glucoside (6), (7S, 8R)-3',9,9'-trihydroxyl-3-methoxyl-7,8-dihydrobenzofunan-1'- propanolneoligan-4-O-beta-D-glucopyranoside. CONCLUSION: Compound 4 and 5 are new compounds.

Benzofurans↗

[Influence of thinning on regeneration in a coastal pinus thunbergii forest].

A coastal forest planted nearby the sea can provide many shelter benefits for the coastal regions. It is ideally if the continuity of the shelter benefits could be preserved through reasonable management. Thinning and regeneration as the most important management techniques for plantations can help the continuity of the shelter benefits of the coastal forest. However, because of the peculiarities of coastal forest, i.e., the coastal plantation nearby the sea is vulnerable to disturbances (thinning as one kind of disturbance), the study on thinning and regeneration within a coastal forest is poorly understood. The purpose of this paper is to give a primary understanding in natural regeneration for the coastal Pinus thunbergii forest with different thinning rates after four growing seasons since thinning. The experiment was carried out at the middle of the shoreline along the Japan Sea, and the investigated sites consisted of four thinning treatments (control, 0% thinned, 20% thinned, 30% thinned and 50% thinned) in a coastal P. thunbergii forest. After thinning, the regenerated seedlings, soil water content, light condition (canopy openness or canopy density), wind regime, and litter depth and quantity were investigated for four growing seasons. The relationships between the regenerated seedlings and light condition, litter, wind profile and soil water content were examined. The results showed that thinning could improve the light condition on the forest floor, increase the exchange of airflow (wind speed) in the coastal forest stand, and ameliorate the water content of the forest soil. These factors accelerated the decomposition of litters, and provided necessary conditions for natural regeneration. The results of regeneration observation indicated that the most intensively thinned treatment (50% thinned with density of about 1500 stems.hm-2) could provide a better condition for regeneration during the four growing seasons. The density and growth of seedling (greater than 1 year) increased significantly with increasing thinning intensities, and the establishment of seedlings was obviously succeeded in the most intensively thinned treatment, but failed in less thinned treatments and understory. The thinned intensity of 50% did not induce wind damage to the coastal forest in the four years after thinning, and did not cause the loss of shelter functions of the coastal forest such as sand blocking, wind breaking and salt preventing etc.. On the contrary, it could provide the suitable conditions for natural regeneration of the pine coastal forest, or for the immigration of other species. Therefore, thinning as the silviculturally created openness is very important for the establishment of seedlings in the coastal forests, which provides a mechanism for the coastal forest from even aged stands dominated by P. thunbergii to stands containing multiple size classes and canopy layers.

China↗

[Studies on the lignan chemical constituents from pine needles of Pinus massoniana Lamb].

AIM: To study the chemical constituents of the pine needles from Pinus massoniana Lamb.. METHODS: Various chromatographic techniques were used for the separation and purification. The physicochemical properties and spectral data were used to elucidate the structures. RESULTS: Three compounds were isolated from the n-BuOH fraction of water-extracts. The structures were identified as (7S,8R)-4,9'-dihydroxyl-3, 3'-dimethyoxyl-7, 8-dihydrobenzofunan-1'-propanolneolignan-9-O-alpha- L-rhamnopyranoside (massonianoside D, 7), 4,4',9,9'-tetrahydroxyl-3,3'-dimethyoxyl-tetrahydrocyclolignan ((+)-isolariciresinol, 8) and 4,4',9'-trihydroxyl-3,3'-dimethyoxyl-tetrahydrocyclolignan-9-O-beta-D- xylopyranoside (isolariciresinol-9-O-xyloside, 9) on the basis of spectral data (ORD, UV, IR, MS, 1HNMR, 13CNMR, 1H-1HCOSY, HMQC and HMBC etc.). CONCLUSION: Compound 7 is a new compound, while compounds 8 and 9 were isolated from this plant for the first time.

Drugs, Chinese Herbal↗