Genetic diversity and population structure analysis of Pistacia species revealed by phenylalanine ammonia-lyase gene markers and implications for conservation

Information on population genetic structure and crop genetic diversity is important for genetically improving crop species and conserving threatened species.The PAL gene sequence is part of a multigene family that can be utilized to design DNA marker systems for genetic diversity and population structure investigation.In the current study, genetic diversity and population structure of100 accessions of wild Pistacia species were investigated with 78 PAL markers.A protocol for using PAL sequences as DNA markers was developed.A total of 313 PAL loci were recognized, showing 100% polymorphism for PAL markers.The PAL markers produced relatively more observed and effective alleles in Pistacia falcata and Pistacia atlantica, with a higher Shannon's information index and expected heterozygosity in P.atlantica, Pistacia vera and Pistacia mutica.Pairwise assessment of Nei's genetic distance and genetic identity between populations revealed a close association between geographically isolated populations of Pistacia khinjuk and Pistacia chinensis.The accessions of wild Pistacia species had more genetic relationship among studied groups of species.Analysis of molecular variance indicated 19% amongpopulation variation and 81% within-population variation for the PAL gene based DNA marker.Population structure analysis based on PAL revealed four groups with high genetic admixture among populations.The results establish PAL markers as a functional DNA marker system and provide important genetic information about accessions from wild populations of Pistacia species.     

Genetic diversity in two threatened species in Vietnam: <Emphasis Type="Italic">Taxus chinensis</Emphasis> and <Emphasis Type="Italic">Taxus wallichiana</Emphasis>

Taxus chinensis and T. wallichiana in have been threatened in their distribution areas in recent decades because of their over-exploitation and reduction and destruction of native habitats. Determining the genetic diversity in populations of the two species will provide guidelines for their protection and preservation. Two hundred and fifteen trees from six populations of T. chinensis and 150 sampled trees of T. wallichiana were sampled. Six microsatellite primer pairs selected from 16 primer pairs were used to investigate genetic variation at the population and species levels. Five yielded polymorphic alleles, and among the 13 putative alleles amplified, 11 were polymorphic (accounting for 76.33 %).Shannon’s information index (I) and percentage of polymorphic bands (PPB) (I = 0.202 and PPB = 67.22 % for T. chinensis; I = 0.217 and PPB = 65.03 % for T. wallichiana). Both species had low levels of genetic diversity (mean H _o = 0.107, H _e = 0.121 for T. chinensis; H _o = 0.095, H _e = 0.109 for T. wallichiana). Genetic differentiation among populations was higher (F _ST = 0.189) for T. chinensis and lower (0.156) for T. wallichiana, indicating limited gene flow (Nm) among populations for T. chinensis (0.68) and T. wallichiana (0.65). Variation among individuals of T. chinensis was 63.59 and 73.12 % for T. wallichiana. Thus, the threatened status of the two conifers is related to a lack of genetic diversity. All populations are isolated in small forest remnants. An ex situ conservation site should be established with a new population for these species that comprises all the genetic groups for the best chance to improve their fitness under environmental stresses.     

Successful <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Populus tomentosa</Emphasis> with apple <Emphasis Type="Italic">SPDS</Emphasis> gene

The problem of salinized soils has become one of the most serious constraints to agricultural and forest productivity. With the purpose of enhancing salt stress tolerance of Populus tomentosa, we transformed this tree species with spermidine synthase （SPDS） genes derived from an apple by an Agrobacterium-mediated method. Four transgenic clones were confu＇med by PCR and Southern blot analysis. As well, the expression of introduced SPDS genes was analyzed by real-time quantitative PCR.     

Genetic diversity analysis in a seed orchard of <Emphasis Type="Italic">Eucalyptus tereticornis</Emphasis>

India is the largest grower of Eucalyptus with an area of 3.943 million hectares under plantations and E. tereticornis is the predominant species in the plains of southern India with an average productivity of 12–25 m³ ha⁻¹ year⁻¹. With the aim to establish seedling seed orchards of the species, seed lots of fifteen provenances were imported from Australia and a trial was laid. In the present study the genetic diversity existing in the seed orchard was estimated using ISSR–PCR. Seven ISSR primers amplified 663 amplicons in the size ranging from 255 to 2,711 bp. The total number of polymorphic bands varied from 59 to 123 with 100% polymorphic banding profiles. The average gene diversity (Hj) of all the provenances ranged from 0.0589 to 0.1109 and the total gene diversity estimated was low (H _T = 0.130) when compared to the earlier reports from other eucalypts species. Analysis of Molecular Variance partitioned the ISSR variation into inter and intra population components. The inter population component accounted for 55.2% of the variation and the intra population component accounted for 46.3% (P < 0.001). A neighbour-joining analysis was done using the dissimilarity matrix to determine the aggregation of the individuals into clusters. Existence of population structure among the populations was revealed in STRUCTURE analysis but geographical region based clustering was not observed. The assessment of intra and inter genetic variation documented in the present study suggests that, along with the phenotypic traits, knowledge about genetic diversity measured at the DNA level in individuals of seed orchards provide an objective guide for selective culling of trees for maintaining optimal diversity for enhanced genetic gains.     

Cloning and expression analysis of a homologous expansin gene <Emphasis Type="Italic">EXP2</Emphasis> in <Emphasis Type="Italic">Picea wilsonii</Emphasis>

Expansins are cell-wall-loosening proteins that have multiple roles during plant development and stress-related processes. In this study, a novel expansin gene PwEXP2 was cloned by rapid amplification of cDNA ends based on the cDNA library of Picea wilsonii and EST fragment of PwEXP2. It was found that PwEXP2 coded 253 amino acids, and putative signal peptides exist at the N-terminal, followed by 8 cysteines, a HFD (His-Phe-Asp) conserved domain, and 4 tryptophan residues at the C-terminal. PwEXP2 was located in cytoplasm and nucleus when transformed in an onion epidermal cell. Quantitative real-time PCR assays showed that PwEXP2 was expressed in various tissues with a relatively high level in needles and low level in mature pollen. The expression level of PwEXP2 dramatically increased after seed germination. Gene expression profiles in abiotic stresses showed that PwEXP2 was induced by high temperature and osmotic stress but not involved in ABA-dependent signaling pathway. These results display the important roles of the PwEXP2 in plant development and multiple adversity stresses.     

Insecticidal properties of <Emphasis Type="Italic">Thymus persicus</Emphasis> essential oil against <Emphasis Type="Italic">Tribolium castaneum</Emphasis> and <Emphasis Type="Italic">Sitophilus oryzae</Emphasis>

Red flour beetle Tribolium castaneum (Herbst) and rice weevil Sitophilus oryzae (L.) are considered to be the major insect pests in storage. Essential oils from aromatic plants are recognized as proper alternatives to fumigants. Thymus persicus (Ronniger ex Rech. f.) is one of these plants that have medicinal properties and is indigenous to Iran. The essential oil was obtained from aerial parts of the plant and analyzed by GC and GC–MS. Carvacrol (44.69%) and thymol (11.05%) were the major constituents of the oil extracted. In this experiment, fumigant toxicity of the essential oil was studied against T. castaneum, S. oryzae at 27 ± 1°C and 60 ± 5% RH in dark condition. The adult insects were exposed to the concentrations of 51.9, 111.1, 207.4 and 370.4 μl/l air to estimate median lethal time (LT₅₀) values. The fumigant toxicity was increased in response to increased essential oil concentrations. The LT₅₀ values at the lowest and the highest concentrations tested were ranged from 28.09 to 13.47 h for T. castaneum, and 3.86 to 2.30 h for S. oryzae. It was found that S. oryzae adults were much more susceptible to the oil than T. castaneum. After 24 h of exposure, the LC₅₀ values (95% fiducial limit) for T. castaneum and S. oryzae were estimated to be 236.9 (186.27–292.81) and 3.34 (2.62–4.28) μl/l air, respectively. These results suggest that T. persicus essential oil merits further study as potential fumigant for the management of these stored-product insects.     

一个新的柽柳类萌芽素基因ThGLP的结构及其表达分析

萌芽素和类萌芽素蛋白在不同植物的各个生长阶段和胁迫相关过程中起到不同的作用。本研究首次从刚毛柽柳cDNA文库中获得类萌芽素蛋白全长基因ThGLP,该基因编码225个氨基酸,含有植物萌芽素和类萌芽素蛋白的功能序列。通过进化树分析发现该基因屑于真正萌芽素亚家族。利用实时定量PCR方法研究了该基因在PEG、NaCl、低温、CdCl2和ABA胁迫下不同时间的表达模式。结果显示PEG、NaCl、低温、CdCl2和ABA处理均能诱导ThGLP基因在柽柳的根和叶中的表达。结果表明ThGLP在柽柳根和叶中表达,参与非生物胁迫应答并由ABA依赖的信号传导途径调控。     

<Emphasis Type="Italic">Ophiostoma</Emphasis> species associated with bark beetles infesting three <Emphasis Type="Italic">Abies</Emphasis> species in Nikko,Japan

Ophiostoma species were isolated from bark beetles and Abies mariesii, A. veitchii and A. homolepis attacked by the beetles in Nikko, Tochigi, central Honshu, Japan. One to two Ophiostoma species were frequently isolated from each species of bark beetle. Ophiostoma subalpinum was the most common associate of Cryphalus montanus. Ophiostoma sp. B as well as O. subalpinum was a common fungus associated with Polygraphus proximus. Ophiostoma europhioides was isolated from Dryocoetes hectographus and D. autographus as one of the common associates. Ophiostoma sp. J and Ophiostoma sp. S were frequently isolated from D. autographus and D. striatus, respectively. These fungi seem to have specific relationships with particular bark beetles. Ophiostoma sp. B, Ophiostoma sp. J and Ophiostoma sp. S have unique morphological characteristics and appear to be new species. Five trees of A. veitchii, approximately 43 years old, were inoculated with five Ophiostoma species to assess the relative virulence of the fungi. Ophiostoma subalpinum, Ophiostoma sp. B, and O. europhioides had relatively higher virulence than the other species studied.     

斑背大尾莺汉口亚种(Locustella pryeri sinensis)遗传多样性及种群结构研究

采用线粒体控制区（807bp）序列分析对中国境内斑背大尾驾3个繁殖种群及1个越冬种群的遗传多样性及种群遗传结构,结果表明：斑背大尾莺的单倍型歧义度（Hd）为0.759&#177;0.056,核苷酸多样性较（π）为0.002。三个地理种群的FST值以及繁殖种群同越冬种群之间的ΦST值表明不同地理单元之间无显著遗传分化。对不同单倍型的聚类分析（UPGMA）结果以及网络图（Network picture）结果也支持不同种群之间无显著分化。分子变异分析 （AMOVA）显示斑背大尾莺汉口亚种不同地理种群间遗传差异不大,98.5%的差异源自种群内部,仅1.5%源自种群间。中性检验结果Fu’sFS值为负值,错配分布分析结果呈单峰,表明斑背大尾驾在我国的进化史经历了种群扩张。这一假设也得到Tajima’D检验和Fu’s检验结果的支持（D=-1.80,p=0.02;Fs=-22.11,p=0.001）,该扩张大约发生在28,700年前。     

Developing conservation strategies for <Emphasis Type="Italic">Pinus koraiensis</Emphasis> and <Emphasis Type="Italic">Eleutherococcus senticosus</Emphasis> by using model-based geographic distributions

We described potential changes in the geographic distribution and occurrence probability of Pinus koraiensis Sieb. et Zucc. and Eleutherococcus senticosus (Rupr. et Maxim.) Maxim. in the counties of northeast China. This information was used to identify priority areas for protection and provide protection and management recommendations within each studied county. The two species were mapped in 2884 study plots throughout this region over a 4-year period (38°40′N–53°30′N, 115°05′E–135°02′E). We used the species distribution models (Maxent), systematic conservation planning models (Marxan), and Geographic Information Systems (ArcGIS 10.0). The distributions of two species were correlated in the study area, enabling unique and economically viable joint conservation measures to be implemented. Three models were combined to identify feasible priority conservation sites. We used local spatial statistics to assess all identified conservation areas in relation to potential climate change based shifts in the geographic distribution of the two species. Model-based conservation strategies were used to identify effective measures to protect and utilize these two tree species in the study region. This study presents a novel technique for assessing wild plant distributions, in addition to serving as a model for the conservation of other species within the framework of general forest management, ecological construction, and geographical surveying.     

Bioremediation of CCA-treated wood by brown-rot fungi <Emphasis Type="Italic">Fomitopsis palustris</Emphasis>, <Emphasis Type="Italic">Coniophora puteana</Emphasis>, and <Emphasis Type="Italic">Laetiporus sulphureus</Emphasis>

This study evaluated oxalic acid accumulation and bioremediation of chromated copper arsenate (CCA)-treated wood by three brown-rot fungi Fomitopsis palustris, Coniophora puteana, and Laetiporus sulphureus. The fungi were first cultivated in a fermentation broth to accumulate oxalic acid. Bioremediation of CCA-treated wood was then carried out by leaching of heavy metals with oxalic acid over a 10-day fermentation period. Higher amounts of oxalic acid were produced by F. palustris and L. sulphureus compared with C. puteana. After 10-day fermentation, oxalic acid accumulation reached 4.2 g/l and 3.2 g/l for these fungi, respectively. Fomitopsis palustris and L. sulphureus exposed to CCA-treated sawdust for 10 days showed a decrease in arsenic of 100% and 85%, respectively; however, C. puteana remediation removed only 18% arsenic from CCA-treated sawdust. Likewise, chromium removal in F. palustris and L. sulphureus remediation processes was higher than those for C. puteana. This was attributed to low oxalic acid accumulation. These results suggest that F. palustris and L. sulphureus remediation processes can remove inorganic metal compounds via oxalic acid production by increasing the acidity of the substrate and increasing the solubility of the metals.An erratum to this article can be found at     

High level expression of glucose-6-phosphate dehydrogenase gene <Emphasis Type="Italic">PsG6PDH</Emphasis> from <Emphasis Type="Italic">Populus suaveolens</Emphasis> in <Emphasis Type="Italic">E. coli</Emphasis>

In order to investigate the functions of the gene PsG6PDH and the mechanisms underlying freezing tolerance of Populus suaveolens, the recombinant expression vector pET-G (pET30a-G6PDH), which contained full encoding region of PsG6PDH gene, was established. The recombinant was identified by lawn-PCR and double enzyme digestion and then transformed into expression host XA90 and induced by isopropyl-â-D-thiogalactoside (IPTG) to express 100 kD polypeptide of G6PDH fusion protein. The results showed that the expressed amount of the fusion protein culminated after 1 mmol&#8226;L^–1 IPTG treatment for 4 h and that pET-G product was predominately soluble and not extra-cellular secreting.     

Enzymatic saccharification and ethanol production of <Emphasis Type="Italic">Acacia mangium</Emphasis> and <Emphasis Type="Italic">Paraserianthes falcataria</Emphasis> wood,and <Emphasis Type="Italic">Elaeis guineensis</Emphasis> trunk

We examined the saccharification and fermentation of meals from Acacia mangium wood, Paraserianthes falcataria wood, and Elaeis guineensis trunk. The levels of enzymatic hydrolysis of cellulose and ethanol production were highest for P. falcataria wood and lowest for A. mangium wood. Ultrasonication pretreatment of meal further increased the rates of hydrolysis and ethanol production in meal from P. falcataria wood. Through this pretreatment, hemicelluloses (xylan and xyloglucan) and cellulose were released in the meal from P. falcataria wood. Loosening of hemicellulose associations can be expected to make P. falcataria wood more useful for bioethanol production.     

Genetic diversity and mating system of <Emphasis Type="Italic">bracatinga</Emphasis> (<Emphasis Type="Italic">Mimosa scabrella</Emphasis>) in a re-emergent agroforestry system in southern Brazil

Bracatinga (Mimosa scabrella) is a legume tree species common in the early stages of succession in Araucaria angustifolia forests in southern Brazil. Bracatinga can form high-density monospecific stands called bracatingais. Its traditional management for charcoal production involves maintenance of the seed bank. Our objective was to analyze the genetic diversity and structure of bracatingais to understand the mechanisms by which intraspecific diversity of M. scabrella is created and maintained in landscapes managed by family farmers in their agroforestry mosaics. We analyzed 14 bracatingais using 8 allozyme loci. We compared parental and progeny generation indices (7 loci) and described the mating system (9 loci) of two progenies. Overall diversity was high: A = 2.69, H _o = 0.257, H _e = 0.382 and similar between populations. Overall fixation (F = 0.364) was similar to the fixation index (f = 0.329). The genetic divergence among populations was low (Θ_p = 0.052) but significant. The progenies’ genetic diversity values were similar to those of the previous generation (H _{e pop11} = 0.342 vs. 0.420/H _{e pop10} = 0.432 vs. 0.400). Progenies were compatible with half-sib and full-sib crossing expectations (θ _xy  = 0.204 and 0.194). Our data showed that there is a tendency for genetic structuring caused not only by the reproductive system but also by genetic drift. It is very likely that the high genetic diversity is amplified by internal migration within each bracatingal. This study showed that current landscape management can contribute to maintaining high levels of bracatinga genetic diversity, which contributes to its regional conservation.     

Leaf-level responses to light in two co-occurring <Emphasis Type="Italic">Quercus</Emphasis> (<Emphasis Type="Italic">Quercus ilex</Emphasis> and <Emphasis Type="Italic">Quercus suber</Emphasis>): leaf structure,chemical composition and photosynthesis

We studied morphological, biochemical and physiological leaf acclimation to incident Photon-Photosynthetic-Flux-Density (PPFD) in Quercus ilex (holm oak) and Quercus suber (cork oak) at Mediterranean evergreen oak woodlands of southern Portugal. Specific leaf area (SLA) decreased exponentially with increasing PPFD in both species. Q. ilex had lower SLA values than Q. suber. Leaf nitrogen, cellulose and lignin concentration (leaf area-based) scaled positively with PPFD. Maximum rate of carboxylation (V_cmax), capacity for maximum photosynthetic electron transport (J_max), rate of triose-P utilization (V_TPU) and the rate of nonphotorespiratory light respiration (Rd) were also positively correlated with PPFD in both Quercus species, when expressed in leaf area but not on leaf mass basis. Q suber showed to have higher photosynthetic potential (V_cmax, J_max^m and V_TPU^m) and a higher nitrogen efficient nitrogen use than Q.ilex. Leaf chlorophyll concentration increased with decreasing PPFD, improving apparent quantum use efficiency (Φ) in both Quercus species. We concluded that, in Q.ilex and Q.suber, leaf structural plasticity is a stronger determinant for leaf acclimation to PPFD than biochemical and physiological plasticity.     

Cloning of <Emphasis Type="Italic">XET</Emphasis> gene from <Emphasis Type="Italic">Anthocephalus chinensis</Emphasis> and its plant expression vector construction

A full-length cDNA sequence of xyloglucan endotransglycosylase gene (XET), abundantly expressed in the cambium of Anthocepha-lus chinensis was cloned by conserved PCR, rapid-amplification of cDNA ends and by chromosome walking. Analytical results of the DNA sequence show that a 912 bp complete open reading frame (ORF) encoded a 303-amino acid protein was in the 1205 bp full cDNA sequence. The deduced amino acid sequence of AcXET, which contained the conserved specific EIDFE catalytic site sequence to XETs was homologous to the other known XET proteins. In order to study the gene function of AcXET and obtain transgenic plants, a plant expression vector pBIAcXET was constructed by recombinating the AcXET fragment from the cloning vector pMD19AcXET and the binary vector pBI121 between the XbaI and SmaI sites. The fragment of AcXET gene was inserted between the CaMV 35S promotor and the coding region of the GUS gene in pBI121. The identification results show that the plant expression binary vector pBIAcXET was constructed successfully. These results lay the foundation for studying the molecular mechanism of AcXET gene during wood formation.     

At5g54160 gene encodes <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> 5-hydroxyconiferaldehyde <Emphasis Type="Italic">O</Emphasis>-methyltransferase

The function of an Arabidopsis thaliana gene, At5g54160 annotated as a caffeic acid O-methyltransferase CAOMT gene was characterized. The recombinant enzyme of this gene (AtOMT1) catalyzed the O-methylation of phenylpropanoid and flavonoid substrates. The specificity constants (k _cat/K _m) for 5-hydroxyconiferaldehyde (5-HCAld) and quercetin were both 0.11 μM⁻¹·min⁻¹. On the other hand, lignins of At5g54160-knockout Arabidopsis mutants lacked syringyl units. In addition, we showed that the gene silencing also resulted in significant accumulation of caffeyl alcohol (CaAlc). These results strongly suggested that At5g54160 gene is involved in syringyl lignin synthesis for the methylation of both 5-hydroxyconiferaldehyde and 3,4-dihydroxyphenyl compound(s). Part of this work was presented at the Annual Meeting of Japan Society for Bioscience, Biotechnology, and Agrochemistry, March 24–27, 2007