Genetic variability and interrelationship of Siberian and Far Eastern larches from RAPD-analysis data

Genetic diversity of larches from six geographically isolated regions, Tomsk, Irkutsk, Ulan-Ude (Siberia), and Blagoveshchensk, Khabarovsk, Yuzhno-Sakhalinsk (Far East) was examined by means of RAPD analysis. Tree DNA samples were compared using 457 RAPD loci (97% of which were polymorphic), identified with 17 primers of random sequences. In the samples examined, 32 to 49% of the genes were in heterozygous state, mean expected heterozygosity (Hexp) varied from 0.1373 to 0.1891, and the genetic distances (DN) for different sample pairs varied from 0.0361 to 0.1802. The main population parameters were determined for Larix sibirica Ledeb., L. gmelinni (Rupr.) Rupr., and L. kamtschatica (Rupr.) Carr. Analysis of the genetic relationships showed that L. kamtschatica was characterized by highest genetic differentiation from the other larches examined, while larches from Primorskii krai were genetically close to L. sibirica.     

Study of the acid hydrolysis of polysaccharides from larch wood for obtaining crystalline glucose

This study has been conducted within the framework of the realization of complex wasteless technology for processing the biomass of the Siberian larch (Larix sibirica Ledeb.) and the Dahurian larch (L. gmelini (Rupr.) Rupr.). Experimental data on the acid hydrolysis of polysaccharides from the larch wood cellolignin residue have been summarized with the aim to develop the technology of manufacturing crystalline glucose.     

Genetic Differentiation of Pedunculate Oak Quercus robur L. in the European Part of Russia Based on RAPD Markers

Using randomly amplified polymorphic DNA markers (RAPD), genetic variation and differentiation in four populations of pedunculate oak Quercus robur L. were examined. The populations occupy a large part of the Quercus robur range in the European Russia (Voronezh and Novgorod oblasts; Republics of Mordovia and Bashkortostan). With each of six random primers (A02, A09, A17, B01, B08, B11), 96 DNA samples were analyzed by PCR. In all, 48 putative polymorphic RAPD loci were detected. We failed to reveal population-specific DNA fragments for any primer although the frequencies of 14 fragments were significantly different among populations. The oak populations studied exhibited high variability: 73–90% of genes were polymorphic and the effective allele number was about 1.4. The total genetic variation varied from 0.202 (Vor) to 0.245 (Nov), which corresponded to the estimates for populations of this species from Central and Western Europe. The populations examined showed low among-population differentiation (G _ST = 0.098); gene flow N _e m was 4.61. The proportion of among-population variation of the RAPD loci studied accounted for 7% of the total variability; more than 93% of the total variability was explained by individual and within-population variation.     

Heterozygosity and hybrid performance in larch

Random Amplified Polymorphic DNAs (RAPD) were used for estimating genetic distances between 12 European larches (Larix decidua) and 12 Japanese larches (L. kaempferi) that were the parents in a factorial mating design. One hundred and eleven fragments were used for establishing genetic distances based on Jaccard's coefficient between parents. Thirteen fragments differentiated the larch species. The genetic distance between individuals of the same species (D _J =0.39 in the Japanese larch and 0.45 in the European larch) was lower than the genetic distance between species (D _J =0.72). A UPGMA dendrogram based on genetic distances clearly clustered each larch species, confirming the speciation at a molecular level. Correlations between genetic distances of the parents and performances of the hybrid families were established for various quantitative traits. Significant values were found for growth characters and branch insertion angle, which suggested an effect of general heterozygosity level on hybrid traits. These correlations also evolved with tree age: the maximal correlation was noticed on 6-year-old trees for height. The lack of correlation between parental genetic distances and hybrid performances for the other quantitative traits suggested that these characters were controlled by fewer genes. The results of this study show that crosses between genetically distant parents produce hybrids with excellent growth performances; this represents a potential selection criterion of the genitors.     

利用正交设计优化兴安落叶松RAPD-PCR反应体系

以兴安落叶松针叶DNA为模板,对影响落叶松RAPD-PCR 扩增的重要参数进行了优化试验,以期建立兴安落叶松RAPD PCR反应的最佳体系。通过采用正交设计L₁₆(4⁵)对兴安落叶松RAPD-PCR反应的5因素(Taq酶、Mg²⁺、dNTP、模板DNA、引物)在4个水平上进行优化试验,结果表明兴安落叶松最佳的RAPD-PCR的反应体系(20 μL)中含有模板90 ng,0.5 μmol·L^-1的引物,1×反应缓冲液,DNTP各为0.25 mmol·L^-1,1 U的Taq DNA聚合酶,Mg²⁺ 2.5 mmol·L^-1。在此基础上筛选出20个扩增稳定、多态性丰富的RAPD引物,并通过梯度 PCR试验,确定了引物最佳退火温度。     

Nitrogen Fixation Associated with Sand Grain Root Sheaths (Rhizosheaths) of Certain Xeric Grasses

Nitrogen fixation (acetylene reduction) was found to be associated with sand grain root sheaths (rhizoseaths) occurring on the following xeric grasses: Oryzopsis hymenoides (Roem. and Shult.) Ricker, Agropyron dasystachyum (Hook.) Scrib., Stipa comata Trin. and Rupr., and Aristida purpurea Nutt. Acetylene reduction rates associated with whole plant specimens of these species varied from 515 to 920 nmol C₂H₄/(g dry wt.) × (6 days). Nitrogenase activity was shown to be associated with the rhizosheaths. Bacillus polymyxa-like nitrogen fixers were isolated from the rhizosheaths of each grass. The isolates reduced acetylene and assimilated ¹⁵N₂.     

Investigation of physicochemical properties of arabinogalactan of different larch species

Samples of arabinogalactan (AG) were isolated by aqueous extraction from wood of different larch species (Larix sibirica Ledeb., Larix gmelinii (Rupr.) Rupr., Larix cajanderi Mayr., and Larix olgensis var. Koreana) and studied by HPLC, IR spectroscopy, and quantitative ¹³C NMR spectroscopy. The wood of the studied larch species was shown to contain significant amount of arabinogalactan (10–17% of the a.d.w. mass) and could be a source of its industrial production. The studied AG samples had similar structural and molecular-mass characteristics. This fact could facilitate the product standardization during its industrial production both from the wood of a single larch species and from mixed raw materials.     

Bacterial (Bacillus thuringiensis subsp. thuringiensis and B. thuringiensis subsp. kurstaki) Entomocidal Preparations Decrease Chlorophyll Accumulation in Larch Needles

Chlorophyll a plus b content and absorption spectra of the homogenates from the cotyledonary leaves of 30-day-old seedlings of two larch species, Larix gmelinii (Rupr.) Rupr. and L. sibirica Ldb. were studied. The seedlings were grown on Perlite containing aqueous solutions of entomocidal biopreparations isolated from Bacillus thuringiensis subsp. thuringiensis (bitoxybacillin) and B. thuringiensis subsp. kurstaki (lepidocide) at various final concentrations (2, 6, and 12 g/l). Changes in the form of chlorophyll absorption spectra induced by biopreparations were established. A marked inhibition of pigment accumulation in the needles dependent on the biopreparation concentration was noted. At a low concentration (2 g/l), the biopreparations virtually did not affect the chlorophyll content; an increase in their concentrations resulted in a decrease in chlorophyll content in leaves by 20% (at 6 g/l) and 40% (at 12 g/l). It is concluded that bitoxybacillin and lepidocide inhibited the chlorophyll accumulation in larch needles to a similar extent.     

The diversity of chloroplast microsatellite loci in Siberian fir (Abies sibirica Ledeb.) and two Far East fir species A. nephrolepis (Trautv.) Maxim. and A. sachalinensis Fr. Schmidt

The genetic variability in 29 populations of Abies sibirica, three of A. nephrolepis, and seven of A. sachalinensis was studied using SSR markers of chloroplast DNA. Among ten primer pairs examined, pairs Pt71936 and Pt30204 gave stable amplification and polymorphic products (with nine and fourteen alleles, respectively). Totally, 70 haplotypes were found, 43 in A. sibirica, 49 in A. sachalinensis, and 31 in A. nephrolepis. The highest values of genetic diversity parameters were observed in A. sachalinensis, and the lowest in A. sibirica. The Siberian fir differs from Far East species by the uneven multimodal frequency distributions of allele length in both cpSSR loci, which is explained by the presence of few separated from each other dominating haplotypes. This fact indicates that A. sibirica and the Far East species have different demographic histories. In A. sibirica, the proportion of diversity between populations in the total genetic diversity, calculated taking into account the differences between haplotypes (R _ST) was 8.34 and 4.42% without accounting for haplotypes differences (R _ST > G _ST, P = 0.01). The pairwise G _ST correlate significantly with geographic distances between the populations A. sibirica and with genetic distances D calculated from allozyme data. No such correlations were found with the R _ST parameter. The results of cpSSR variability analysis strongly support the conclusions inferred from allozyme data: several geographic groups of comparatively genetically close populations are identified, which may be explained by the history of colonization of the present-day Siberian fir range. Original Russian Text ? S.A. Semerikova, V.L. Semerikov, 2007, published in Genetika, 2007, Vol. 43, No. 12, pp. 1637–1646.     

Using RAPD for Estimating Genetic Polymorphism in and Phylogenetic Relationships among Species of the Genus Lycopersicon (Tourn.) Mill.

RAPD genome analysis of 53 species and cultivars of the genusLycopersicon (Tourn.) Mill. revealed their high genetic polymorphism (Tourn.) Mill., based on which their phylogenetic relationships were inferred. In total, 248 polymorphic DNA fragments were amplified. Intraspecific polymorphism was maximum (79%) in L. peruvianum and minimum (9%) in L. parviflorum. In general, genome divergence among cross-pollinating tomato species was substantially higher than in self-pollinating species. An UPGMA dendrogram constructed from the RAPD patterns was consisted with the Lycopersicon phylogeny inferred from the molecular data of RFLP, ISSR, and microsatellite analyses and with a classification based on morphological characters. The relationships of taxa within the genus Lycopersicon are discussed.     

Stomatal control of photosynthesis in detached leaves of woody and herbaceous plants

Photosynthesis and transpiration were assayed in leaves and needles of some woody species (Pinus sibirica Du Tour, P. sylvestris L., Larix sibirica Ledeb., Betula pendula Roth., Quercus robur L.), annual herbaceous plants (Amaranthus cruentus L. cv. Tampala, Celosia argentea L. f. cristata (L.), Gomphrena dispersa Standl., Solanum tuberosum L., Helianthus annuus L., H. tuberosus L.), and perennial herbs (Inula helenium L., Poligonum weyrichii F. Schmidt, Polymnia sonchifolia Poepp. & Endl.). A high-precision portable gas-analyzing system GFS-3000 with a climate-controlled chamber was used for measurements on leaves both before and after leaf detachment from the shoot under conditions optimal for photosynthesis: photosynthetically active radiation of 2000 μE/(m² s), 22–25°C, and a relative humidity of 65–70%. The steady-state gas exchange in illuminated leaves of all plant species examined was characterized by a directly proportional relationship between photosynthesis and transpiration (R ₂ = 0.87). This means that the temporal course of H₂O and CO₂ gas exchange in detached leaves suffices to characterize the status of stomatal control of photosynthesis. The general trend in the effect of leaf detachment, observed in herbaceous and woody plants, is that the stomatal control of photosynthesis was retained within first 3–5 min after leaf excision. By contrast, the increase in transpiration after leaf detachment was species-specific. Because of this circumstance, the measurements of transpiration by rapid weighing method may result in overestimation of transpiration rates by 10–15% for some plant species, compared to steady-state rates of gas exchange in undetached leaves.     

外源亚精胺对盐胁迫下白刺幼苗叶片抗氧化酶系统的影响

为进一步阐明盐生植物白刺耐盐性与多胺的关系,通过水培试验研究了叶面喷施亚精胺(Spd)对不同浓度NaCl胁迫下西伯利亚白刺幼苗叶片丙二醛(MDA)和超氧阴离子(O2)产生速率,以及抗氧化物酶系统和根系活力的影响.结果表明:叶面喷施0.1 mmol·L1 Spd 5 d后,可显著提高100和200 mmol·L1 NaCl胁迫下白刺幼苗叶片超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的活性以及根系活力,降低了叶片MDA含量和O2的产生速率;而在0、50、300 mmol·L-1 NaC1处理下,外施Spd对白刺幼苗叶片上述指标无显著影响.研究结果证实,在100～200 mmol·L-1 NaCl胁迫范围内,外施亚精胺可能通过增强体内保护酶活性来显著降低活性氧水平,有效减轻盐胁迫对盐生植物白刺幼苗造成的过氧化伤害,从而增强白刺对盐环境的适应性.     

Lipids of the meristems of the main coniferous edificators from central siberia under low-temperature adaptation: 1. The characteristics of the fatty acid composition of phospholipids from winter meristems of Larix sibirica Ledeb., Picea obovata L., and Pinus sylvestris L.

Trienoic fatty acids (FAs) have been found to be the dominant group of unsaturated fatty acids in the composition of phospholipids (PLs) from Larix sibirica Ledeb., whereas dienoic FAs have been established to prevail in PLs from Picea obovata L. and Pinus sylvestris L. Bud swelling in spring is accompanied by a decrease in the content of unsaturated FAs by approximately 30%. The analysis of the change in the content of individual C₁₈ FAs in the PL structure upon the transition of the tree from winter dormancy to vegetation has revealed the important functional role of acyl-lipid ω₆ desaturase, which is responsible for the synthesis of linoleic acid (C_{18: 2}), in the formation of the cryoprotected state of meristem cell membranes of Larix sibirica Ledeb., Picea obovata L., and Pinus sylvestris L. In addition, the particular significance of acyl-lipid ω₃ desaturase for the cryoadaptation of meristem cells in Larix sibirica Ledeb., as it catalyzes the transformation of linoleic acid (C_{18: 2}) to linolenic acid (C_{18: 3}) in PLs of larch 1.5–3 times more intensively than in the case of spruce and pine, is shown.     

Rates of spontaneous hybridization and hybrid recruitment in co-existing exotic and native mature larch populations

Exotic larch (Larix decidua Mill., Larix kaempferi (Lamb.) Carr. and Larix sibirica Ledeb.) and their hybrids have been introduced into Canada to test adaptation and growth for reforestation purposes. For tree breeding operations, provenance trials and seed orchards of exotic larches and their hybrids have been established in arboreta adjacent to natural forest stands of the indigenous species Larix laricina (Du Roi) K. Koch. This proximity offers an opportunity to measure spontaneous hybridization and the recruitment of hybrids. Using a combination of two chloroplast and three mitochondrial DNA markers, we estimated the rate of spontaneous hybridization taking place between native (L. laricina) and exotic (L. decidua, L. kaempferi, L. sibirica) species of larch in two arboreta. More than 5,000 seeds and 1,000 recruits from the two experimental sites were sampled and genetically identified. The occurrence of spontaneous hybridization between L. laricina and the exotics was found both in seeds and from recruits freely established near the plantations. A low hybridization rate (3 % or less) was observed among the seeds of L. laricina maternal trees. Spontaneous native-exotic hybrids were also observed to establish and survive in the environment under the current climatic conditions at a similarly low rate. However, a much higher and variable rate of establishment was observed for recruits with exotic components (up to 34.6 % at one site). These results indicate that the spontaneous hybridization and the recruitment of hybrid and exotic recruits do occur and should be taken into consideration in the management and establishment of plantations of fast-growing species with exotic components.     

Influence of temperature on fractional composition of proteins and respiration of germinating seeds of Gmelin and Siberian larch

The influence of optimal and contrasting temperatures on the fractional composition of proteins and respiration of germinating seeds of Gmelin larch (Larix gmelinii (Rupr.) Rupr.) and Siberian larch (Larix sibirica Ledeb.) was revealed. The dominance of globulins (salt-soluble fraction) and insoluble proteins was noted in viable air-dry seeds of larch. Optimal and contrasting temperatures of germination of seeds caused significant changes in the content and dynamics of separate fractions of protein, the contrasting temperatures causing greater changes: increasing in the content of insoluble proteins, intense consumption of globulins at separate stages of germination. Respiration of the seeds during germination at contrasting temperatures also increased more than at an optimal temperature. Thermal regime of germination affected the character of relations between the respiration rate and contents of different protein fractions.     

Genotyping with RAPD and microsatellite markers resolves pathotype diversity in the ascochyta blight pathogen of chickpea

 The poor definition of variation in the ascochyta blight fungus (Ascochyta rabiei) has historically hindered breeding for resistance to the chickpea (Cicer arietinum L.) blight disease in West Asia and North Africa. We have employed 14 RAPD markers and an oligonucleotide probe complementary to the microsatellite sequence (GATA)₄ to construct a genotype-specific DNA fragment profile from periodically sampled Syrian field isolates of this fungus. By using conventional pathogenicity tests and genome analysis with RAPD and microsatellite markers, we demonstrated that the DNA markers distinguish variability within and among the major pathotypes of A. rabiei and resolved each pathotypes into several genotypes. The genetic diversity estimate based on DNA marker analysis within pathotypes was highest for the least-aggressive pathotype (pathotype I), followed by the aggressive (pathotype II) and the most-aggressive pathotype (pathotype III). The pair-wise genetic distance estimated for all the isolates varied from 0.00 to 0.39, indicating a range from a clonal to a diverse relationship. On the basis of genome analysis, and information on the spatial and temporal distribution of the pathogen, a general picture of A. rabiei evolution in Syria is proposed. Received: 10 January 1998 / Accepted: 23 January 1998     

Genetic diversity of larch at the north of Primorskii Krai and limits of Larix olgensis A. Henry distribution

The genetic diversity of four mtDNA fragments and five microsatellite loci of cpDNA was examined in six larch population samples from the territory of the Sikhote-Alin Nature Reserve (northern Primor’e). In order to identify possible species-specific differences between the samples, the latter were collected at transects along the shore of the Sea of Japan and at different distances from the sea. Based on a number of morphological characters, some authors suggest that Olgan larch (Larix olgensis A. Henry) grows in the shoreline part of the reserve and, moving inland, it is replaced by Gmelin larch (Larix gmelinii (Rupr.) Rupr.). According to the other data, the northern border of the Olgan larch range does not reach the reserve territory. The data obtained were compared to those obtained previously for three samples from the south of Primor’e, including those for the locus classicus of Olgan larch. In the examined larch individuals (186, for mtDNA and 200, for cpDNA), a total of five mitotypes and 52 chlorotypes were recovered. According to the results of the AMOVA test, the proportion of variations accounted for the differences among all Larix population samples examined over both types of markers was higher (N _ST = 0.435, for mtDNA and R _ST = 0.041, for cpDNA) than that for the differences among the population samples within the reserve (N _ST = 0.079, for mtDNA and R _ST = 0.005, for cpDNA). No differences were detected between the groups of shoreline and continental populations.     

Application of a Coupled Photosynthesis–Stomatal Conductance Model to Analysis of Carbon Assimilation by Spruce and Larch Trees in the Forests of Russia

Photosynthetic activities of common spruce (Picea abies (L.) Karst.) and Dahurian larch (Larix gmelinii (Rupr) Rupr ex Kuzen) were analyzed on the basis of datasets obtained for 110- to 130-year-old forest stands in Middle Russia and East Siberia. Using a Li-Cor 6200 gas analyzer, photosynthesis was measured in parallel with transpiration, stomatal conductance, CO₂ concentrations in ambient air and intercellular spaces, the photosynthetically active radiation, air temperature, and air humidity. The data were examined within the framework of a biochemical model of photosynthesis of Farquhar et al. [1] in combination with the stomatal conductance model proposed by Jarvis [2]. The species-specific differences in carbon assimilation rates were discovered, and dependences of photosynthesis on the needle age, light regime, and growth conditions were revealed. The model parameters obtained were used to simulate the photosynthetic rates in spruce and larch trees at various weather conditions.     

Assessment of genetic diversity and relationships of Krachaai Sayam,an endemic plant in Thailand using microsatellite markers

Nineteen microsatellite markers were developed from Boesenbergia siamensis (Gagnep.) P. Sirirugsa, an endemic plant of Kanchanaburi Province, Thailand and used to determine genetic diversity of this plant. The number of alleles of each locus varied from 4 to 16 in 25 individual samples. The average values of observed and expected heterozygosities were 0.577 and 0.804, respectively. The polymorphic information content value ranged between 0.486 and 0.912. Ten loci significantly deviated from Hardy–Weinberg equilibrium, and eight pairs of loci showed significant evidence of linkage disequilibrium. Moreover, these newly developed microsatellite markers were used to determine cross-species amplification with three samples each of B. thorelii (Gagnep.) Loes., B. rotunda (L.) Mansf. and Kaempferia parviflora Wall. Of the 19 markers developed, 13 (68.42%) primer pairs were able to be used with B. thorelii (Gagnep.) Loes. and K. parviflora Wall., while 12 (63.16%) primer pairs were able to be used with B. rotunda (L.) Mansf. These microsatellite markers will be useful for further genetic analysis of this endemic plant as well as related species.     

Population genetic structure of <Emphasis Type="Italic">Sargassum thunbergii</Emphasis> (Fucales,Phaeophyta) detected by RAPD and ISSR markers

Genetic variation of four populations of Sargassum thunbergii (Mert.) O. Kuntze and one outgroup of S. fusiforme (Harv.) Setchell from Shandong peninsula of China was studied with random amplified polymorphic DNA (RAPD) and inter-simple sequence repeat (ISSR) markers. A total of 28 RAPD primers and 19 ISSR primers were amplified, showing 174 loci and 125 loci, respectively. Calculation of genetic diversity with different indicators (P%, percentage of polymorphic loci; H, the expected heterozygosity; I, Shannon’s information index) revealed low or moderate levels of genetic variations within each S. thunbergii population. High genetic differentiations were determined with pairwise Nei’s unbiased genetic distance (D) and fixation index (F _ST ) between the populations. The Mantel test showed that two types of matrices of D and F _ST were highly correlated, whether from RAPD or ISSR data, r = 0.9310 (P  = 0.008) and 0.9313 (P = 0.009) respectively. Analysis of molecular variance (AMOVA) was used to apportion the variations between and within the S. thunbergii populations. It indicated that the variations among populations were higher than those within populations, being 57.57% versus 42.43% by RAPD and 59.52% versus 40.08% by ISSR, respectively. Furthermore, the Mantel test suggested that the genetic differentiations between the four populations were related to the geographical distances (r > 0.5), i.e., they conformed to the IBD (isolation by distance) model, as expected from UPGMA (unweighted pair group method with arithmetic averages) cluster analysis. As a whole, the high genetic structuring between the four S. thunbergii populations along distant locations was clearly indicated in the RAPD and ISSR analyses (r > 0.8) in our study.