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2014 year, number 4

1.
ABOUT THE INTERNATIONAL WORKSHOP–SEMINAR

A. Onuchin Alexander
V. N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch, Academgorodok, 50/28, Krasnoyarsk, 660036 Russian Federation

Abstract >>
The fourth issue of "Siberian forest magazine" theme, it presents the materials of the first International Workshop-Seminar "Molecular Genetics in forest management: status, problems and prospects of application", held in May 2014 in Moscow at the Russian Center for Forest Protection with the support of Federal Forestry Agency.



2.
ON HOLDING THE FIRST MEETING OF THE INTERNATIONAL GENETIC SEMINAR

V.V. Soldatov1, K. V. Krutovsky2,3,4,5
1Russia Centre for Forest Protection (Roslesozashcita), 141207, Moscow region, Pushkino, Nadsonovskaya str., 13
2Siberian Federal University, Akademgorodok, 50a/2, Krasnoyarsk, 660036 Russian Federation
3N. I. Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkin str., 3, Moscow, 119333 Russian Federation
4University of Gttingen, Bsgenweg, 2, Gttingen, D-37077 Germany
5Texas A&M University, HFSB 305, 2138 TAMU, College Station, Texas, 77843 USA

Abstract >>
In Moscow on 28-29 May 2014 the Russian Center for Forest Protection ("Roslesozashchita") with the support of the Federal Forestry Agency (FFA) held its first international meeting of genetic seminar "Molecular Genetics in forest management: status, problems and prospects of application" with involving a wide range of experts on forest genetics, genomics and biotechnology, business executives, producers, managers of forest federal agencies and academic institutions, mainly the Russian Federation (RF) and Belarus.



3.
PROSPECTS FOR GENOMIC RESEARCH IN FORESTRY

K. V. Krutovsky1,2,3,4
1Siberian Federal University, Akademgorodok, 50a/2, Krasnoyarsk, 660036 Russian Federation
2N. I. Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkin str., 3, Moscow, 119333 Russian Federation
3University of Gttingen, Bsgenweg, 2, Gttingen, D-37077 Germany
4Texas A&M University, HFSB 305, 2138 TAMU, College Station, Texas, 77843 USA
Keywords: genome, de novo sequencing, Siberian larch (Larix sibirica Ledeb.), Siberian stone pine (Pinus sibirica Du Tour), Scots pine (Pinus sylvestris L.), boreal forest, phytopathogens, forestry

Abstract >>
Conifers are keystone species of boreal forests. Their whole genome sequencing, assembly and annotation will allow us to understand the evolution of the complex ancient giant conifer genomes that are 4 times larger in larch and 7–9 times larger in pines than the human genome. Genomic studies will allow also to obtain important whole genome sequence data and develop highly polymorphic and informative genetic markers, such as microsatellites and single nucleotide polymorphisms (SNPs) that can be efficiently used in timber origin identification, for genetic variation monitoring, to study local and climate change adaptation and in tree improvement and conservation programs.



4.
APPLICATION OF MOLECULAR GENETIC METHODS FOR FORESTRY IN BELARUS

V. E. Padutov, O. Yu. Baranov, D. I. Kagan, O. A. Kovalevich, M. Ya. Ostrikova, S. V. Panteleev, S. I. Ivanovskaya, D. V. Kulagin
Institute of Forest, National Academy of Sciences of Belarus, Proletarskaya str., 71, Gomel, 246001 Republic of Belarus
Keywords: molecular genetics, forestry, plantation and population seed growing, breeding, phytopathological monitoring, planting material, Republic of Belarus

Abstract >>
The results of molecular genetic studies, carried out by researchers of the Institute of Forest of the National Academy of Sciences of Belarus are presented in the paper. Examples of their practical use in forestry of Belarus are shown.



5.
DEVELOPMENT OF CYTOPLASMATIC SSR-MARKERS FOR POPULATION GENETIC STUDIES OF THE SIBERIAN STONE PINE (PINUS SIBIRICA DU TOUR)

E. A. Shilkina1, N. V. Oreshkova2,3, A. A. Ibe1,3, K. O. Deych1,3, K. V. Krutovsky4,5,6
1Centre for Forest Protection of Krasnoyarsk Territory, Akademgorodok, 50a/2, Krasnoyarsk, 660036 Russian Federation
2V. N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch, Akademgorodok, 50/28, Krasnoyarsk, 660036 Russian Federation
3Siberian Federal University, Akademgorodok, 50a/2, Krasnoyarsk, 660036 Russian Federation
4N. I. Vavilov Institute of General Genetics, Russian Academy of Sciences, Akademgorodok, 50a/2, Krasnoyarsk, 660036 Russian Federation
5University of Gttingen, Gubkin str., 3, Moscow, 119333 Russian Federation
6Texas A&M University, Bsgenweg, 2, Gttingen, D-37077 Germany
Keywords: DNA markers, PCR, chloroplast and mitochondrial markers, mitotype, locus, allele, haplotype, Siberian stone pine (Pinus sibirica Du Tour)

Abstract >>
Three chloroplast and one mitochondrial DNA markers were developed and used for genotyping of 60 trees in two populations of the Siberian stone pine (Pinus sibirica Du Tour). Two chloroplast loci were monomorphic in both populations, and one polymorphic with two alleles. Therefore, four chloroplast haplotypes were revealed totally. A mitochondrial DNA marker had two alleles or haplotypes (mitotypes).



6.
RELATIONSHIP BETWEEN GENETIC AND BREEDING RESEARCH OF FOREST WOODY PLANTS

L. I. Milyutin
V. N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch, Akademgorodok, 50/28, Krasnoyarsk, 660036 Russian Federation
Keywords: genetics, breeding, phenetics, polymorphism, geographic variability, forest woody plants

Abstract >>
Current and unsolved problems in the relationship of genetic and breeding research of forest woody plants are discussed in the paper. In particular, disadvantages of isoenzymic and DNA polymorphism are analyzed. The tasks aimed at bringing together research of forest geneticists and breeders are discussed.



7.
APPLICATION OF PHENO–GEOGRAPHICAL RESEARCH RESULTS IN FORESTRY OF RUSSIA

A. I. Vidyakin
Institute of Biology, Komi Republic Scientific Centre, Russian Academy of Sciences, Nekrasov str., 65, Kirov, 610035 Russian Federation
Keywords: Scots pine (Pinus sylvestris L.), population, pheno-geographical research, application of the results in forestry, Russia

Abstract >>
The article shows the main results of pheno-geographical research of the Scots pine (Pinus sylvestris L.) population-chorologic structure in the Russian plain, in the Southern Trans-Urals. It also suggests possible practical uses of these results in forestry of Russia. It is recommended to use the maps showing the spatial arrangement outline obtained during the research in developing seed zoning of the species. A system of silvicultural-economic measures aiming at restoring genetic variation and evolutionary-genetic processes in populations is discussed.



8.
MOLECULAR GENETIC METHODS IMPLEMENTATION FOR PHYTOPATHOGEN IDENTIFICATION IN FOREST STANDS AND NURSERIES OF THE RUSSIAN FEDERATION

T. S. Alimova1, V. A. Sivolapov2, N. A. Karpechenko2, O. K. Shishkina1, S. V. Panteleev3, O. A. Kovalevich3
1Russian Centre for Forest Protection, Nadsonovskaya str., 13, Pushkino, Moscow Region, 141200 Russian Federation
2Centre for Forest Protection of Voronezh Region, Lomonosov str., 105, Voronezh, 394000 Russian Federation
3Institute of Forest, National Academy of Sciences of Belarus, Proletarskaya str., 71, Gomel, 246001 Republic of Belarus
Keywords: phytopathogens, DNA analysis of phytopathogens, doughnut fungus (Rhizina undulata Fr. 1815), annosum root rot (Henerobasidion annozum (Fr.) Breff 1889), fusariosis, phomosis, forest stands, nurseries, Russian Federation

Abstract >>
The results of the application of molecular genetics methods for the analysis of the plant pathogens present in forest plantations and nurseries of the Russian Federation, including doughnut fungus and annosum root rot are presented. The prospects and benefits of using DNA analysis for early diagnosis of plant diseases without isolation of the pathogen in pure culture, shortening time of analysis, and the possibility of mass screening are discussed.



9.
MOLECULAR PHYTOPATHOLOGY: CURRENT APPROACHES AND MAIN DIRECTIONS IN DIAGNOSTICS OF WOODY PLANT DISEASES

O. Yu. Baranov
Institute of Forest, National Academy of Sciences of Belarus, Proletarskaya str., 71, Gomel, 246001 Republic of Belarus
Keywords: molecular phytopathology, hytopathogen, DNA markers, epiphytotics, woody plants

Abstract >>
In the article the authors describe the prospects for diagnosis of woody plants diseases based on the use of modern methods of molecular plant pathology. The metagenomic approach based on the analysis of complex pathogens, including non-pathogenic microflora is described. The use the multicopy universal loci characterized by a number of advantages in determining taxonomic affiliation of infectious agents during phytopathological molecular analysis is proposed.



10.
MOLECULAR GENETIC DIAGNOSTICS OF FOREST PLANT MYCOSIS AGENTS, TRANSPORTED BY INSECTS

S. V. Panteleev, O. Yu. Baranov
Institute of Forest, National Academy of Sciences of Belarus, Proletarskaya str., 71, Gomel, 246001 Republic of Belarus
Keywords: DNA, PCR, forest planting material, hytopathogen, Cladosporium, Alternaria, transport by insects

Abstract >>
The aspects of molecular genetic techniques in the diagnostics of fungal species composition in the tissues of insects in order to clarify their role in transport of pathogens of major diseases of forest tree species in nurseries is discussed in the paper. It is established that the insects are one of factors of potential transport and distribution of a number of phytopathogenic fungi, in particular of the genera Cladosporium and Alternaria.



11.
CYTOLOGICAL, MOLECULAR-GENETIC AND SILVICULTURAL-SELECTION RESEARCH OF POLYPLOID POPLARS

A. I. Sivolapov1, D. V. Politov2, O. S. Mashkina3, M. M. Belokon2, V. A. Sivolapov4, Y. S. Belokon2, T. M. Tabatskaya5
1Voronezh State Academy of Forestry and Technology, Timiryazev str., 8, Voronezh, 394087 Russian Federation
2N. I. Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkin str., 3, Moscow, 119991 Russian Federation
3Voronezh State University, Universitetskaya Ploschad, 1, Voronezh, 394006 Russian Federation
4Centre for Forest Protection of Voronezh Region, Lomonosov str., 105, Voronezh, 394087 Russian Federation
5All-Russian Scientific and Research Institute for Forest Genetics, Breeding and Biotechnology, Lomonosov str., 105, Voronezh, 394087 Russian Federation
Keywords: poplar, aspen, polyploids, the number of chromosomes, DNA analysis, microsatellite loci, genetic passportization, productivity of poplar cultures

Abstract >>
The results of cytological, silvicultural and selection research and DNA analysis of polyploid poplars are discussed in the article. Poplar clones were studied for productivity, the number of chromosomes, selection and testing of microsatellite loci for genetic certification of planting material. The study showed a high diagnostic performance of species, of individual genotypes and clones within species and hybrids on the basis of gene markers of polymorphism of microsatellite DNA.



12.
EFFICIENCY OF USING PERMANENT SEED SOURCES FOR CONSERVATION OF GENETIC POOL OF SCOTS PINE (PINUS SYLVESTRIS L.) IN BELARUS

S. I. Ivanovskaya
Institute of Forest, National Academy of Sciences of Belarus, Proletarskaya str., 71, Gomel, 246001 Republic of Belarus
Keywords: Scots pine (Pinus sylvestris L.), permanent seed sources, isoenzyme analysis, conservation of the gene pool, Republic of Belarus

Abstract >>
On the basis of electrophoretic isoenzyme, analysis of permanent seed sources of Scots pine from Belarus is conducted. Research revealed that almost all of the analyzed plus stands, genetic reserves, seed orchards of the I order provenance and 71.5 % of seed orchards of the II order support maintenance of the level of average heterozygosity characteristic of Scots pine in Belarus. It is shown that in the plantation and population, seed from permanent seed sources can ensure the conservation of the species gene pool at higher productivity than created forest plantations.



13.
THE VARIABILITY OF CYTOPLASMIC DNA HAPLOTYPES OF NORWAY SPRUCE IN THE PROVENANCE TRIALS

V. E. Padutov, A. I. Sidor, D. I. Kagan, O. A. Kovalevich, S. N. Veras
Institute of Forest, National Academy of Sciences of Belarus, Proletarskaya str., 71, Gomel, 246001 Republic of Belarus
Keywords: Norway spruce (Picea abies), variability, mitochondrial DNA, chloroplast DNA, haplotype, provenance trails

Abstract >>
A genetic analysis of mitochondrial (mtDNA) and chloroplast (cpDNA) genomes of 27 climatypes of Norway spruce was conducted in the provenance (Cherikov forestry district, Republic of Belarus). It was found that the analyzed climatypes presented boreal and carpathian mtDNA haplotypes. The first of them is found in all regions of growth climatypes (Priuralsky, Northwest, Central, Baltic, Byelorussian, Southwest), the second - only in Belarusian and Southwest regions. It is shown that polymorphism cpDNA significantly higher in comparison with the mtDNA. For a number of alleles of loci cpDNA the clinal variation was found in the frequency of their occurrence. It was revealed that some cpDNA haplotypes, as haplotypes mtDNA, of Norway spruce can be represented as in all regions of the investigated part of the range as on certain of its territories.



14.
STAGE–BYSTAGE CERTIFICATION OF TREES IN PLANTATIONS OF SCOTS PINE GENETIC BREEDING COMPLEX

V. V. Tarakanov1, L. I. Kalchenko2, K. G. Zatcsepina1,2, A. K. Ekart1, D. N. Shuvaev2
1V. N. Sukachev Institute of Forest Russian Academy of Sciences, Siberian Branch, Novosibirsk, Post Box 45, 630082 Russian Federation
2Centre for Forest Protection of Altai Territory, Proletarskaya str., 61, Barnaul, 656056 Russian Federation
Keywords: genetic certification, clone plantations, Scots pine (Pinus sylvestris L.), phens, allozyme analysis, Altai

Abstract >>
In Altai territory in Scots pine (Pinus sylvestris L.) clonal plantations, that include nearly 1.5 thousand trees of 118 clones, the stage-by-stage certification of trees was carried out. Certification process included 3 stages: 1) forestry stage (preliminary), 2) phenetic stage (basic), 3) genetical stage (précising). Accuracy of phenetic certification was 97.1 %. The final stage of certification was carried out taking into account of revealed «phenetic clusters» within of clones. One-ramet and many-ramets (mixed) samples were compared within clusters and these must show identical results if all ramets are truly marked. The stage-by-stage certification method allows less expensive molecular genetics testing in some cases.



15.
MOLECULAR GENETIC ANALYSIS OF SOMACLONAL LINES GENOTYPES OF SILVER BIRCH AND HYBRID BIRCH

A. V. Konstantinov, S. V. Panteleev
Institute of Forest, National Academy of Sciences of Belarus, Proletarskaya str., 71, Gomel, 246001 Republic of Belarus
Keywords: somaclonal variation, silver birch, hybrid birch, RAPD-analysis, genetic passport

Abstract >>
Enrichment of genetic diversity by means of somaclonal variation can allow selection of individuals with increased adaptability to various unfavorable conditions. Twenty shoot cultures differing in organogenesis and morphological features were selected for two studied clones. Multilocus genetic passports of somaclonal lines were developed according to RAPD analysis. Among the samples derived from clone 52-84/8 shoot cultures the changes in RAPD-spectra were detected over primers UBC-106 and UBC-254. In the case of clone 6-161/3 the same changes were detected over primers UBC-268 and UBC-154. UBC-203 primer didnt show any variation.



16.
PRELIMINARY RESULTS OF DE NOVO WHOLE GENOME SEQUENCING OF THE SIBERIAN LARCH (LARIX SIBIRICA LEDEB.) AND THE SIBERIAN STONE PINE (PINUS SIBIRICA DU TOUR)

K. V. Krutovsky1,2,3,4, N. V. Oreshkova1,5, Yu. A. Putintseva1, A. A. Ibe1,6, K. O. Deych1,6, E. A. Shilkina1,6
1Siberian Federal University, Akademgorodok, 50a/2, Krasnoyarsk 660036 Russian Federation
2N. I. Vavilov Institute of General Genetics, Russian Academy of Sciences, Gubkin str., 3, Moscow, 119333 Russian Federation
3University of Gttingen, Bsgenweg, 2, Gttingen, D-37077 Germany
4Texas A&M University, HFSB 305, 2138 TAMU, College Station, Texas, 77843 USA
5V. N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch, Akademgorodok, 50/28, Krasnoyarsk, 660036 Russian Federation
6Centre for Forest Protection of Krasnoyarsk Territory, Akademgorodok, 50a/2, Krasnoyarsk, 660036 Russian Federation
Keywords: genome, de novo sequencing, Siberian larch (Larix sibirica Ledeb.), Siberian stone pine (Pinus sibirica Du Tour)

Abstract >>
The Illumina HiSeq2000 DNA sequencing generated 2 906 977 265 high quality paired-end nucleotide sequences (reads) and 576 Gbp for Siberian larch (Larix sibirica Ledeb.) that corresponds to 48X coverage of the larch genome (12.03 Gbp), and 3 427 566 813 reads and 679 Gbp for Siberian stone pine (Pinus sibirica Du Tour) that corresponds to 29X coverage of its genome (23.6 Gbp). These data are not enough to assemble and annotate whole genomes, but the obtained nucleotide sequences have allowed us to discover and develop effective highly polymorphic molecular genetic markers, such as microsatellite loci that are required for population genetic studies and identification of the timber origin. Sequence data can be used also to discover single nucleotide polymorphisms (SNPs). Genomic studies of Russian boreal forests and major phytopathogens associated with them will also allow us to identify biomarkers that can be used for solving important scientific and economic problems related to the conservation of forest genetic resources and breeding more resilient and fast growing trees with improved timber and resistance to diseases and adverse environmental factors.



17.
THE USE OF GENETIC MARKERS OF VARIOUS TYPES FOR EVALUATION OF INTRASPECIFIC DIFFERENTIATION LEVEL OF THE SIBERIAN SPRUCE

A. K. Ekart1, S. A. Semerikova2, V. L. Semerikov2, A. N. Kravchenko1, O. S. Dymshakova2, A. Ya. Larionova1
1V. N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch, Akademgorodok, 50/28, Krasnoyarsk, 660036 Russian Federation
2Institute of Plant and Animal Ecology, Russian Academy of Sciences, Ural Branch, 8 Marta str., 202, Yekaterinburg, 620144 Russian Federation
Keywords: Siberian spruce (Picea obovata Ledeb.), isoenzymes, nuclear and chloroplast microsatellites, genetic diversity, differentiation

Abstract >>
On the basis of the analysis of variability of isoenzyme loci and microsatellite loci of nuclear and chloroplast DNA the data were obtained about the degree of differentiation of a number populations of Siberian spruce (Picea obovata Ledeb.), including isolated populations located on the island of Olkhon, in the reserve Bogd–Uul (Mongolia) and on the south of the Magadan region. It was shown that the combined use of various types of genetic markers gives an opportunity to get more complete and objective information about a genetic diversity and intraspecific differentiation of this widespread in Siberia species.



18.
INTRASPECIFIC GENETIC DIFFERENTIATION OF THE CAJANDERI LARCH (LARIX CAJANDERI) IN THE NORTH–EAST ASIA

N. V. Oreshkova
V. N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch, Akademgorodok, 50/28, Krasnoyarsk, 660036 Russian Federation
Keywords: Cajanderi larch (Larix cajanderi), markers of a nuclear genome, allelic variants, genetic structure, intraspecific differentiation

Abstract >>
The study of intraspecific genetic differentiation of the Cajanderi larch (Larix cajanderi) was carried out in the north–east of Asia — in Magadan region and on the Kamchatka Peninsula. Genetic diversity of the larch was investigated using eight nuclear microsatellite loci. SSR analysis revealed high level of genetic diversity of the Cajanderi larch in the Far North-East of Asia. Significant genetic differences between L. cajanderi samples from Magadan region and Kamchatka suggests early isolation of the larch on the peninsula.



19.
ABOUT SAMPLES IN THE RESEARCH OF INTRASPECIFIC GENETIC DIVERSITY OF SCOTS PINE (PINUS SYLVESTRIS L.)

I. V. Tikhonova1, V. L. Semerikov2, S. A. Semerikova2, O. V. Dymshakova2, K. G. Zatcsepina1
1V. N. Sukachev Institute of Forest, Russian Academy of Sciences, Siberian Branch, Akademgorodok, 50/28, Krasnoyarsk, 660036 Russian Federation
2Institute of Plant and Animal Ecology, Russian Academy of Sciences, Ural Branch, 8 Marta str., 202/3, Yekaterinburg, 620144 Russian Federation
Keywords: the sample, genetic polymorphism, Scots pine (Pinus sylvestris L.)

Abstract >>
The influence of sample size on the results of the assessments of the genetic diversity of populations of Scots pine on the variability of 12 polymorphic loci isoenzymes and 4 chloroplast microsatellite loci (cpSSR). It was shown that increasing the sample size (to over 100 trees in the population) leads to significant increase in estimates of allozyme diversity (number of alleles and genotypes) and haplotype diversity of chloroplast DNA (microsatellite loci cpSSR) and to the reduction of inter-population genetic distance Nei without significantly changing the parameters characterizing the genetic structure of the species. Standard sample sizes allow reliable estimation of the population genetic structure of the species, but is not sufficient to study its intra-population diversity.



20.
GENETIC VARIABILITY AND DIFFERENTIATION OF THE KAMCHATKA, SAKHALIN AND KURIL ISLANDS LARCHES

I. Yu. Adrianova
Institute of Biology and Soil Science, Russian Academy of Sciences, Far Eastern Branch, Prospekt Stoletiya Vladivostoka, 159, Vladivostok, 690022 Russian Federation
Keywords: larch (Larix), RAPD analysis, genetic variability, genetic differentiation, AMOVA, Kamchatka, Sakhalin, Kuril Islands

Abstract >>
The genetic variability and differentiation of the 10 larch populations from Kamchatka, Sakhalin and Iturup Island (Kuril archipelago) were investigated by RAPD method. On the basis of analysis of 319 RAPD loci identified by means of nine random primers it was indicated that the samples from Kamchatka have higher parameters of genetic variability (He = 0.172, P95 = 43.0 %) as compared with the samples from Sakhalin and Iturup (He = 0.142, P95 = 39.5 % and He = 0.143, P95 = 41.1 %, correspondingly). Significant genetic differentiation for the samples from Sakhalin (GST = 0.24) and Kamchatka (GST = 0.25) was revealed. Reliable correlation between genetic and geographic distances between samples was found (r = 0.68, p < 0.005).



21.
Resolution of the First International Conference and Workshop on "Molecular Genetics in Forestry: the Status, Problems, and Prospects for Application", to be held 28–29 May, 2014 in Moscow, Russia