Приведены результаты исследования генетической дифференциации популяций лиственницы сибирской (Larix sibirica Ledeb.) в широтном градиенте климатических условий, полученные на основе генотипирования генома с помощью высокопроизводительного секвенирования геномных районов ДНК, ассоциированных с сайтами рестрикции (ddRADseq). Изучена также корреляция пяти основных климатических переменных с изменчивостью 47 929 генетических маркеров - однонуклеотидных полиморфизмов или “снипов” (от английского SNPs - single nucleotide polymorphisms). Всего изучено 125 деревьев: 61 дерево в четырех популяциях вдоль западной географической трансекты и 64 дерева в четырех популяциях вдоль восточной географической трансекты. Выявлен 21 SNPsс признаками отбора, включая 9 SNPsаутлайеров, чья изменчивость не может быть объяснена селективно-нейтральными процессами, и 12 SNPs, чья изменчивость коррелировала с изменчивостью некоторых климатических факторов. Семь SNPsрасположены в интронах митохондриальных генов, три расположены вблизи митохондриальных генов, кодирующих NAD2 и рибосомальные белки S7 и S11, один на отдалении от ядерного гена, кодирующего белок, гомологичный связанному с микротрубочками futsch-подобному белку Arabidopsis thaliana, два в белковых генах неизвестной природы и три в контигах, не содержащих гены, и для которых не найдены гомологичные последовательности в NCBI GenBank. The genetic differentiation of Siberian larch (Larix sibirica Ledeb.) populations in the latitudinal gradient of climatic conditions was studied based on high-throughput double digest restriction-site associated DNA sequencing (ddRADseq) data. We studied the correlation of five main climatic variables with the variability of 47,929 single nucleotide polymorphisms (SNPs). A total of 125 trees were studied: 61 trees in four populations along the western geographic transect and 64 trees in four populations along the eastern geographic transect. 21 SNPs with signatures of selection were identified, including 9 outlier SNPs whose variability cannot be explained by selectively neutral processes, and 12 SNPs whose variability correlated with the environmental factors. Seven SNPs are located in the introns of mitochondrial genes, three are located relatively close to the mitochondrial genes encoding NAD2 and ribosomal proteins S7 and S11, one is located at a distance from the nuclear gene encoding a protein homologous to the microtubule-associated futsch-like protein of Arabidopsis thaliana, two in the protein genes of an unknown nature and three in contigs containing no genes, and for which no homologous sequences were found in the NCBI GenBank.

Twenty-two highly variable SSR markers were developed in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] from five SSR-enriched genomic libraries. Fifteen PCR primer pairs amplified a single codominant locus, while seven primer pairs occasionally amplified two loci. The Mendelian inheritance of all 22 SSRs was confirmed via segregation analyses in several Douglas-fir families. The mean observed heterozygosity and the mean number of alleles per locus were 0.855 (SE=0.020) and 23 (SE=1.6), respectively. Twenty markers were used in genetic linkage analysis and mapped to ten known linkage groups. Because of their high polymorphism and unambiguous phenotypes, 15 single-locus markers were selected as the most suitable for DNA fingerprinting and parentage analysis. Only three SSRs were sufficient to achieve an average probability of exclusion from paternity of 0.998 in a Douglas-fir seed orchard block consisting of 59 parents.

We isolated PTD, a member of the DEFICIENS (DEF) family of MADS box transcription factors, from the dioecious tree, black cottonwood (Populus trichocarpa). In females, in situ hybridization experiments showed that PTD mRNA was first detectable in cells on the flanks of the inflorescence meristem, before differentiation of individual flowers was visually detectable. In males, the onset of PTD expression was delayed until after individual flower differentiation had begun and floral meristems were developing. Although PTD was initially expressed throughout the inner whorl meristem in female and male flowers, its spatial expression pattern became sex-specific as reproductive primordia began to form. PTD expression was maintained in stamen primordia, but excluded from carpel primordia, as well as vegetative tissues. Although PTD is phylogenetically most closely related to the largely uncharacterized TM6 subfamily of the DEF/APETELA3(AP3)/TM6 group, its spatio-temporal expression patterns are more similar to that of DEF and AP3 than to other members of the TM6 subfamily.

We studied nuclear gene diversity and population differentiation using 91-98 randomly amplified polymorphic DNA (RAPD) loci in the California closed-cone pines knobcone (Pinus attenuata Lemm.), bishop (P. muricata D. Don), and Monterey (P. radiata D. Don) pines. A total of 384 trees from 13 populations were analyzed for RAPDs and another sample of 242 trees from 12 of these 13 populations were analyzed at 32-36 allozyme loci, using a published data set. Twenty-eight of 30 (93%) comigrating RAPD fragments tested were found to be homologous by Southern hybridization in all three species. Using an enriched mitochondrial DNA (mtDNA) preparation and a chloroplast DNA (cpDNA) library as probes, two fragments of cpDNA origin, and one of mtDNA origin present among RAPD profiles were excluded from analysis of nuclear gene diversity. RAPD markers revealed moderately higher intrapopulation gene diversity and substantially higher total genetic diversity and population differentiation than did allozyme markers for each species. We performed a simulation study using allozyme data, which showed that the dominant and biallelic nature of RAPD markers could explain the differences observed in differentiation parameters, but not in gene diversity; RAPD phenotypes appear to represent more underlying gene diversity than do allozyme phenotypes. Results of joint phylogenetic analyses of both the RAPD and allozyme markers strongly supported a common ancestor for P. radiata and P. attenuata pines, and south-to-north migration histories for all three species.Key words: allozymes, dominance, gene diversity, Pinus attenuata, Pinus muricata, Pinus radiata, phylogeny, RAPDs.

We examined mitochondrial DNA polymorphisms via the analysis of restriction fragment length polymorphisms in three closely related species of pines from western North America: knobcone (Pinus attenuata Lemm.), Monterey (P. radiata D. Don), and bishop (P. muricata D. Don). A total of 343 trees derived from 13 populations were analyzed using 13 homologous mitochondrial gene probes amplified from three species by polymerase chain reaction. Twenty-eight distinct mitochondrial DNA haplotypes were detected and no common haplotypes were found among the species. All three species showed limited variability within populations, but strong differentiation among populations. Based on haplotype frequencies, genetic diversity within populations (HS) averaged 0.22, and population differentiation (GST and theta) exceeded 0.78. Analysis of molecular variance also revealed that >90% of the variation resided among populations. For the purposes of genetic conservation and breeding programs, species and populations could be readily distinguished by unique haplotypes, often using the combination of only a few probes. Neighbor-joining phenograms, however, strongly disagreed with those based on allozymes, chloroplast DNA, and morphological traits. Thus, despite its diagnostic haplotypes, the genome appears to evolve via the rearrangement of multiple, convergent subgenomic domains.

We developed a method of screening RAPD markers for the presence of organelle DNA products using enriched organelle DNA probes, then used these markers to compare the structure of nuclear and mitochondrial RAPD diversity in Douglas fir. Of 237 screened RAPD fragments from 25 primers, 16% were identified as originating in the mitochondrial genome and 3% in the chloroplast genome. The mitochondrial DNA probe correctly distinguished fragments with known maternal inheritance (which is exclusive for the mitochondrial genome in the Pinaceae), and neither of the organelle probes hybridized to biparentally inherited fragments. Mitochondrial RAPD markers exhibited low diversity within populations compared to nuclear RAPD diversity ( H S = 0.03 and 0.22, respectively), but were much more highly differentiated than were fragments of nuclear origin at both the population ( G ST = 0.18 and 0.05, respectively) and racial levels ( G ST = 0.72 and 0.25, respectively). Both nuclear and mitochondrial DNA based phylogenetic analyses identified the varieties as monophyletic groups; the nuclear RAPD markers further separated the north and south interior races.

We present linkage maps and estimate genome length for two hybrid individuals of Douglas-fir [Pseudotsuga menziesii (Mirb.) France], a coniferous tree species of wide distribution in the western United States. The hybrids were produced by crosses between the coastal (var, menziesii) and interior (var. glauca) varieties. Haploid megagametophytes from 80 seeds of each individual were analyzed using 81 10-bp random amplified polymorphic DNA (RAPD) primers selected for polymorphism, fragment strength, and repeatability in preliminary segregation analysis. Most (82-90%) of the segregating fragments followed the expected 1:1 Mendelian segregation; however, 10-18% showed significant segregation distortion (P <.05) among megagametophytes of the two trees. In one tree, 201 of 221 segregating loci analyzed were combined into 16 major linkage groups of 4 or more loci (plus 1 group of 3 loci, 3 groups with pairs of loci, and 11 unlinked loci); in the other, 238 of 250 segregating loci were combined into 18 major groups (plus 2 groups of 3 loci each and 6 unlinked loci). Analyses of the distribution of markers indicated highly significant clustering in both trees (P <.001). Including flanking regions and unlinked loci, both trees had linkage maps of similar length, 2600 cM and 3000 cM; expected total map size ranged from 2800 to 3500 cM based on method-of-moments estimation. When a common RAPD protocol was used, more than one-third of the RAPD markers segregated in both hybrids. These maps of Douglas-fir are among the largest reported for conifers, a possible consequence of its 13 haploid chromosomes.

Stratified seeds of coastal Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco var. menziesii) were germinated, sown in soil, and seed coats and megagametophytes were removed at various stages of early seedling development. Yield and quality of DNA extracted from the megagametophytes were related to several morphological traits of the seedlings after 2 months of growth in a controlled environment chamber. Regression and analysis of variance demonstrated nonlinear associations between stage of megagametophyte removal and seedling size traits, DNA yield and quality, and RNA presence. Megagametophyte removal when cotyledons had extended one-quarter of their length (about 4 mm) outside the seed coat (our stage 4) resulted in sufficient DNA for construction of saturated PCR- (polymerase chain reaction) based genome maps and had little effect on seedling development.

Approximately 4000 mature seeds from 350 trees in nine populations (12–75 trees per population) of Siberian stone pine were investigated for multiple embryos (polyembryony). Haploid megagametophytes and embryos were genotyped for eight allozyme loci. Eight-yone seeds (2.11%) had more than 1 embryo. Of these, 71 seeds had 2 embryos (1.85%), 6 seeds had 3 embryos (0.16%), 3 seeds had 4 embryos (0.08%) and 1 seed had 6 embryos (0.026%). Allozyme comparison of megagametophytes and embryos could distinquish two types of polyembryony in 56 of the 81 seeds. In 28 seeds (50%) the polyembryony was polyzygotic (independent fertilizations of more than one egg cell in the ovule); 25 seeds (45%) had most likely monozygotic polyembryony (genetically identical embryos resulting from the cleavage of a single proembryo) and 3 seeds had both genetically different and genetically identical embryos. To the best of our knowledge, this is the first genetic evidence for the form of polyembryony in conifer seeds.

Genetic control of 14 enzyme systems was studied in seeds of Scots pine from a Zaural'e natural population. Electrophoretic separation for allele products of 24 loci was obtained. Loci Acp, Adh-1, Adh-2, Fe, Gdh, Got-2, Got-3, Lap-2, Mdh-2, Mdh-3, Pgi-2, Pgm-1 Skdh-1, Skdh-2, 6-Pgd-1, 6-Pgd-2, and Fdh were polymorphic, whereas loci ldh, Pgi-1, and Pgi-2, were monomorphic. At loci Cat, Got-1, Lap-1, and Mdh-1, rare alleles were detected. A modifier gene for malate dehydrogenase (Mdh-m) was found. Forty-five out of 49 heterozygotes showed segregation ratios conforming to those expected for Mendelian loci 1:1). The results obtained were compared to published data on genetic control of the above isozyme systems in Scots pine from other parts of the range.

With the aid of allozyme polymorphism genetic structure of populations of four cembrae pines species (genus Pinus, subsection Cembrae) was studied. A respectively high level of genetic variability in all species studied was found. Average values of expected heterozygosity were 0.156 in Pinus sibirica, 0.109 in P. cembra, 0.249 in P. punila, 0.124 in P. koraiensis. All measures of genetic variability are characteristic of wide-distributed conifer species. An analysis of genotypic distributions has shown the tendency to heterozygote deficiency in embryos (obviously) as a consequence of self-pollination) and slight heterozygote excess in adult trees (probably as a consequence of selection against inbred progeny and balanced selection). The levels of intra- and interpopulation variability in cembrae pines were 96-98% and 2-4%, respectively. Slight geographic differentiation may be explained taking into account the ecological traits of comifers. Clusterization based on the Nei's genetic distances reflects the spatial localization of the populations studied.