[From population genetics to population genomics of forest trees: integrated population genomics approach].

Early works by Altukhov and his associates on pine and spruce laid the foundation for Russian population genetic studies on tree species with the use of molecular genetic markers. In recent years, these species have become especially popular as nontraditional eukaryotic models for population and evolutionary genomic research. Tree species with large, cross-pollinating native populations, high genetic and phenotypic variation, growing in diverse environments and affected by environmental changes during hundreds of years of their individual development, are an ideal model for studying the molecular genetic basis of adaptation. The great advance in this field is due to the rapid development of population genomics in the last few years. In the broad sense, population genomics is a novel, fast-developing discipline, combining traditional population genetic approaches with the genomic level of analysis. Thousands of genes with known function and sometimes known genomic localization can be simultaneously studied in many individuals. This opens new prospects for obtaining statistical estimates for a great number of genes and segregating elements. Mating system, gene exchange, reproductive population size, population disequilibrium, interaction among populations, and many other traditional problems of population genetics can be now studied using data on variation in many genes. Moreover, population genomic analysis allows one to distinguish factors that affect individual genes, alleles, or nucleotides (such as, for example, natural selection) from factors affecting the entire genome (e.g., demography). This paper presents a brief review of traditional methods of studying genetic variation in forest tree species and introduces a new, integrated population genomics approach. The main stages of the latter are : (1) selection of genes, which are tentatively involved in variation of adaptive traits, by means of a detailed examination of the regulation and the expression of individual genes and genotypes, with subsequent determination of their complete allelic composition by direct nucleotide sequencing; (2) examination of the phenotypic effects of individual alleles by, e.g., association mapping; and (3) determining the frequencies of the selected alleles in natural population for identification of the adaptive variation pattern in the heterogeneous environment. Through decoding the phenotypic effects of individual alleles and identification of adaptive variation patterns at the population level, population genomics in the future will serve as a very helpful, efficient, and economical tool, essential for developing a correct strategy for conserving and increasing forests and other commercially valuable plant and animal species.

Highly variable SSR markers in Douglas-fir: Mendelian inheritance and map locations.

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.

A DEFICIENS homolog from the dioecious tree black cottonwood is expressed in female and male floral meristems of the two-whorled, unisexual flowers.

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.

Structure and expression of duplicate AGAMOUS orthologues in poplar.

To investigate the homeotic systems underlying floral development in a dioecious tree, and to provide tools for the manipulation of floral development, we have isolated two Populus trichocarpa genes, PTAG1 and PTAG2, homologous to the Arabidopsis floral homeotic gene AGAMOUS (AG). PTAG1 and PTAG2 are located on separate linkage groups, but their non-coding regions are highly similar, consistent with a phylogenetically recent duplication. Intron/exon structure is conserved in relation to AG and the Antirrhinum AG orthologue, PLENA (PLE), and low-stringency Southern analysis demonstrated the absence of additional genes in the poplar genome with significant PTAG1/2 homology. PTAG1 and PTAG2 exhibit an AG-like floral expression pattern, and phylogenetic analysis of the AG subfamily strongly supports evolutionary orthology to C-class organ identity genes. The high degree of similarity shared by PTAG1 and PTAG2 in both sequence (89% amino acid identity) and expression indicates that they are unlikely to be functionally associated with specification of tree gender. Unexpectedly, PTAG transcripts were consistently detected in vegetative tissues.

Abundant mitochondrial genome diversity, population differentiation and convergent evolution in pines.

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.

Allozyme evidence for polyzygotic polyembryony in Siberian stone pine (Pinus sibirica Du Tour).

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.

[Study of the breed-forming process in sheep using multivariate methods of analysis of genetic and morphologic distances]. / Izuchenie porodoobrazovatel'nogo protsessa u ovets s pomoshch'iu mnogomernykh metodov analiza geneticheskikh i morfologicheskikh rasstoianii.

Comparison of biochemical genetic variation and morphological variation during formation of a new breed of sheep by means of multi-breed mating has been carried out. Genetic distances calculated by biochemical markers using various methods (by M. Nei, J.S. Rogers, I.I. Cavalli-Sforza and A.W.F. Edvards) have been discovered to significantly correlate with Manhattan and average taxonomic distances calculated on the basis of six morphological traits (r = 0.841-0.889, t = 2.98-2.99, P = 0.999). Nevertheless, it appeared that relationships between crossbred animals revealed with the help of methods for multidimensional analysis (principle component analysis and multidimensional scaling) on the basis of genetic distances matrix more precisely correspond to the real scheme and direction of breeding work than relationships described while using the given methods on the basis of morphological distances matrix. Greater correspondence of data on biochemical genetic variations of the animal groups studied to real processes of breeding, as compared with the data on morphological traits, is supposed to be connected with direct involvement of non-accounted phenotypic characters into the breeding work.