Comparative Genomics of Seasonal Senescence in Forest Trees.

In the course of evolution, both flowering plants and some gymnosperms have developed such an adaptation to winter and unfavorable living conditions as deciduousness. Of particular interest is Siberian larch (Larix sibirica Ledeb.), which is the only species in the pine family (Pinaceae) with a seasonal deciduousness. New generation sequencing technologies make it possible to study this phenomenon at the genomic level and to reveal the genetic mechanisms of leaf and needle aging in angiosperms and gymnosperms. Using a comparative analysis of the genomes of evergreen and deciduous trees, it was found that the genes that control EXORDIUM LIKE 2 (EXL2) and DORMANCY-ASSOCIATED PROTEIN 1 (DRM1) proteins are most represented in Siberian larch, while an excess of genes that control proteins acting as immune receptors were found in evergreens. Orthologs from the family of genes that control leucine-rich repeat receptor-like kinases (LRR-RLK) contributed mostly to the distinction between evergreens and deciduous plants.

Sequence Capture of Mitochondrial Genome with PCR-Generated Baits Provides New Insights into the Biogeography of the Genus Abies Mill.

Mitochondrial DNA (mtDNA), being maternally inherited in plants of the family Pinaceae, is an important source of phylogeographic information. However, its use is hindered by a low mutation rate and frequent structure rearrangements. In the present study, we tested the method of genomic libraries enrichment with mtDNA via the sequence capture method yielding mtDNA data which were further used to reconstruct the phylogenetic tree of the genus Abies. The baits for hybrid capture were obtained by long-range PCR using primers designed on the basis of the assembly of Abies sibirica Ledeb. mitochondrial genome. Mitochondrial genomes of Picea sitchensis (Bong.) Carr., Larix sibirica Ledeb., and Keteleeria davidiana (Bertrand) Beissn. were used as an outgroup. The resulting phylogenetic tree consists of two sister branches, including the Eurasian and American species, respectively, with some exceptions. The subclade of A. sachalinensis (F. Schmidt) Mast. and A. veitchii Lindl. (Japan and Sakhalin islands) occupies a basal position in the branch of American firs, probably due to the complex history of fir migrations from North America to Eurasia. The tree has high support for majority of clades. For species represented by more than one sample an intraspecific variability was found which is suitable to design mtDNA markers for phylogeographic and population studies.

Comparative Genomics Analysis of Repetitive Elements in Ten Gymnosperm Species: "Dark Repeatome" and Its Abundance in Conifer and Gnetum Species.

Repetitive elements (RE) and transposons (TE) can comprise up to 80% of some plant genomes and may be essential for regulating their evolution and adaptation. The "repeatome" information is often unavailable in assembled genomes because genomic areas of repeats are challenging to assemble and are often missing from final assembly. However, raw genomic sequencing data contain rich information about RE/TEs. Here, raw genomic NGS reads of 10 gymnosperm species were studied for the content and abundance patterns of their "repeatome". We utilized a combination of alignment on databases of repetitive elements and de novo assembly of highly repetitive sequences from genomic sequencing reads to characterize and calculate the abundance of known and putative repetitive elements in the genomes of 10 conifer plants: Pinus taeda, Pinus sylvestris, Pinus sibirica, Picea glauca, Picea abies, Abies sibirica, Larix sibirica, Juniperus communis, Taxus baccata, and Gnetum gnemon. We found that genome abundances of known and newly discovered putative repeats are specific to phylogenetically close groups of species and match biological taxa. The grouping of species based on abundances of known repeats closely matches the grouping based on abundances of newly discovered putative repeats (kChains) and matches the known taxonomic relations.

Siberian larch (Larix sibirica Ledeb.) mitochondrial genome assembled using both short and long nucleotide sequence reads is currently the largest known mitogenome.

BACKGROUND: Plant mitochondrial genomes (mitogenomes) can be structurally complex while their size can vary from ~ 222 Kbp in Brassica napus to 11.3 Mbp in Silene conica. To date, in comparison with the number of plant species, only a few plant mitogenomes have been sequenced and released, particularly for conifers (the Pinaceae family). Conifers cover an ancient group of land plants that includes about 600 species, and which are of great ecological and economical value. Among them, Siberian larch (Larix sibirica Ledeb.) represents one of the keystone species in Siberian boreal forests. Yet, despite its importance for evolutionary and population studies, the mitogenome of Siberian larch has not yet been assembled and studied. RESULTS: Two sources of DNA sequences were used to search for mitochondrial DNA (mtDNA) sequences: mtDNA enriched samples and nucleotide reads generated in the de novo whole genome sequencing project, respectively. The assembly of the Siberian larch mitogenome contained nine contigs, with the shortest and the largest contigs being 24,767 bp and 4,008,762 bp, respectively. The total size of the genome was estimated at 11.7 Mbp. In total, 40 protein-coding, 34 tRNA, and 3 rRNA genes and numerous repetitive elements (REs) were annotated in this mitogenome. In total, 864 C-to-U RNA editing sites were found for 38 out of 40 protein-coding genes. The immense size of this genome, currently the largest reported, can be partly explained by variable numbers of mobile genetic elements, and introns, but unlikely by plasmid-related sequences. We found few plasmid-like insertions representing only 0.11% of the entire Siberian larch mitogenome. CONCLUSIONS: Our study showed that the size of the Siberian larch mitogenome is much larger than in other so far studied Gymnosperms, and in the same range as for the annual flowering plant Silene conica (11.3 Mbp). Similar to other species, the Siberian larch mitogenome contains relatively few genes, and despite its huge size, the repeated and low complexity regions cover only 14.46% of the mitogenome sequence.

De novo sequencing, assembly and functional annotation of Armillaria borealis genome.

BACKGROUND: Massive forest decline has been observed almost everywhere as a result of negative anthropogenic and climatic effects, which can interact with pests, fungi and other phytopathogens and aggravate their effects. Climatic changes can weaken trees and make fungi, such as Armillaria more destructive. Armillaria borealis (Marxm. & Korhonen) is a fungus from the Physalacriaceae family (Basidiomycota) widely distributed in Eurasia, including Siberia and the Far East. Species from this genus cause the root white rot disease that weakens and often kills woody plants. However, little is known about ecological behavior and genetics of A. borealis. According to field research data, A. borealis is less pathogenic than A. ostoyae, and its aggressive behavior is quite rare. Mainly A. borealis behaves as a secondary pathogen killing trees already weakened by other factors. However, changing environment might cause unpredictable effects in fungus behavior. RESULTS: The de novo genome assembly and annotation were performed for the A. borealis species for the first time and presented in this study. The A. borealis genome assembly contained ~ 68 Mbp and was comparable with ~ 60 and ~ 79.5 Mbp for the A. ostoyae and A. mellea genomes, respectively. The N50 for contigs equaled 50,544 bp. Functional annotation analysis revealed 21,969 protein coding genes and provided data for further comparative analysis. Repetitive sequences were also identified. The main focus for further study and comparative analysis will be on the enzymes and regulatory factors associated with pathogenicity. CONCLUSIONS: Pathogenic fungi such as Armillaria are currently one of the main problems in forest conservation. A comprehensive study of these species and their pathogenicity is of great importance and needs good genomic resources. The assembled genome of A. borealis presented in this study is of sufficiently good quality for further detailed comparative study on the composition of enzymes in other Armillaria species. There is also a fundamental problem with the identification and classification of species of the Armillaria genus, where the study of repetitive sequences in the genomes of basidiomycetes and their comparative analysis will help us identify more accurately taxonomy of these species and reveal their evolutionary relationships.

Mobile genetic elements explain size variation in the mitochondrial genomes of four closely-related Armillaria species.

BACKGROUND: Species in the genus Armillaria (fungi, basidiomycota) are well-known as saprophytes and pathogens on plants. Many of them cause white-rot root disease in diverse woody plants worldwide. Mitochondrial genomes (mitogenomes) are widely used in evolutionary and population studies, but despite the importance and wide distribution of Armillaria, the complete mitogenomes have not previously been reported for this genus. Meanwhile, the well-supported phylogeny of Armillaria species provides an excellent framework in which to study variation in mitogenomes and how they have evolved over time. RESULTS: Here we completely sequenced, assembled, and annotated the circular mitogenomes of four species: A. borealis, A. gallica, A. sinapina, and A. solidipes (116,443, 98,896, 103,563, and 122,167 bp, respectively). The variation in mitogenome size can be explained by variable numbers of mobile genetic elements, introns, and plasmid-related sequences. Most Armillaria introns contained open reading frames (ORFs) that are related to homing endonucleases of the LAGLIDADG and GIY-YIG families. Insertions of mobile elements were also evident as fragments of plasmid-related sequences in Armillaria mitogenomes. We also found several truncated gene duplications in all four mitogenomes. CONCLUSIONS: Our study showed that fungal mitogenomes have a high degree of variation in size, gene content, and genomic organization even among closely related species of Armillara. We suggest that mobile genetic elements invading introns and intergenic sequences in the Armillaria mitogenomes have played a significant role in shaping their genome structure. The mitogenome changes we describe here are consistent with widely accepted phylogenetic relationships among the four species.

Stepwise large genome assembly approach: a case of Siberian larch (Larix sibirica Ledeb).

BACKGROUND: De novo assembling of large genomes, such as in conifers (~ 12-30 Gbp), which also consist of ~ 80% of repetitive DNA, is a very complex and computationally intense endeavor. One of the main problems in assembling such genomes lays in computing limitations of nucleotide sequence assembly programs (DNA assemblers). As a rule, modern assemblers are usually designed to assemble genomes with a length not exceeding the length of the human genome (3.24 Gbp). Most assemblers cannot handle the amount of input sequence data required to provide sufficient coverage needed for a high-quality assembly. RESULTS: An original stepwise method of de novo assembly by parts (sets), which allows to bypass the limitations of modern assemblers associated with a huge amount of data being processed, is presented in this paper. The results of numerical assembling experiments conducted using the model plant Arabidopsis thaliana, Prunus persica (peach) and four most popular assemblers, ABySS, SOAPdenovo, SPAdes, and CLC Assembly Cell, showed the validity and effectiveness of the proposed stepwise assembling method. CONCLUSION: Using the new stepwise de novo assembling method presented in the paper, the genome of Siberian larch, Larix sibirica Ledeb. (12.34 Gbp) was completely assembled de novo by the CLC Assembly Cell assembler. It is the first genome assembly for larch species in addition to only five other conifer genomes sequenced and assembled for Picea abies, Picea glauca, Pinus taeda, Pinus lambertiana, and Pseudotsuga menziesii var. menziesii.

Siberian larch (Larix sibirica Ledeb.) chloroplast genome and development of polymorphic chloroplast markers.

BACKGROUND: The main objectives of this study were sequencing, assembling, and annotation of chloroplast genome of one of the main Siberian boreal forest tree conifer species Siberian larch (Larix sibirica Ledeb.) and detection of polymorphic genetic markers - microsatellite loci or simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs). RESULTS: We used the data of the whole genome sequencing of three Siberian larch trees from different regions - the Urals, Krasnoyarsk, and Khakassia, respectively. Sequence reads were obtained using the Illumina HiSeq2000 in the Laboratory of Forest Genomics at the Genome Research and Education Center of the Siberian Federal University. The assembling was done using the Bowtie2 mapping program and the SPAdes genomic assembler. The genome annotation was performed using the RAST service. We used the GMATo program for the SSRs search, and the Bowtie2 and UGENE programs for the SNPs detection. Length of the assembled chloroplast genome was 122,561 bp, which is similar to 122,474 bp in the closely related European larch (Larix decidua Mill.). As a result of annotation and comparison of the data with the existing data available only for three larch species - L. decidua, L. potaninii var. chinensis (complete genome 122,492 bp), and L. occidentalis (partial genome of 119,680 bp), we identified 110 genes, 34 of which represented tRNA, 4 rRNA, and 72 protein-coding genes. In total, 13 SNPs were detected; two of them were in the tRNA-Arg and Cell division protein FtsH genes, respectively. In addition, 23 SSR loci were identified. CONCLUSIONS: The complete chloroplast genome sequence was obtained for Siberian larch for the first time. The reference complete chloroplast genomes, such as one described here, would greatly help in the chloroplast resequencing and search for additional genetic markers using population samples. The results of this research will be useful for further phylogenetic and gene flow studies in conifers.

Complete mitochondrial genome of a woolly mammoth (Mammuthus primigenius) from Maly Lyakhovsky Island (New Siberian Islands, Russia) and its phylogenetic assessment.

We present a complete sequence and an annotation of the mitochondrial genome of the woolly mammoth (Mammuthus primigenius) found in 2012 on Maly Lyakhovsky Island (North-Eastern Siberia, Russia). The genome was 16,851 bp long and contained 13 protein-coding, 22 tRNA, and 2 rRNA genes. It was AT reach (61.3%) with A = 32.9%, T = 28.4%, C = 25.3%, and G = 13.4%.

De novo assembling and primary analysis of genome and transcriptome of gray whale Eschrichtius robustus.

BACKGROUND: Gray whale, Eschrichtius robustus (E. robustus), is a single member of the family Eschrichtiidae, which is considered to be the most primitive in the class Cetacea. Gray whale is often described as a "living fossil". It is adapted to extreme marine conditions and has a high life expectancy (77 years). The assembly of a gray whale genome and transcriptome will allow to carry out further studies of whale evolution, longevity, and resistance to extreme environment. RESULTS: In this work, we report the first de novo assembly and primary analysis of the E. robustus genome and transcriptome based on kidney and liver samples. The presented draft genome assembly is complete by 55% in terms of a total genome length, but only by 24% in terms of the BUSCO complete gene groups, although 10,895 genes were identified. Transcriptome annotation and comparison with other whale species revealed robust expression of DNA repair and hypoxia-response genes, which is expected for whales. CONCLUSIONS: This preliminary study of the gray whale genome and transcriptome provides new data to better understand the whale evolution and the mechanisms of their adaptation to the hypoxic conditions.