Aleksey A. Penin

De novo sequencing and characterization of floral transcriptome in two species of buckwheat ( Fagopyrum )

BackgroundTranscriptome sequencing data has become an integral component of modern genetics, genomics and evolutionary biology. However, despite advances in the technologies of DNA sequencing, such data are lacking for many groups of living organisms, in particular, many plant taxa. We present here the results of transcriptome sequencing for two closely related plant species. These species, Fagopyrum esculentum and F. tataricum, belong to the order Caryophyllales - a large group of flowering plants with uncertain evolutionary relationships. F. esculentum (common buckwheat) is also an important food crop. Despite these practical and evolutionary considerations Fagopyrum species have not been the subject of large-scale sequencing projects.ResultsNormalized cDNA corresponding to genes expressed in flowers and inflorescences of F. esculentum and F. tataricum was sequenced using the 454 pyrosequencing technology. This resulted in 267 (for F. esculentum) and 229 (F. tataricum) thousands of reads with average length of 341-349 nucleotides. De novo assembly of the reads produced about 25 thousands of contigs for each species, with 7.5-8.2× coverage. Comparative analysis of two transcriptomes demonstrated their overall similarity but also revealed genes that are presumably differentially expressed. Among them are retrotransposon genes and genes involved in sugar biosynthesis and metabolism. Thirteen single-copy genes were used for phylogenetic analysis; the resulting trees are largely consistent with those inferred from multigenic plastid datasets. The sister relationships of the Caryophyllales and asterids now gained high support from nuclear gene sequences.Conclusions454 transcriptome sequencing and de novo assembly was performed for two congeneric flowering plant species, F. esculentum and F. tataricum. As a result, a large set of cDNA sequences that represent orthologs of known plant genes as well as potential new genes was generated.

Active chromatin and transcription play a key role in chromosome partitioning into topologically associating domains

Recent advances enabled by the Hi-C technique have unraveled many principles of chromosomal folding that were subsequently linked to disease and gene regulation. In particular, Hi-C revealed that chromosomes of animals are organized into topologically associating domains (TADs), evolutionary conserved compact chromatin domains that influence gene expression. Mechanisms that underlie partitioning of the genome into TADs remain poorly understood. To explore principles of TAD folding in Drosophila melanogaster, we performed Hi-C and poly(A)(+) RNA-seq in four cell lines of various origins (S2, Kc167, DmBG3-c2, and OSC). Contrary to previous studies, we find that regions between TADs (i.e., the inter-TADs and TAD boundaries) in Drosophila are only weakly enriched with the insulator protein dCTCF, while another insulator protein Su(Hw) is preferentially present within TADs. However, Drosophila inter-TADs harbor active chromatin and constitutively transcribed (housekeeping) genes. Accordingly, we find that binding of insulator proteins dCTCF and Su(Hw) predicts TAD boundaries much worse than active chromatin marks do. Interestingly, inter-TADs correspond to decompacted inter-bands of polytene chromosomes, whereas TADs mostly correspond to densely packed bands. Collectively, our results suggest that TADs are condensed chromatin domains depleted in active chromatin marks, separated by regions of active chromatin. We propose the mechanism of TAD self-assembly based on the ability of nucleosomes from inactive chromatin to aggregate, and lack of this ability in acetylated nucleosomal arrays. Finally, we test this hypothesis by polymer simulations and find that TAD partitioning may be explained by different modes of inter-nucleosomal interactions for active and inactive chromatin.

Selection and validation of reference genes for quantitative real-time PCR in buckwheat (Fagopyrum esculentum) based on transcriptome sequence data.

Quantitative reverse transcription PCR (qRT-PCR) is one of the most precise and widely used methods of gene expression analysis. A necessary prerequisite of exact and reliable data is the accurate choice of reference genes. We studied the expression stability of potential reference genes in common buckwheat (Fagopyrum esculentum) in order to find the optimal reference for gene expression analysis in this economically important crop. Recently sequenced buckwheat floral transcriptome was used as source of sequence information. Expression stability of eight candidate reference genes was assessed in different plant structures (leaves and inflorescences at two stages of development and fruits). These genes are the orthologs of Arabidopsis genes identified as stable in a genome-wide survey gene of expression stability and a traditionally used housekeeping gene GAPDH. Three software applications – geNorm, NormFinder and BestKeeper - were used to estimate expression stability and provided congruent results. The orthologs of AT4G33380 (expressed protein of unknown function, Expressed1), AT2G28390 (SAND family protein, SAND) and AT5G46630 (clathrin adapter complex subunit family protein, CACS) are revealed as the most stable. We recommend using the combination of Expressed1, SAND and CACS for the normalization of gene expression data in studies on buckwheat using qRT-PCR. These genes are listed among five the most stably expressed in Arabidopsis that emphasizes utility of the studies on model plants as a framework for other species.

Sequencing and Analysis of Plastid Genome in Mycoheterotrophic Orchid Neottia nidus-avis

Plastids are the semiautonomous organelles that possess their own genome inherited from the cyanobacterial ancestor. The primary function of plastids is photosynthesis so the structure and evolution of plastid genomes are extensively studied in photosynthetic plants. In contrast, little is known about the plastomes of nonphotosynthetic species. In higher plants, plastid genome sequences are available for only three strictly nonphotosynthetic species, the liverwort Aneura mirabilis and two flowering plants, Epifagus virginiana and Rhizanthella gardneri. We report here the complete sequence of a plastid genome of nonphotosynthetic mycoheterotrophic orchid Neottia nidus-avis, determined using 454 pyrosequencing technology. It was found to be reduced in both genome size and gene content; this reduction is however not as drastic as in the other nonphotosynthetic orchid, R. gardneri. Neottia plastome lacks all genes encoding photosynthetic proteins, RNA polymerase subunits but retains most genes of translational apparatus. Those genes that are retained have an increased rate of both synonymous and nonsynonymous substitutions but do not exhibit relaxation of purifying selection either in Neottia or in Rhizanthella.

Paratrypanosoma Is a Novel Early-Branching Trypanosomatid

The kinetoplastids are a widespread and important group of single-celled eukaryotes, many of which are devastating parasites of animals, including humans. We have discovered a new insect trypanosomatid in the gut of Culex pipiens mosquitoes. Glyceraldehyde-3-phosphate dehydrogenase- and SSU rRNA-based phylogenetic analyses show this parasite to constitute a distinct branch between the free-living Bodo saltans and the obligatory parasitic clades represented by the genus Trypanosoma and other trypanosomatids. From draft genome sequence data, we identified 114 protein genes shared among the new flagellate, 15 trypanosomatid species, B. saltans, and the heterolobosean Naegleria gruberi, as well as 129 protein genes shared with the basal kinetoplastid Perkinsela sp. Individual protein phylogenies together with analyses of concatenated alignments show that the new species, here named Paratrypanosoma confusum n. gen., n. sp., branches with very high support at the base of the family Trypanosomatidae. P. confusum thus represents a long-sought-after missing link between the ancestral free-living bodonids and the derived parasitic trypanosomatids. Further analysis of the P. confusum genome should provide insight into the emergence of parasitism in the medically important trypanosomatids.

Exploring the limits for reduction of plastid genomes: a case study of the mycoheterotrophic orchids Epipogium aphyllum and Epipogium roseum.

The question on the patterns and limits of reduction of plastid genomes in nonphotosynthetic plants and the reasons of their conservation is one of the intriguing topics in plant genome evolution. Here, we report sequencing and analysis of plastid genome in nonphotosynthetic orchids Epipogium aphyllum and Epipogium roseum, which, with sizes of 31 and 19 kbp, respectively, represent the smallest plastid genomes characterized by now. Besides drastic reduction, which is expected, we found several unusual features of these “minimal” plastomes: Multiple rearrangements, highly biased nucleotide composition, and unprecedentedly high substitution rate. Only 27 and 29 genes remained intact in the plastomes of E. aphyllum and E. roseum—those encoding ribosomal components, transfer RNAs, and three additional housekeeping genes (infA, clpP, and accD). We found no signs of relaxed selection acting on these genes. We hypothesize that the main reason for retention of plastid genomes in Epipogium is the necessity to translate messenger RNAs (mRNAs) of accD and/or clpP proteins which are essential for cell metabolism. However, these genes are absent in plastomes of several plant species; their absence is compensated by the presence of a functional copy arisen by gene transfer from plastid to the nuclear genome. This suggests that there is no single set of plastid-encoded essential genes, but rather different sets for different species and that the retention of a gene in the plastome depends on the interaction between the nucleus and plastids.

Comparative genome sequencing reveals genomic signature of extreme desiccation tolerance in the anhydrobiotic midge

Anhydrobiosis represents an extreme example of tolerance adaptation to water loss, where an organism can survive in an ametabolic state until water returns. Here we report the first comparative analysis examining the genomic background of extreme desiccation tolerance, which is exclusively found in larvae of the only anhydrobiotic insect, Polypedilum vanderplanki. We compare the genomes of P. vanderplanki and a congeneric desiccation-sensitive midge P. nubifer. We determine that the genome of the anhydrobiotic species specifically contains clusters of multi-copy genes with products that act as molecular shields. In addition, the genome possesses several groups of genes with high similarity to known protective proteins. However, these genes are located in distinct paralogous clusters in the genome apart from the classical orthologues of the corresponding genes shared by both chironomids and other insects. The transcripts of these clustered paralogues contribute to a large majority of the mRNA pool in the desiccating larvae and most likely define successful anhydrobiosis. Comparison of expression patterns of orthologues between two chironomid species provides evidence for the existence of desiccation-specific gene expression systems in P. vanderplanki.

The Plastid Genome of Mycoheterotrophic Monocot Petrosavia stellaris Exhibits Both Gene Losses and Multiple Rearrangements

Plastid genomes of nonphotosynthetic plants represent a perfect model for studying evolution under relaxed selection pressure. However, the information on their sequences is still limited. We sequenced and assembled plastid genome of Petrosavia stellaris, a rare mycoheterotrophic monocot plant. After orchids, Petrosavia represents only the second family of nonphotosynthetic monocots to have its plastid genome examined. Several unusual features were found: retention of the ATP synthase genes and rbcL gene; extensive gene order rearrangement despite a relative lack of repeat sequences; an unusually short inverted repeat region that excludes most of the rDNA operon; and a lack of evidence for accelerated sequence evolution. Plastome of photosynthetic relative of P. stellaris, Japonolirion osense, has standard gene order and does not have the predisposition to inversions. Thus, the rearrangements in the P. stellaris plastome are the most likely associated with transition to heterotrophic way of life.

Comparative chloroplast genomics and phylogenetics of Fagopyrum esculentum ssp. ancestrale– A wild ancestor of cultivated buckwheat

BackgroundChloroplast genome sequences are extremely informative about species-interrelationships owing to its non-meiotic and often uniparental inheritance over generations. The subject of our study, Fagopyrum esculentum, is a member of the family Polygonaceae belonging to the order Caryophyllales. An uncertainty remains regarding the affinity of Caryophyllales and the asterids that could be due to undersampling of the taxa. With that background, having access to the complete chloroplast genome sequence for Fagopyrum becomes quite pertinent.ResultsWe report the complete chloroplast genome sequence of a wild ancestor of cultivated buckwheat, Fagopyrum esculentum ssp. ancestrale. The sequence was rapidly determined using a previously described approach that utilized a PCR-based method and employed universal primers, designed on the scaffold of multiple sequence alignment of chloroplast genomes. The gene content and order in buckwheat chloroplast genome is similar to Spinacia oleracea. However, some unique structural differences exist: the presence of an intron in the rpl2 gene, a frameshift mutation in the rpl23 gene and extension of the inverted repeat region to include the ycf1 gene. Phylogenetic analysis of 61 protein-coding gene sequences from 44 complete plastid genomes provided strong support for the sister relationships of Caryophyllales (including Polygonaceae) to asterids. Further, our analysis also provided support for Amborella as sister to all other angiosperms, but interestingly, in the bayesian phylogeny inference based on first two codon positions Amborella united with Nymphaeales.ConclusionComparative genomics analyses revealed that the Fagopyrum chloroplast genome harbors the characteristic gene content and organization as has been described for several other chloroplast genomes. However, it has some unique structural features distinct from previously reported complete chloroplast genome sequences. Phylogenetic analysis of the dataset, including this new sequence from non-core Caryophyllales supports the sister relationship between Caryophyllales and asterids.

Fast Evolution from Precast Bricks: Genomics of Young Freshwater Populations of Threespine Stickleback Gasterosteus aculeatus

Adaptation is driven by natural selection; however, many adaptations are caused by weak selection acting over large timescales, complicating its study. Therefore, it is rarely possible to study selection comprehensively in natural environments. The threespine stickleback (Gasterosteus aculeatus) is a well-studied model organism with a short generation time, small genome size, and many genetic and genomic tools available. Within this originally marine species, populations have recurrently adapted to freshwater all over its range. This evolution involved extensive parallelism: pre-existing alleles that adapt sticklebacks to freshwater habitats, but are also present at low frequencies in marine populations, have been recruited repeatedly. While a number of genomic regions responsible for this adaptation have been identified, the details of selection remain poorly understood. Using whole-genome resequencing, we compare pooled genomic samples from marine and freshwater populations of the White Sea basin, and identify 19 short genomic regions that are highly divergent between them, including three known inversions. 17 of these regions overlap protein-coding genes, including a number of genes with predicted functions that are relevant for adaptation to the freshwater environment. We then analyze four additional independently derived young freshwater populations of known ages, two natural and two artificially established, and use the observed shifts of allelic frequencies to estimate the strength of positive selection. Adaptation turns out to be quite rapid, indicating strong selection acting simultaneously at multiple regions of the genome, with selection coefficients of up to 0.27. High divergence between marine and freshwater genotypes, lack of reduction in polymorphism in regions responsible for adaptation, and high frequencies of freshwater alleles observed even in young freshwater populations are all consistent with rapid assembly of G. aculeatus freshwater genotypes from pre-existing genomic regions of adaptive variation, with strong selection that favors this assembly acting simultaneously at multiple loci.