M. A. Filyushin

De novo transcriptome assembly of the mycoheterotrophic plant Monotropa hypopitys

Monotropa hypopitys (pinesap) is a non-photosynthetic obligately mycoheterotrophic plant of the family Ericaceae. It obtains the carbon and other nutrients from the roots of surrounding autotrophic trees through the associated mycorrhizal fungi. In order to understand the evolutionary changes in the plant genome associated with transition to a heterotrophic lifestyle, we performed de novo transcriptomic analysis of M. hypopitys using next-generation sequencing. We obtained the RNA-Seq data from flowers, flower bracts and roots with haustoria using Illumina HiSeq2500 platform. The raw data obtained in this study can be available in NCBI SRA database with accession number of SRP069226. A total of 10.3 GB raw sequence data were obtained, corresponding to 103,357,809 raw reads. A total of 103,025,683 reads were filtered after removing low-quality reads and trimming the adapter sequences. The Trinity program was used to de novo assemble 98,349 unigens with an N50 of 1342 bp. Using the TransDecoder program, we predicted 43,505 putative proteins. 38,416 unigenes were annotated in the Swiss-Prot protein sequence database using BLASTX. The obtained transcriptomic data will be useful for further studies of the evolution of plant genomes upon transition to a non-photosynthetic lifestyle and the loss of photosynthesis-related functions.

Polymorphism of sequences and the secondary structure of b/c intron of mitochondrial gene nad1 in Monotropa hypopitys and related ericaceae species

Nucleotide sequences of b/c intron of the mitochondrial gene nad1 were determined for the first time and analyzed in 24 plants of Monotropa hypopitys and in three related Ericaceae species. Two mitotypes of b/c intron of the nad1 gene, differing in sequence lengths and the presence of a number of single nucleotide substitutions and indels, were revealed in M. hypopitys. For the first time, a possible pre-mRNA secondary structure of M. hypopitys b/c intron was determined and IBS/EBS binding sites and the borders of six functional domains were identified.

Intraspecific variability of ITS sequences in the parasitic plant Monotropa hypopitys L. from the European Russian populations

Intraspecific sequence polymorphism in the ITS region of the nuclear ribosomal operon was examined in ten populations of the parasitic plant Monotropa hypopitys from Belgorod region, Vologda region, and Kaluga region of Russia. Based on an analysis of the identified nucleotide substitutions and indels in the ITS regions, the M. hypopitys populations were divided into two groups (A and B). Four new ITS haplotypes of M. hypopitys were identified.

Transcriptome-Wide Characterization of the MADS-Box Family in Pinesap Monotropa hypopitys Reveals Flowering Conservation in Non-photosynthetic Myco-Heterotrophs

Pinesap Monotropa hypopitys is a myco-heterotrophic non-photosynthetic higher angiosperm perennial plant devoid of vegetative organs and with standard flower arrangement. The MADS-domain transcription factor family has multiple regulatory functions in plant life cycle and is implicated in flower evolution and diversity. This study is the first to describe the MADS-box genes in a myco-heterotrophic eudicot. By performing the M. hypopitys transcriptome-wide analysis, we identified 30 MADS-box genes belonging to the major clades in the MIKCc and MIKC* lineages. Among them, MhyMADS18 is suggested as an ancestral gene of the cluster comprising AP1/FUL, FLC, SEP, and AGL6 clades. The RNA-seq profiling of the MhyMADS expression in the flowering plant revealed mRNAs specific to bracts, flowers, and roots with adventitious buds. Our results represent systematic and expression analysis of the pinesap MADS-box genes that may be involved in seed dormancy regulation, flowering time control, and flower organ identity specification. The obtained data should further our understanding of the roles played by the MADS-box genes in developmental regulation of myco-heterotrophic plants.

5.8S rDNA variability in Allium species belonging to the third evolutionary group

Sequence analysis of 5.8S rDNA in 67 accessions of the subgenus Allium and six other subgenera belonging to the third evolutionary group of Allium genus (Friesen et al., 2006) was performed. Nucleotide substitutions in 5.8S rDNA sequences of Allium accessions were identified and studied for the first time. The probable secondary structure of 5.8S rRNA was constructed. It was shown that mutations in 5.8S rDNA do not involve conserved motifs, and they did not significantly affect the secondary structure of the RNA molecule in Allium accessions.

Homeobox genes encoding WOX transcription factors in the flowering parasitic plant Monotropa hypopitys

The formation and maintenance of plant stem cell populations are controlled by the WOX family of homeobox-containing transcription factors. The evolution of WOX genes is considered one of the main reasons for the morphology of flowers and the diversity in plant architecture. The stem cell regulation mechanism is considered to be conserved among flowering plants and most thoroughly studied in Arabidopsis thaliana as a model. The angiosperms morphological diversity implies that there are species-specific features inherent in this mechanism, while the basic signaling is maintained. The unique flowering achlorophyllous mycoheterotrophic plant Monotropa hypopitys obtains nutrients from the tree roots through the mycorrhizal symbiosis. In inductive conditions, the reproductive stem with bracts and an inflorescence at the top is developed from an adventitious root bud. Like other plants, M. hypopitys forms the inflorescence, flower and root meristems, presumably using the conserved mechanisms regulating the stem cell niche. The study of M. hypopitys homeobox genes should contribute to the knowledge about the function of WOX transcription factors and the further understanding of the stem cells’ control mechanisms in the mycoheterotrophic species. The aim of this study is to analyze the M. hypopitys root, bracts, and flower transcriptomes obtained from two individual flowering plants. In total, five WOX genes have been identified and characterized by their structure, phylogeny, expression pattern, and possible functions. The assumption is that the MhyWUS1 and MhyWUS2 genes maintain the stem cell population in the inflorescence and flower meristems; MhyWOX13 has a role in controlling the root stem cell niche, seed pod formation, flowering initiation, and basic cellular processes; MhyWOX4 controls the cambium stem cells; and MhyWOX2 participates in the differentiation of egg cells and zygotes.

Analysis of the ITS1/ITS2 nuclear spacers and the secondary structure of 5.8S rRNA gene in endemic species Bellevalia sarmatica (Pall. ex Georgi) Woronow and related species of the subfamily scilloideae

Sequence variability of the ITS spacers and 5.8S rRNA gene was examined in 11 accessions of the subfamily Scilloideae, including seven accessions of rare and endangered species Bellevalia sarmatica from Volgograd region. The intraspecific polymorphism level of the examined ITS1–5.8S–ITS2 sequence of B. sarmatica accessions constituted 1.3%. The phylogenetic position of B. sarmatica within the genus Bellevalia was determined. It was demonstrated that B. sarmatica belonged to the section Nutantes, and the most closely related species were B. webbiana and B. dubia. Nucleotide substitutions in the 5.8S rRNA gene sequence of the analyzed Scilloideae accessions were identified and studied. The predicted secondary structure of 5.8S rRNA gene was constructed. It was demonstrated that in the examined accessions, mutations in the 5.8S rRNA gene were mainly localized in the third hairpin region and had no effect on the secondary structure of the 5.8S rRNA molecule.

The complete plastid genome sequence of garlic Allium sativum L

Abstract The complete plastid genome sequence of garlic Allium sativum was determined using Illumina sequencing. The plastid DNA is 153,172 bp in length and includes a large single copy region (LSC) of 82,035 bp and a small single copy region (SSC) of 18,015 bp, which are separated by a pair of 26,561 bp inverted repeat regions (IRs). In total, 134 genes are identified, containing 82 protein-coding genes, 38 tRNA genes, eight rRNA genes and six pseudogenes. Most of genes occur as a single copy, while 19 genes are duplicated in IRs. Among 15 intron-containing genes, clpP and ycf3 contain two introns and the rest have one intron.

5.8S rRNA sequence and secondary structure in Monotropa hypopitys and related Ericaceae species

This is the first study to investigate the secondary structure of 5.8S rRNA in M. hypopitys and related Ericaceae species. The identified nucleotide substitutions are localized mostly at the 3′ and 5′ ends of the gene, in the region of the third hairpin, and do not significantly affect the topology of the secondary structure of the 5.8S rRNA molecule.

Analysis of the Sequences of Internal Transcribed Spacers ITS1, ITS2 and the 5.8S Ribosomal Gene of Species of the Amaranthus Genus

Analysis of the sequence ITS1-5.8S-ITS2 in 11 samples of the amaranth species (Amaranthus caudatus, A. cruentus, A. hybridus, A. tricolor, A. paniculatus, A. hypohondriacus) was performed. It has been shown that the variability of the sequences of the intergenic spacers ITS1, ITS2 and 5.8S rRNA gene of the amaranth species analyzed is extremely low. A possible secondary structure of the 5.8S rRNA molecule was determined for the first time; three conservative motifs were identified. A single nucleotide substitution found in A. hybridus did not change the loop topology. In the sample of Celosia cristata taken as an external group, a four-nucleotide insertion in the 5′-end of the gene and a one-nucleotide deletion in the fourth hairpin not affecting the general topology of the 5.8S rRNA molecule were found.