Perspectives of using Illumina MiSeq for identification of arbuscular mycorrhizal fungi.

Arbuscular mycorrhiza fungi (AMF) form one of the most common symbiosis with the majority of land plants. AMF supply the plant with various mineral elements, primarily phosphorus, and improve the water supply. The search for the most effective AMF strains for symbiosis and the creation of microbial preparations on that basis is an important task for modern biology. Owing to the difficulties of cultivation without a host plant and their high genetic polymorphism, identifying AMF is very difficult. A high number of cryptic species often makes morphological identification unreliable. Recent years have seen a growth in the number of AMF biodiversity studies performed by modern NGS-based methods, Illumina MiSeq in particular. Currently, there are still many questions that remain for the identification of AМF. The most important are whether conservative or variable sequences should be used to select a marker for barcoding and whether universal primers or those specific to AMF should be used. In our work, we have successfully used universal primers ITS3 and ITS4 for the sequencing in Illumina MiSeq of the 5.8S rDNA - ITS2 region of the 35S rRNA genes, which contain both a conservative and variable regions. The molecular genetic approach for AMF identification was quite effective and allowed us to reliably identify eight of nine isolates to the species level: five isolates of Rhizophagus irregularis, and one isolate of R. invermaius, Paraglomus laccatum, and Claroideoglomus etunicatum, respectively. For all five R. irregularis isolates, high variability in the ITS region and the absence of ecotopic-related molecular characters in the ITS2 region were demonstrated. The NCBI data is still insufficient for accurate AMF identification of Acaulospora sp. isolates from the genus to the species level.

[Polymorphic sites in transcribed spacers of 35S rRNA genes as an indicator of origin of the Paeonia cultivars].

Region ITS1­5.8S rDNA­ITS2 is sequenced in 27 varieties of cultivated ornamental peonies, ten of which presumably originate from Paeonia lactiflora, one from P. officinalis, 13 from hybridization of P. lactiflora and P. peregrina, or P. officinalis, and three are Itoh hybrids. Comparative analysis of distribution patterns of polymorphic sites (PS) for the obtained DNA sequences and data from GenBank is carried out. Hypotheses of origin of the studied varieties, except for two, which, as previously assumed, originate from hybridization of P. lactiflora and P. peregrina, are confirmed. It is shown that the sequence ITS1­5.8S rDNA­ITS2 is a good genetic marker for cultivars of the P. lactiflora group and Itoh hybrids, and that the PS distribution patterns in these sequences can provide valuable information on the kinship and origin of individual varieties. However, insufficient knowledge of wild species from the P. officinalis kinship group limits the use of this marker in the study of varieties obtained through interspecific hybridization within the Paeonia section.

[Interspecific hybridization in the genus Paeonia (Paeoniaceae): polymorphic sites in transcribed spacers of the 45S rRNA genes as indicators of natural and artificial peony hybrids].

The ITS1-5.8S rDNA-ITS2 regions of 33 accessions belonging to 16 species and five natural and garden interspecific hybrids of the genus Paeonia L. were sequenced. Chromatograms of the peony hybrids demonstrated the presence of the signals, corresponding to two different nucleotides at the positions differing in the parents, indicating that in the hybrids, no rDNA isogenization usually occurred, and they preserved rDNA of both parents. Analysis of these polymorphic sites (PS) showed that P. x majkoae was interspecific hybrid between P. tenuifolia and P. caucasica. The ITS of P. hybrida differs from ITS of P. x majkoae in 19 mutations. Because of this, P. x majkoae is definitely not synonymous to P. hybrida. Comparative analysis of ITS 1-5.8S rDNA-ITS2 showed that species diversity in section Paeonia was based on recombination as a result of intraspecific hybridization of three haplotype families. Specifically, haplotypes A, typical of the P. tenuifolia and P. anomala genomes, haplotypes B, typical of P. mlokosewitschii and P. obovata, and haplotypes of family C, currently represented in rDNA of diploid and tetraploid forms of some Caucasian and Mediterranean species. The ITS regions many diploid peonies contain no dimorphic sites, while P. oreogeton, P. cambessedesii, P. rhodia, and P. daurica carry from ten to 17 PS, and supposed to be the interspecific hybrids. Most of the tetraploid peonies contain from six to 18 PS in the ITS regions. These are alloploids with one of the parental genomes similar to that of P. mlokosewitschii (B1), or P. obovata (B3). The second parental genome in P. banatica, P. peregrina, and P. russii is represented by the genome, close to that of P. tenuifolia (A). P. macrophylla, P. mascula, P. coriacea, P. wittmanniana, and P. tomentosa carry genome of series B and genome of series C, which slightly resembles genome A.

[The origin of polyploid genomes of bluegrasses Poa L. and gene flow between Northern Pacific and sub-Antarctic islands].

The involvement of present-day diploid bluegrass species in the formation of polyploid genomes was investigated using comparison of sequences of internal transcribed spacers ITS1 and ITS2, and the 5.8S rDNA sequence. It was demonstrated that highly polyploid New Zealand bluegrasses, P. cita (2n = 84; ca. 96 to 100), P. chathamica (2n = 112), and P. litorosa (2n = 263 to 266) formed separate highly supported clade together with tetraploids (2n = 28) P. intrusa, P. anceps, and P. trioides (Austrofestuca littoralis). Among the diploid species (2n = 14), the closest relatives of these species, as well as of the polyploid species of section Poa, are the genomes of Eurasian species P. remota, P. chaixcii (sect. Homalopoa), P densa (Bolbophorum), and P. sibirica (sect. Macropoa). Nuclear genomes of polyploid Stenopoa, Tichopoa, Oreinos, and Secundae are definitely related to the genome of Arctic species P. pseudabbreviata (sect. Abbreviatae). On the contrary, judging by the genes for nuclear 45S rRNA, genomes of diploid P. trivialis (sect. Pandemos), P. annua, and P. supina (sect. Ochlopoa both) are only remotely related to the genomes of highly polyploid species (distances p between them and other bluegrass species from different sections of subgenus Poa constitute 6-10% and 11-15%, respectively). The conclusion on the relationships between highly polyploid and diploid bluegrass species was tested using analysis of synapomorphic mutations in the 5.8S rRNA gene. It was demonstrated that genomes of Poa eminens (2n = 42) and P. schischkinii (2n = 70) (sect. Arctopoa both) were noticeably different in ITS regions from the genomes of the members of the type subgenus Poa. A comparison of the Arctopoa ITS regions showed that the differences between them constituted only 0.2%. At the same time, p distances between the Arctopoa ITS and those from the species belonging to other sections of the genus Poa varied from 5 to 14%. South American species P chonotica (sect. Andinae) (=Ncoraepoa chonotica) (2n = 42) was found to be related to Arctagrostis, Festucella, and Hookerochloa, being at the same time quite distant from the other species of the genus Poa. Polymorphic in chromosome number highly polyploid species of Northern Hemisphere, P. arctica (2n = 42 to 106), P. turneri (2n = 42, 63 to 64), and P. smirnovii (2n = 42, 70) (sect. Malacanthae) are relative to a large group of tetraploid (2n = 28) endemic bluegrass species from New Zealand and sub-Antarctic islands (P. novae-zelandiae and allied species).

[Genomic structure of the autotetraploid oat species Avena macrostachya inferred from comparative analysis of the ITS1 and ITS2 sequences: on the oat karyotype evolution during the early stages of the Avena species divergence].

To examine the genomic structure of Avena macrostachya, internal transcribed spacers, ITS1 and ITS2, as well as nuclear 5.8S tRNA genes from three oat species with AsAs karyotype (A. wiestii, A. hirtula, and A. atlantica), and those from A. longiglumis (AlAl), A. canariensis (AcAc), A. ventricosa (CvCv), A. pilosa, and A. clauda (CpCp) were sequenced. All species of the genus Avena examined represented a monophyletic group (bootstrap index = 98), within which two branches, i.e., species with A- and C-genomes, were distinguished (bootstrap indices = 100). The subject of our study, A. macrostachya, albeit belonging to the phylogenetic branch of C-genome oat species (karyotype with submetacentic and subacrocentric chromosomes), has preserved an isobrachyal karyotype, (i.e., that containing metacentric chromosomes), probably typical of the common Avena ancestor. It was suggested to classify the A. macrostachya genome as a specific form of C-genome, Cm-genome. Among the species from other genera studied, Arrhenatherum elatius was found to be the closest to Avena in ITS1 and ITS structure. Phylogenetic relationships between Avena and Helictotrichon remain intriguingly uncertain. The HPR389153 sequence from H. pratense genome was closest to the ITS1 sequences specific to the Avena A-genomes (p-distance = 0.0237), while the differences of this sequence from the ITS1 of A. macrostachya reached 0.1221. On the other hand, HAD389117 from H. adsurgens was close to the ITS1 specific to Avena C-genomes (p-distance = 0.0189), while its differences from the A-genome specific ITS1 sequences reached 0.1221. It seems likely that the appearance of highly polyploid (2n = 12-21x) species of H. pratense and H. adsurgens could be associated with interspecific hybridization involving Mediterranean oat species carrying A- and C-genomes. A hypothesis on the pathways of Avena chromosomes evolution during the early stages the oat species divergence is proposed.

Cell cycle and its parameters in flowering plants.

Data on the duration of cell cycle and its phases in meristems are reviewed for 170 species from 93 genera of 38 families of higher plants. The reviewed cell cycle parameters are submitted in tabulated form, including taxonomic and anatomical characteristics of particular subjects, methods, experimental conditions, duration of cell cycle and its phases, and references. The influence of environmental factors on the cell cycle and temperature dependence of cell cycle parameters are considered in addition to certain features and causes of daily dynamics of mitotic index. Special attention is paid to the problem of comparability of different results of determination of cell cycle duration. As shown below, the only correct comparison of cell cycle parameters in different species is that, which is based on the evidence provided at species-specific optimum temperatures. A rather simple method for determining the optimum temperature of cell division and growth is based on the analysis of root growth rate. Critical temperature points are defined to serve for determination of optimum temperature for the cell cycle. As shown below, retardation of growth rate at low temperatures results from the proportional increase in the duration of cell cycle phases, while at the minimum temperature the morphological characteristics of meristem remain unchanged. Cell division anomalies or morphogenesis disruption that occur as cell cycle parameters change may be due presumably to the shock temperature action within the tolerant limits. Our experiments have suggested that the rhythm of illumination may exert essential influence on the parameters, structure and stationarity of the cell cycle.