The version of microevolutionary relationships between the Siberian species Elymus margaritae, E. komarovii, and E. transbaicalensis (Poaceae) according to sequencing of the nuclear gene GBSS1 (waxy)

A comparative study was made of the sequence of the GBSS1 gene fragment in accessions of closely related species Elymus margaritae, E. komarovii, and E. transbaicalensis from Siberia. Nucleotide sequences of the gene were determined for St subgenomes in accessions of E. margaritae and E. komarovii from classical habitats. The StH-genomic constitution was confirmed, and microevolutionary relationships between species were evaluated by constructing the NJ dendrogram. It was shown that variants of the St subgenomes in accessions E. margaritae GUK-1009 and E. komarovii AUK-9803 belong to the North American ancestral line St2 of the genus Pseudoroegneria, in contrast to accessions E. margaritae AUK-0650 and E. komarovii GAR-0501. The latter belong to the Asian branch of St1, together with variants of subgenomes in the species E. gmelinii and E. pendulinus. That is, according to the differentiation levels of the St subgenome, accession E. margaritae GUK-1009 (holotype) is significantly distanted from the accession AUK-0650 (paratype), which in turn is close to accessions of E. komarovii and E. transbaicalensis from East Sayan. According to levels of differentiation of the H subgenome, the studied species did not show noticeable differences.


Introduction
The species Elymus margaritae A.V. Agaf., Kobozeva et B. Salomon was described in collectings from the Altai Republic (holotype: Ust-Koksinsky District, Krasnaya Gora; paratype: Kosh-Agachsky District, Ukok Plateau), but initially these accessions were referred to E. komarovii (Nevski) Tzvelev [1]. Before the description of the new species E. margaritae, plants were observed in conditions of open ground and climate chamber when generations changing.
The conclusion was drawn, that specimens of the supposed new species maintained natural morphology, which is significantly different from the typical E. komarovii forms. Actual questions when studying a new E. margaritae species are its origination, variability, genetic specificity and crossability with other species.
Previously results were presented of the study of GBSS1 gene sequence in Elymus species from Siberian and Russian Far East areas to determine their genomic constitution and assess the phylogenetic differentiation [2]. Here the accession numbers of clones in GenBank NCBI are indicated. But no sequences from St genome were revealed in the sets of 8 sequenced clones of both E. komarovii GAR-0501 and E. margaritae GUK-1009 accessions. Therefore additional procedures of isolation and sequencing of GBSS1 genes from St genomes in these key accessions were performed, because GAR-0501 accession was gathered in the classical habitat of E. komarovii [3], and GUK-1009 accession -in the point of collecting of E. margaritae holotype [1,4].
The purpose of this research was to study the relationships of E. margaritae with morphologically close E. komarovii and E. transbaicalensis species, to reveal their specificity and possibility of mutual introgression.

Materials and Methods
Besides accessions of three mentioned species, the comparative research included clones of previously studied reference species from different regions of Asian Russia, which we assigned to two different groups according to St subgenome classification: E. kamczadalorum and E. jacutensis (St 1 ), E. kronokensis and E. caninus (St 2 ) ( Table 1). Locations of accessions and authors of collectings were added in the publication [4]. Sequences of GBSS1 gene from species with St, Н and Y genomes were drawn from GenBank NCBI (http://www.ncbi.nlm.nih.gov/nuccore) ( Table 2). Techniques of preparation of probes and GBSS1 sequences were carried out as described earlier [2]. The construction of dendrograms by Neighbor-Joining method was performed with TREECON ver. 1.3b software [5]. To avoid erroneous results of St clones sequencing in accessions E. komarovii GAR-0501 and E. margaritae GUK-1009, additional St clones of these accessions were used to construct the dendrogram.

Results and Discussions
The dendrogram built on the basis of nucleotide sequences of GBSS1 gene in St and H subgenomes is shown in Fig. 1  Clones of E. kamczadalorum, E. jacutensis, E. kronokensis and E. caninus with Нsubgenome have an insertion of 2 bp in 9 intron, which is absent in all other clones, but presents in Hordeum jubatum clone. Н-subgenomic clone of E. caninus have substitutions of 2, 3, 4, 6 bp and two deletions of 4 and 5 bp in 10 intron, which are identical to those of H. jubatum clone and absent in all other clones. This probably influenced the isolation of clone canABZ54_1 with H. jubatum clone into the separate clade within the common clade with H-subgenomic accessions. Clone canABZ54_1 have substitutions of 3, 3 and 4 bp in 12 intron compared to all other clones, among which first two substitutions are identical to those of E. kronokensis clone, what also has been reflected on dendrogram.
Clones of E. gmelinii and E. pendulinus -gme7727_Y and pen7732_Y -have a substitution of 3 bp (GAT) in 12 intron and insertion of 2 bp (CA) in 13 intron, due to what they separated into Y-subgenomic clade.