Open Access
Issue
BIO Web Conf.
Volume 24, 2020
International Conferences “Plant Diversity: Status, Trends, Conservation Concept” 2020
Article Number 00001
Number of page(s) 5
DOI https://doi.org/10.1051/bioconf/20202400001
Published online 21 September 2020

© The Authors, published by EDP Sciences, 2020

Licence Creative Commons
This is an Open Access article distributed under the terms of the Creative Commons Attribution License 4.0, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

1 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.

2 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 (St1), E. kronokensis and E. caninus (St2) (Table 1). Locations of accessions and authors of collectings were added in the publication [4]. Sequences of GBSS1 gene from species with St, H 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.

Table 1

Clones of E. margaritae, E. komarovii, E. transbaicalensis accessions and 4 reference species of the genus Elymus, included in a comparative study of GBSS1 gene sequences.

Table 2

Species accessions and their clones drawn from the GenBank NCBI.

3 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. It confirms the StH-genomic constitution of studied species. It is shown, that variants of St subgenomes of E. margaritae GUK-1009 and E. komarovii AUK-9803 accessions belong to the North American ancestral line St2 of Pseudoroegneria genus together with reference species E. caninus and E. kronokensis, unlike the accessions of E. margaritae AUK-0650 and E. komarovii GAR-0501. The latter accessions most probably belong to Asian St1 clade with St subgenome variants of E. gmelinii and E. pendulinus species [2]. Studied species showed no noticeable differences on levels of H subgenome differentiation, but they confirmed their belonging to the evolutional H1 variant in contrast to the reference E. kamczadalorum and E. jacutensis species, related to H2 variant.

The main differences between subgenome variants St1 and St2 were in 11, 12 and 13 introns of GBSS1 gene and are as follows. Clones with St2 variant have insertions of 8 bp (GCCTCCTC), 3 bp (CAG), 2 bp (TT), 1 bp (C and A), which are lack in St1, and they doesn’t have insertions of 3 bp (GTA) and 1 bp (T), which present in St1-subgenomic clones. The two variants of St subgenome also differ from each other by substitutions: GT in St2 was substituted by AG in St1, TAT was substituted by CCG, CT – by TC, GT – by CA, and AT – by CC. In addition, clones with St1 and St2 variants differ by substitutions of single nucleotides: 17 transitions (3 A/G and 14 C/T) and 10 transversions (1 A/T, 5A/C, 2 C/G and 2 G/T). These distinctions allowed clones with St1 and St2 subgenome variants to be separated into different clades.

The main differences between H and St subgenomes of GBSS1 gene are in the presence of sequences in St-subgenomic clones, which are absent in H-subgenomic carriers: 17 bp (with single distinctions in it between St1 and St2 subgenomes), 2 bp (TT), 3 bp (AGT) and 4 bp (CATT). H-subgenomic clones also differ from St-subgenomic ones by a number of substitutions of 2, 3, 4, 5 bp, and by 52 single nucleotide substitutions: 32 transitions (16 C/T and 16 A/G) and 20 tansversions (8 A/C, 4 G/T, 5 G/C and 3 A/T).

H-subgenomic clones have several short sequences, identical with St2-subgenomic clones, but differing from St1-subgenomic ones. So, H and St2 subgenomes have sequences 6 bp (GCCTCC), 2 bp (TT) and 1 bp (C and A), which are absent in Sf. Also H and St2 subgenomes have substitutions compared to St1 subgenome: GT in H and St2 substituted by AG in St1, TAT – by CCG, CT – by TC, and GT – by CA.

Clones of E. kamczadalorum, E. jacutensis, E. kronokensis and E. caninus with H-subgenome have an insertion of 2 bp in 9 intron, which is absent in all other clones, but presents in Hordeum jubatum clone. H-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.

Thus, according to differentiation levels of the St subgenome, the accession E. margaritae GUK-1009 (holotype) is significantly separated from the AUK-0650 accession (paratype), which, in turn, is close to E. komarovii and E. transbaicalensis accessions from East Sayan.

At the same time our previous research has shown that accession AUK-0650 of E. margaritae contains clones of GBSS1 gene both with St1 and St2 subgenome variants [2]. This phenomenon demonstrates the appearance of controversial results when comparing data of morphology and molecular genetics. Nevertheless, close clustering of clones of studied species on the dendrogram is consistent with obtained preliminary data about the tendency of species to mutual introgression.

This work was supported by the state project “Estimation of the morphogenetic potential of the North Asian plant population by experimental methods” (state registration number: AAAA-A17-117012610051-5) for the Central Siberian Botanical Garden (CSBG) SB RAS, with partial financial support of the Russian Foundation for Basic Research (project No. 18-04-01030). Materials of the bioresource scientific collection of the CSBG SB RAS “Collections of living plants in open and closed ground”, USU No. 440534 were used.

thumbnail Fig. 1

The NJ dendrogram based on the analysis of GBSS1 gene sequences (exons and introns from 9 through 14) of the Siberian species E. komarovii, E. transbaicalensis and E. margaritae in comparison with the reference species from the NCBI GenBank. Asterisks indicate species — monogenomic carriers of the St and H subgenomes.

References

All Tables

Table 1

Clones of E. margaritae, E. komarovii, E. transbaicalensis accessions and 4 reference species of the genus Elymus, included in a comparative study of GBSS1 gene sequences.

Table 2

Species accessions and their clones drawn from the GenBank NCBI.

All Figures

thumbnail Fig. 1

The NJ dendrogram based on the analysis of GBSS1 gene sequences (exons and introns from 9 through 14) of the Siberian species E. komarovii, E. transbaicalensis and E. margaritae in comparison with the reference species from the NCBI GenBank. Asterisks indicate species — monogenomic carriers of the St and H subgenomes.

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