Microevolutionary relationships between biotypes of Elymus confusus, E. peschkovae, and E. sibiricus (Poaceae) according to hybridization and sequencing of the nuclear gene GBSS1 (waxy)

According to descriptions, the Siberian species E. peschkovae and E. confusus differ in the presence or absence of trichomes on lemma and rachilla surfaces only. Two methods were used f or study microevolutionary relationships between these speci es: hybridization of biotypes from different locations and analysis of n ucleotide sequences of the low-copy nuclear gene GBSS1. Created and grown hybrids in combinations AMU-8804 × BER-0807 and AMU-8804 × BUK-1109 showed complete seed sterility in two field vegetat ions. Taking into account wide species areas, 3 hybrids were created between Magadan biotypes from a common habitat in the combination E. confusus MOL1887 × E. peschkovae MOL-1882. The plants showed seed fertility (SF) of 0–3 %. This result does not obscure the possibil ity to assess SF in the F 2 generation and the character of inheritance of diag nostic traits. A comparative study of the GBSS1 gene sequences in accessions was carried out in comparison with clones of reference species. Differentiation of clones by the St2 subgenome in E. confusus, E. peschkovae, E. sibiricus and E. caninus did not reveal a clear relationships between the s pecies. Meanwhile, a certain species specificity for the H1 subgenome was noted, confirming the existence of microevolutionary isola tion of these species.


Introduction
Elymus sibiricus L. and E. confusus (Roshev.) Tzvel. are spreaded everywhere in southern regions of Siberia [1]. In the Russian Far East E. confusus area is shifted to the North, where it totally replaces E. sibiricus [2]. Both species are characterized by glabrous lemmas, but E. sibiricus rarely have shortly pilous lemmas. It was shown in the previous experiment that interspecific introgression is possible under the joint growth of these two species in Siberia, as a result of sexual hybridization [3].
Created and analyzed hybrids of East-Sajan biotypes in combination E. sibiricus BUR-0569 × E. confusus BUM-0505 in generation F 1 showed mostly opened anthers, values of seed fertility (SF) accounted for 2-7 seeds per spike.
Among 20 plants of the F 2 generation, grown on the opened plot, 17 corresponded to E. sibiricus on the trait of doubled spikelets on spike ledge, 3 plants carried single spikelets on all spikes. These results gave reason to refer E. sibiricus and E. confusus to a unified recombination gene pool with reproductive compatibility at the α α α α2 level at least within the Southern Siberia area.
Elymus peschkovae Tzvelev (-Agropyron confusum var. pubiflorum Roshev. -Elymus confusus var. pubiflorus (Roschev.) Tzvelev -E. pubiflorus (Roshev.) Peschkova) differs from E. confusus by pilose lemmas (Fig. 1). In Siberia the area of E. peschkovae is distinctly shifted to the North-East part in comparison with E. confusus. The purpose of this research was to study microevolutionary relationships between E. peschkovae and E. confusus biotypes. Two experimental methods were used: hybridization of biotypes from different points of species area, and analysis of nucleotide sequences of low-copy nuclear gene waxy (granule-bound starch synthase 1, GBSS1).

Materials and Methods
Parental biotypes for hybridization were grown from seeds of wild-growing accessions collected at different points of areas, or from seeds of their offspring grown under conditions of self-pollination. Plants were hybridized according to the previously developed technique [4]. Techniques of probe preparation and sequences study of the GBSS1 gene in three species were performed as described earlier [5]. The origin of accessions and clone numbers submitted to the GenBank NCBI (http://www.ncbi.nlm.nih.gov/nuccore) are shown in Table 1. In addition to accessions of three species and monogenomic markers, clones of E. caninus (L.) L. from GenBank NCBI, as a morphologically related species, were included in the study of GBSS1 sequences ( Table 2). The construction of dendrograms by the Neighbor-Joining method was carried out in the TREECON ver. 1.3b soft [6].

Results and Discussions
Hybrids between E. peschkovae (Amur region) and E. confusus (The Republic of Buryatia), created and grown by us in combinations AMU-8804 × BER-0807 (2 plants) and AMU-8804 × BUK-1109 (2 plants), have shown total seed sterility. This phenomenon forced us to return to the problem of phylogenetical relationships between three species, especially considering their wide distribution areas within the Asian part of Russia.
In vegetation period 2020 we have created 3 hybrids between Magadan biotypes from the common ecotope in the valley of river Ola in combination E. confusus MOL-1887 × E. peschkovae MOL-1882. In the greenbox all three plants had a little part of opened anthers and showed low fertility (SF) 0-3%, which was confirmed on the open experimental plot. This result does not obscure the possibility to estimate inheritance of the distinctive (diagnostic) traits and dynamics of SF in the F 2 generation.
At the same time we carried out a comparative research of the GBSS1 gene sequences in collection accessions E. peschkovae and E. confusus from the different points of area compared to clones of reference species. Dendrogram, built on the data of GBSS1 gene sequences (exons from 9 to 14 only), is shown on the Fig. 2.
Based on these results, the following conclusions were made: 4. Three of the four E. peschkovae clones belonging to the H subgenome were separated from the E. confusus clones into a particular clade, which is probably not an accidental event and supports the microevolutionary isolation of the two species. We can suppose an independent origination of the trait «pilose lemmas» in the mountane biotype E. peschkovae JTO-1708, which is located in the other separated branch E. confusus -E. sibiricus. 5. The largest group of H 1 subgenome comprised all E. sibiricus clones except the one from Yakutia-Sakha, and four E. confusus clones. This fact can hypothetically explain the high reproductive compatibility of the E. confusus BUM-0505 accession when hybridized with the accession E. sibiricus BUR-0569 from Buryatia (Agafonov, Gerus, 2009). This work was performed in the scope of state assignment for CSBG SB RAS «Estimation of the morphogenetic potential of the North Asian plant population by experimental