New Insights into the Genomic Structure of Avena L.: Comparison of the Divergence of A-Genome and One C-Genome Oat Species

Abstract

We used next-generation sequencing analysis of the 3′-part of 18S rDNA, ITS1, and a 5′-part of the 5.8S rDNA region to understand genetic variation among seven diploid A-genome Avena species. We used 4−49 accessions per species that represented the As genome (A. atlantica, A. hirtula, and wiestii), Ac genome (A. canariensis), Ad genome (A. damascena), Al genome (A. longiglumis), and Ap genome (A. prostrata). We also took into our analysis one C-genome species, A. clauda, which previously was found to be related to A-genome species. The sequences of 169 accessions revealed 156 haplotypes of which seven haplotypes were shared by two to five species. We found 16 ribotypes that consisted of a unique sequence with a characteristic pattern of single nucleotide polymorphisms and deletions. The number of ribotypes per species varied from one in A. longiglumis to four in A. wiestii. Although most ribotypes were species-specific, we found two ribotypes shared by three species (one for A. damascena, A. hirtula, and A. wiestii, and the second for A. longiglumis, A. atlantica, and A. wiestii), and a third ribotype shared between A. atlantica and A. wiestii. A characteristic feature of the A. clauda ribotype, a diploid C-genome species, is that two different families of ribotypes have been found in this species. Some of these ribotypes are characteristic of Cc-genome species, whereas others are closely related to As-genome ribotypes. This means that A. clauda can be a hybrid between As- and C-genome oats.

Keywords: grasses; hybridization; molecular phylogeny; next-generation sequencing; rDNA; rRNA genes.

Figure 1

rDNA cluster and studied region (showed by a rectangle).

Figure 2

Variable sites of major ribotypes of the A-genome diploid Avena species: SNPs and a single nucleotide deletion. Positions with SNPs are indicated by a number. Dots indicate the identity of similar nucleotide bases with the reference sequence A. sativa KX872934 (A genome from the hexaploid). Position 1 of our alignment corresponded to nucleotide 218 of the reference sequence. D—deletion. #—single nucleotide deletion in the reference sequence between nucleotide 351 and 352.

Figure 3

Comparisons of 16 ribotypes (#1–16) network from seven diploid A-genome Avena species with five ribotypes (#17–21) in a C-genome species (A. clauda) based on ITS1 sequences. The five A. clauda ribotypes were included for comparison purposes and because it had some A-genome ribotypes in its rDNA. For each ribotype, the size of the circles represents the percentage of reads as shown in Table 1. The smallest circles correspond to ITS1 variants that have been read fewer than 1000 times. See Figure 2 for details of each ribotype.

Figure 4

Neighbor-joining tree based on sequences of 296 accessions of A-genome oats and Avena clauda (C genome). The phylogenetic tree was constructed in MEGA X using the pairwise genetic distance matrix, Maximum Composite Likelihood algorithm. Font colors are as follows: A. canariensis (green), A. damascena (pink), A. longiglumis (purple), A. prostrata (olive), A. atlantica (red), A. hirtula (black), A. wiestii (orange), and A. clauda (blue).

Figure 5

(A). Multidimensional scaling based on sequences of 165 of 169 accessions from seven Avena species. Each species was represented by 4–49 accessions. Two outlier accessions in A. canariensis (OM004573 and OM004575) and A. prostrata (OM004633 and OM004639) were found to be quite different from all other accessions in the phylogenetic tree and multidimensional scaling analysis (Figure S1) and are excluded here. The multidimensional scaling plot was plotted using CurlyWhirly v1.19.09.04. (B). Multidimensional scaling based on 296 accessions of eight oat species—A-genome oats and A. clauda (C genome). Font colors are as follows: A. canariensis (green), A. damascena (pink), A. longiglumis (purple), A. prostrata (olive), A. atlantica (red), A. hirtula (black), A. wiestii (orange), and A. clauda (blue).

Figure 6

Possible causes of intragenomic rDNA heterogeneity in Avena with A genomes (only chromosomes carrying major and minor NORs are shown [7]). (A) Diploid species have two major and one minor NORs (as in A. damascena [8]). The rDNA of both main NORs belong to the same ribotype as a result of concerted evolution. In the minor locus, 35S rDNA belongs to another ribotype. (B) A diploid having a single main ribotype, like A. longiglumis. (C) F1 hybrid between species A and B. (D–F) Chromosomes of Fn hybrids—chromosomes after recombination (D,E) or without recombination (F). (G) Diploid hybrids with different chromosome sets as a result of selection pressure. (H) rDNA loci of the minor site are homogenized towards the major ribotype. This figure is published with the kind permission of E. D. Badaeva and colleagues. It is reproduced from [7], with modifications.