*2.2. Genome Structure and Gene Content*

The result of the assembly was the set of 10 contigs with total length 404,063 bp. Their coverage ranges from 713 to 988× (Table 1). The coverage along all 10 contigs is rather uniform (Figure S1).


**Table 1.** Length and coverage of buckwheat mitochondrial chromosomes.

Moving 10 kbp from the end of each contig to its start and mapping reads to such forms of contigs also indicates a uniform coverage, implying that these contigs correspond to circular sequences. Long reads generated by the Pacific Bioscience and Oxford Nanopore Technologies platforms are a great tool for the detection of alternative structural variants in mitochondrial genomes (see, e.g., [20]). We did not identify the alternative variants at high frequency; only two variants were supported by >10% reads. The most frequent is an inversion within the chromosome mito9; it is supported by approximately 16% of reads. All other variants were found at a much lower level (see Table S1). The predominant type of the structural variation is the chromosome merge (25 out of 42 variants with frequency higher than 1%). Buckwheat mitochondrial contigs have a number of repeats; in particular the largest contig, mito1, carries a large direct repeat (~10 Kb). There is also a number of smaller repeats, both direct and inverted (see Figure 1). The repeats are known to be a hotspot for the recombination (see, for example, [21,22]). Indeed, we found that many structural variants, predominantly chromosome merges, are associated with the repeats (Table S1). This shows that the buckwheat mitochondrial genome undergoes recombination, which generates a diversity of subgenomic forms.

However, these alternative forms have a lower frequency (there are no structural variants supported by more than 50% of reads). This suggests that 10 independent circular chromosomes are the predominant form of the mitochondrial genome of *F. esculentum*. The chromosomes mito1–10 carry a complete set of genes typical for plant mitogenomes: Nine *nad* genes, two *sdh* genes, *cob*, *cox1*–*3*, five *atp* genes, four cytochrome c maturation factors, *matR*, *mttB*, and ribosomal protein (RP) genes (Figure 1).

**Figure 1.** Map of the buckwheat mitochondrial genome representing genes and repeats. Genes shown in the outer circle are transcribed clockwise; in the inner circle, counterclockwise. Repeats with length >500 bp and similarity >95% are represented.

The latter are variable within angiosperms; virtually any of the RP genes are lost in one or more angiosperm lineages [23]. In particular, the loss of *rps13* occurred in the common ancestor of rosids and *rps8*, in the common ancestor of seed plants. Congruent with this, in *F. esculentum*, the typical mitochondrial *rps8* is absent while *rps13* is present, as well as *rps1*, *3*, *4*, *7*, *10*, *12*, and *14*. Concerning large subunit RP genes, *rpl5* and *rpl16* are present while *rpl2* and *rpl10* are absent. *rpl2* is present in *Nepenthes* [24] but absent in *Fallopia* [7] and *Beta* [25], indicating multiple losses in Caryophyllales. *rpl10* is a pseudogene in *Nepenthes* and completely absent in *Beta* and *Fallopia*. It is not uncommon for mitochondrial RP genes to be replaced by their plastid counterparts, either nucleus-encoded [26] or integrated in the mitochondrial genome [27]. The *F. esculentum* mitogenome carries large insertions of DNA of plastid origin (MIPT) (see Table S2). They are especially abundant in chromosome 7 where they constitute as much as 42% of its the total length. We assume that the MIPT are recent, based on their high level of similarity with the plastome (94–100%) and on the fact that they are not shared with other Caryophyllales. The MIPTs include a number of plastid genes with intact ORFs (e.g., *rpl16*, *infA*) while many others have internal stop codons (*rps3*, *rpl22*, *rpl14*, *rps8*), making them unlikely to have functional significance. Not only the gene content but the intron content is also variable in mitochondrial genes. There are several mechanisms responsible for this, in particular, retroprocessing—the integration in the genome of the cDNA corresponding to the processed transcripts [28] and horizontal DNA transfer [29]. In *F. esculentum*, *cox1* intron, which was frequently acquired via HGT in many groups of angiosperms [30], is absent, as well as in other Caryophyllales. Five out of nine *nad* genes have introns, and three (*nad1*, *nad5*, *nad2*) of them have interchromosomal trans-splicing. In *nad1*, *atp6*,

and *nad4L*, we found an atypical start codon ACG, suggesting RNA editing. The ACG start codon in these genes is also observed in most angiosperms, including Caryophyllales; editing converting it to a typical start codon was shown experimentally in several species [31,32]. Regarding RNA-coding genes, we found 3 rRNA genes and 24 tRNA genes. The latter divide into two groups, typical mitochondrial and chloroplast-like genes. There are 15 native mitochondrial tRNA genes (12 are unique) and 9 chloroplast-like genes (8 are unique). The genes coding for trna-Met (elongator) are duplicated.
