*3.8. Mapping and SNP Calling*

As a reference for mitochondrial and plastid genome analysis, we used mitochondrial and plastid contigs of the cultivar Dasha generated in this study. Before mapping, the Illumina reads were downsampled to 50–200 million of paired-and reads, in order to minimize the representation of the numts and nupts. The mapping was performed using CLC Genomics Workbench v.9.5.4 with the following settings: match score 1, mismatch cost 3, linear gap cost, insertion cost 3, deletion cost 3, length fraction required to be aligned 1, similarity fraction 0,97, non-specific match handling—map randomly.

SNP calling was performed using the same program with the following options: ploidy 1, ignore positions with coverage above 10,000 (this mean actually no upper limit because in all mappings the highest coverage was lower than 10,000×), ignore broken pairs, minimum coverage 20, minimum count 15, minimum frequency (%) 75, base quality filter = yes, neighborhood radius 5, minimum central quality 20, minimum neighborhood quality 15, read direction filter = yes, direction frequency (%) 30, relative read direction filter = yes, significance (%) = 1, read position filter = yes, significance (%) = 1.

The buckwheat mitochondrial genome contains large inserts of plastid origin. Due to the 10-fold higher coverage of the plastid genome compared to the mitogenome, the mapping of plastid reads on these regions will interfere with the variant calling. In order to get rid of the false variants originated from the mapping of plastid reads, we excluded all variants found in the regions of plastid origin (as defined in Table S2).

**Supplementary Materials:** The following are available online at http://www.mdpi.com/2223-7747/9/5/618/s1, Figure S1: Read coverage of buckwheat mitochondrial chromosomes, Figure S2: Phylogenetic trees based on ML analysis of single mitochondrial gene sequences; Table S1: Structural variants detected in the *F. esculentum* mitogenome (the variants with frequency >1% are reported) and their association with repeats; Table S2: Segments of plastid origin in the mitogenome of *F. esculentum*; Table S3: Sequence variants that differ in the mitogenome of buckwheat cultivars and *F. esculentum* ssp. *ancestrale*; Table S4: Sequence variants that differ in the plastome of Dasha cultivar and *F. esculentum* ssp. *ancestrale*.

**Author Contributions:** Conceptualization, M.D.L.; methodology, M.I.S., M.D.L.; software, M.I.S.; validation, M.I.S., A.S.K.; formal analysis, M.I.S., A.S.K.; investigation, M.D.L., A.A.P.; resources, A.N.F.; data curation, M.I.S., M.D.L.; writing—original draft preparation, M.D.L.; writing—review and editing, M.I.S.; visualization, M.I.S., A.A.P.; supervision, M.D.L.; project administration, M.D.L.; funding acquisition, M.D.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by Russian Science Foundation, grant number 18-76-10018.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
