(2022) 22:302
Sanetomo et al. BMC Plant Biology
https://doi.org/10.1186/s12870-022-03669-8

Open Access

RESEARCH

Discovery of a novel mitochondrial DNA
molecule associated with tetrad pollen sterility
in potato
Rena Sanetomo1*, Kotaro Akai2 and Akito Nashiki1,3

Abstract
Background: Tetrad sterility in potato is caused by a specific cytoplasm, called ­TSCsto, derived from the Mexican wild
tetraploid species Solanum stoloniferum. Different S. stoloniferum accessions crossed as females with S. tuberosum
resulted in 12 fertile hybrids and 27 sterile hybrids exhibiting tetrad sterility.
Results: Whole-mitochondrial-genome sequencing was performed for two fertile hybrids and three hybrids exhibiting tetrad sterility. Two to seven contigs, with the total assembly lengths ranging from 462,716 to 535,375 bp, wereassembled for each hybrid. Unlike for the reference mitochondrial genome (cv. Désirée), two different recombinanttype contigs (RC-I and RC-II) were identified. RC-I featured by the rpl5-ψrps14 gene joined to the nad6 gene, generating a novel intergenic region. Using a PCR marker (P-3), we found that this intergenic region occurred exclusively ininterspecific hybrids exhibiting tetrad sterility and in their parental S. stoloniferum accessions. A part of this intergenic
sequence was expressed in the pollen. From a large survey in which P-3 was applied to 129 accessions of 27 mostly
Mexican wild species, RC-I was found in diploid S. verrucosum and polyploid species. From eight accessions of S. verrucosum used as females, 92 interspecific hybrids were generated, in which only those carrying RC-I exhibited tetradsterility.
Conclusions: RC-I was clearly associated with tetrad sterility, and the RC-I-specific intergenic region likely contains a
causal factor of tetrad sterility.
Keywords: Tetrad sterility, Potato, Cytoplasmic male sterility, Mitochondrial genome, Solanum stoloniferum, Nanopore
sequencing
Background
Plant mitochondrial genomes (mitogenomes) are large
and complex compared with those of other eukaryotes [1–4]. Mitochondrial genes are mostly conserved
and are essential for respiration and metabolism, but
mitogenome sizes vary greatly depending on plant
species because mitogenomes contain noncoding*Correspondence: sanetomo@obihiro.ac.jp
1
Potato Germplasm Enhancement Laboratory, Obihiro Universityof Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080‑8555, Japan
Full list of author information is available at the end of the article

sequences and sequences of unknown origin [5]. Copy
number variation in plant mitochondria results fromstoichiometric or heteroplasmic differences [6]. The
plant mitogenome sometimes experiences mutations
by recombination, insertion, or nuclear invasion in aregion favorable for expression. However, the recombinant mitochondrial molecule does not immediately
increase in abundance intracellularly. A nuclear genethat controls replication and recombination of themitogenome exists, and expression of this gene results
in increased mitogenome abundance [7]. The interaction between nuclear and mitochondrial genes has
an important role in the function and development of© The Author(s) 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to theoriginal author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images orother third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line
to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory
regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of thislicence, visit http://​creat​iveco​mmons.​org/​licen​ses/​by/4.​0/. The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​
mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Sanetomo et al. BMC Plant Biology(2022) 22:302

reproductive organs. Cytoplasmic male sterility (CMS)
is one of the phenomena caused by these interactions[8].
The full structure of potato (Solanum tuberosum L.)
mitogenomes in the varieties Cicero and Désirée wererevealed by Varré et al. [9]. The potato mitogenome has
a total length of approximately 474 kb and consists ofat least three independent molecules (subgenomes): a
312.5 kb linear molecule and 112.8 kb and 49.2 kb circular molecules. The potato mitogenome is not present
as a master circular molecule like it does in other crop
species. Instead, different sizes of circular and linear molecules have formed through multiple repeat sequences[9]. Achakkagari et al. [10] compared the mitogenome
sequences of 13 accessions of nine cultivated species andone wild species with the previously reported mitogenomes of S. tuberosum [9, 11] and S. commersonii [12].
Each mitogenome is represented by three independentcircular molecules, except in a wild species (S. bukasovii), which has a single circular molecule. Some genes
are duplicated via repeat sequences in several species,
but the number of core genes is similar across all accessions. Another study by Achakkagari et al. [13] revealed
that each mitogenome in nine diploid potato clones(mostly S. tuberosum) and a wild diploid species (S.
okadae) consisted of multiple circular molecules withsimilar structures and gene organization. On the other
hand, comparing the mitogenomes of wild and cultivated potatoes, researchers detected DNA polymorphisms in several genes, such as atp6, nad3, rps10, cob,and rpl5-ψrps14 [14], which allowed classification of
mitochondrial DNA into α, β and γ types [15]. In addition, stoichiometric variation causing marked copy
number differences has been reported for the recombination-derived 10,794-bp sequence harboring the “Band1” sequence with unknown function existing only in the
Mexican wild diploid species S. verrucossum and in Mexican polyploid species [16].
Different types of CMS and related genes have been
discovered in plants [7, 17–19]. Various CMS types havealso been reported in tuber-bearing Solanum species[20–23]. Nine cytoplasmic factors in cultivated potato (S.
tuberosum ssp. tuberosum) are involved in sterility traits
such as indehiscence, shriveled microspores, no sporadic
formation, anther–style fusion, ventral-styled anthers,
and thin anthers [24]. The short anther locus (Sa), whichcauses CMS by interacting with S. phureja cytoplasm[25], was rediscovered and mapped to chromosome 6 in
an inbred line-derived ­F2 population [26]. Seven types ofmale sterility were detected in interspecific hybrids possessing the S. verrucosum cytoplasm, which were likely
caused by the interaction of at least four cytoplasmic factors and specific plasmon-sensitive nuclear genes [27].

Page 2 of 16

Tetrad sterile-type cytoplasmic male sterility (T-CMS),
in which pollen development is stopped at the tetradstage and clumps of four premature pollen grains are
released, has been observed in interspecific hybrids withcytoplasm derived from the Mexican wild diploid species S. verrucosum (2n = 2x = 24) [25, 27–29] and in cultivars with cytoplasm derived from the Mexican wild
tetraploid species S. stoloniferum (2n = 4x = 48) [15, 30,31]. S. stoloniferum is a highly polymorphic wild species
distributed from the southern United States of America
to Mexico [32]. Among S. stoloniferum accessions, only
those having a specific cytoplasm T­ SCsto exhibit T-CMS
in interspecific hybrids [33]. ­TSCsto is associated with an
859-bp mitochondrial DNA fragment amplified from theintergenic region between rpl5 and rps10 [33]. Within
this intergenic region or in the nearby region, the presence of a part of the cob gene (ψcob), a pseudogene of therps14 gene (ψrps14), and a mutation hotspot are known[34, 35]. However, little is known about the causal gene of
T-CMS or structural differences in the mitogenome characterizing ­TSCsto.
Previously, we obtained interspecific hybrids from S.
tuberosum crossed with different S. stoloniferum accessions as females, resulting in either fertile hybrids ortetrad sterile-type cytoplasmic male-sterile hybrids(hereafter referred to as T-CMS hybrids or clones) [33].
In this study, the mitogenomes of two fertile hybrids andthree T-CMS hybrids were sequenced using Nanopore
sequencing technology. Unlike in the reference mitogenome (cv. Désirée), we identified a recombinant-type
mitochondrial DNA molecule RC-I specifically foundin ­TSCsto. RC-I had a novel intergenic region between
rpl5-ψrps14 and nad6. The same region was found in the
Mexican diploid S. verrucosum and polyploid species.
Consequently, a survey of 92 diploid interspecific hybrids
generated from eight accessions of S. verrucosum as
females indicated that only those carrying this intergenic
region exhibited T-CMS, demonstrating that the RC-Ispecific intergenic region is associated with T-CMS.

Results
Constructed contigs and their homology to the reference
mitogenome

Nanopore sequencing using MinION was performed
for the mitogenomes from two pollen-fertile clones(17H117-9 and 17H131) and three T-CMS clones(15H156, 18H255, and cv. Alwara). All these clones have
S. stoloniferum-derived cytoplasms: 17H131 has D/αtype cytoplasm, 15H156 and 18H225 have D/γ-type
cytoplasm, and 17H117-9 and cv. Alwara have W/γtype cytoplasm [33]. After base calling and eliminationof low-quality reads, the mean quality score (QS) wasapproximately 11.3 for all the samples, and the read N50

Sanetomo et al. BMC Plant Biology(2022) 22:302

Page 3 of 16

ranged from 14.8 kb to 43.6 kb (Table S1). Out of 28,786
to 1,896,605 high-quality reads, 4071 to 9899 randomly
chosen reads mapped to the reference mitogenome of cv.
Désirée, which consisted of a 312.5 kb linear molecule(MN104801, hereafter referred to as M01) and 112.8 kb(MN104802, M02) and 49.2 kb (MN104803, M03) circular molecules [9], were assembled into two to seven contigs for each clone (Table 1). The total assembly lengths
ranged from 462,716 bp for cv. Alwara to 535,375 bp for
17H131, covering 97.5% (cv. Alwara) to 112.8% (17H131)
of the reference mitogenome (Table S1).
Sequence homology with the reference mitogenome
sequences was investigated by dot-plot analysis using
D-GENIES with the default parameters (Fig. S1), and theresults are shown in Fig. 1. The five mitogenomes werestructurally different from the reference mitogenome andTable 1 Constructed contigs and a comparison of them and thereference mitogenome of cv. Désirée
Clone

ContigLength (bp) No. of ­genes1) Corresponding
region present
inPollen-fertile clone
17H117-9 Contig 1

49,826

6

M01

Contig 2

63,042

7

M01

Contig 3

45,101

7

M01

Contig 4

17,176

7

M02

Contig 5

66,955

11

M02

Contig 6 187,226

23 (11)

Contig 7

49,115

7

M01 + M02

Total

478,441

Contig 1

49,807

6

M01

Contig 2

37,27310

M01

Contig 3 245,122

31 (3)

M01

Contig 4 154,071

22 (10)

M02

Contig 5

49,102

7

M03

Total

535,375

Contig 1 125,611

18 (1)

M01

Contig 2 307,654

39 (5)

Contig 3

98,2547 (7)M01 + M02

Total

531,519

17H131

M03

T-CMS clone
15H156

18H225

AlwaraM03

Contig 1 264,271

31 (1)

M01

Contig 2 193,129

34

Contig 3

48,791

6

M01 + M02

Total

506,191

Contig 1 413,590

48 (5)

Contig 2

49,126

7

Total

462,716

M03

M01 + M02
M03

1)
Open-reading frames (orfs) and tRNA genes were not counted. Duplicatedgenes are shown in parentheses.

from each other. Complex rearrangements with frequent
inversions and translocations and some duplications
were visualized. M03 of cv. Désirée was maintained asa single intact molecule (shown in green in Fig. 1) in allclones except 15H156.
Gene annotation

The gene annotation results are presented in Additional
file 3. The total numbers of protein-coding genes, theirtruncated ones, and rRNA genes varied among the clones(Table 1). Of these genes, 36 standard mitochondrial
genes (atp1, atp4, atp6, atp8, atp9, ccmB, ccmC, ccmFC,
ccmFN, cob, cox1, cox2, cox3, matR, mttb, nad1, nad3,
nad4, nad4L, nad5, nad6, nad7, nad9, rpl2, rpl5, rpl10,
rpl16, rps1, rps3, rps4, rps10, rps12, rps13, rps19, sdh3,
and sdh4) and 5S, 18S, and 26S rRNA genes (rrn5, rrn18,
and rrn26) were identified in all five clones except for
15H156, which lacked rpl16 (see Additional file 3). However, nad2, which is common in the potato mitogenome[9, 10], was not found in every clone. In addition to thegenes with complete sequences, duplicated or truncatedand fragmented sequences of these genes were also identified. Four truncated genes, namely, atp8 (72 bp), cox2
(104 bp), nad6 (66 bp), and rps1 (90 bp), were found in all
except 17H117-9, which lacked all these truncated genes.
M03 of cv. Désirée and equivalent molecules, shownin green in Fig. 1, consisted of a set of seven genes (rpl2,
rpl10, atp9, ccmB, nad9, ccmFN, and sdh3), except in thecase of 18H225, which lacked atp9 from this gene set.
15H156 had a double-sized contig (contig 3), in which
the set of seven genes was duplicated.
The other mitochondrial genes were located in one(cv. Alwara) to six contigs (17H117-9) in each clone,with a total of 15 different contigs. Seven of the 15 contigs were terminated with either rpl5-ψrps14, nad6 or
rps10 (see Additional file 3). ψrps14, located downstream of rpl5, is a pseudogene previously described bothin potato and in Arabidopsis [9, 14, 35–37], and it has
been shown that a functional copy of the gene has beentransferred to the nucleus [38] and that the sequence ofthe mitochondrial pseudogene has remained virtuallyunchanged for the past 80 million years [39]. Genes identified in contigs 1, 2 and 3 of 17H117-9; contigs 1, 2 and
3 of 17H131; contig 1 of 15H156, and contig 1 of 18H225
were all found in M01 of cv. Désirée. These contigs are
represented in blue in Fig. 1. Similarly, genes identified
in contigs 4 and 5 of 17H117-9 and contig 4 of 17H131
were all found in M02 of cv. Désirée. These contigs are
represented in red in Fig. 1. Interestingly, recombinanttype contigs that included the genes found in M01 and
M02 were identified (Table 1; Fig. 1). These recombinantcontigs (RCs) were classified into two types (RC-I and
RC-II). RC-I was found in contig 2 of 15H156, contig 2

Sanetomo et al. BMC Plant Biology(2022) 22:302

Page 4 of 16

Fig. 1 Schematic comparisons of assembled contigs, with Désirée showing an abundance of genomic rearrangements. The blue, red, and green
regions are homologous to the M01, M02, and M03 reference mitogenome regions, respectively, of cv. Désirée. The arrows indicate RC-I-specificintergenic regionsof 18H225, and contig 1 of cv. Alwara (all of which are
T-CMS clones), which contained a 108.3 kb highly conserved sequence consisting of 15 protein-coding genes(rps10, cox1, ccmFN, sdh3, rps1, atp6, atp8, cox3, sdh4,
cob, ccmFC, nad3, rps12, atp9, and rpl5-ψrps14). Thisconserved sequence contained a set of genes similar toa set within M02 of cv. Désirée but differed by the two
inversions (Fig. 1). The rpl5-ψrps14-end of this sequence(shown by arrows in Fig. 1) was always joined to the last468 bp of nad6, a member gene of M01 (Fig. 2a). The fulllength nad6 (654 bp) was present in the fertile clones
and one T-CMS clone (18H225), while the truncated468 bp nad6 was found only in T-CMS clones and joinedto rpl5-ψrps14 (see Additional file 3). The other end of
this conserved sequence was terminated at 15H156 orjoined to different member genes of M01 in 18H225 andcv. Alwara (Fig. 2b). The second recombinant type (RCII) was found in contig 6 of 17H117-9 (pollen fertile), in
which the conserved sequence found in RC-I was fragmented into different contigs, and the rpl5-ψrps14 end
was not joined to nad6 (Additional file 3).
Characterization of the rpl5‑ψrps14–nad6 connecting
region specific to RC‑I

Since we previously found a diagnostic PCR marker for­TSCsto in a location near rpl5 [33], the RC-I-specificintergenic region between rpl5-ψrps14 and nad6 in cv.
Alwara was specifically investigated. According to theresults of a nucleotide homology search using BLASTN(NCBI), there were 108-bp and 105-bp noncodingSanetomo et al. BMC Plant Biology(2022) 22:302

Page 5 of 16

Fig. 2 Schematic representation of the recombinant contig RC-I. a The P-3 region in cv. Alwara, amplified using the primer pair rpl5midR/nad6(1992 bp). The positions of nucleotide substitutions and insertions/deletions described in Table 2 are shown in Italic figures. Homologous regionswith four reads (a to d) from pollen transcripts are shown by blue lines. A108-bp region and a 105-bp region with high homology (> 94%) to thosein M02 are shown by striped boxes. The 108-bp sequence contained the first 25 bp sequence of rps10. b RC-I in three T-CMS clones. c Hypothesizedcircular molecule that amplified the 859-bp band and coexisted substoichiometrically with RC-I

regions highly homologous (> 94%) to those in M02(Fig. 2a, striped boxes). The 108-bp sequence ended withthe first 25-bp sequence of rps10. There is a truncatedcopy of cob (ψcob) between rpl5-ψrps14 and rps10 in cv.
Désirée [9] and in nine diploid breeding clones except
S. okadae [13]. Like in S. okadae, there was no cob-likesequence in this region in our materials. Interestingly, the105-bp noncoding region was 83% homologous to a 541bp region of the mitogenome of the Capsicum annuum
cultivar CMS line FS4401 (Sequence ID: KJ865409.1), theregion of which encodes orf165 [40].
To further investigate the RC-I-specific region, four
PCR primer sets were designed (P-1 to P-4, shown in
Table S2). P-1 and P-2 amplify the intragenic regions ofrpl5 and nad6, respectively, while P-3, using the primer
set rpl5midR/nad6, amplifies the RC-I-specific region
consisting of the last 127 bp of rpl5, ψrps14, the intergenic region, and the first 21 bp of the truncated nad6
gene (Fig. 2a). P-4 amplifies the intergenic region between
rps4 and nad6. rps4 was always joined to nad6 according to the gene annotation data (Additional file 3), andboth genes are in the same transcription unit [9]. Thirtyeight accessions of S. stoloniferum and 39 interspecifichybrids resulting from S. stoloniferum × S. tuberosum
were analyzed via these primer sets. The gene-specificprimer sets P-1 and P-2 and the P-4 primer set amplified single bands with expected sequence lengths (534,468, and 919 bp, respectively) from all the samples (Table
S3). However, P-3 amplified approximately 1.9 kb bands
from 18 accessions of S. stoloniferum and 27 interspecifichybrids derived from 12 accessions of S. stoloniferum. All
these hybrids exhibited either T-CMS or the other typeof pollen sterility [33]. The remaining 20 accessions of S.
stoloniferum and 12 hybrids derived from six accessionsof S. stoloniferum showed no amplified band when P-3
was used; these 12 hybrids produced normal pollen [33].
Therefore, the region amplified by P-3 was specificallyassociated with the T-CMS trait (Table S3).
The P-3-amplified bands from 17 accessions of S.
stoloniferum, 27 interspecific hybrids, and cv. Alwara
were sequenced. The total length ranged from 1992 to2010 bp due to the various lengths of intergenic regions,Sanetomo et al. BMC Plant Biology(2022) 22:302

Page 6 of 16

which ranged from 1844 to 1862 bp. Four nucleotide substitutions, one 3-bp insertion/deletion, and one 15-bp
duplication were detected (Table 2). Based on these polymorphisms, five different sequences were identified. Cv.
Alwara had the shortest sequence (1992 bp; see Fig. S2),
which was the most frequent sequence in S. stoloniferum(8 of 17 accessions).
Homology between the P‑3‑amplified region
and the 859‑bp band

Previously, we identified the 859-bp band, which was
amplified using the primer pair rp15rps14outF/ALM_5;
this band ultimately served as a diagnostic marker for­TSCsto [33]. The sequence of the first 711 bp of the 859bp band completely matched the region from 393 to
1103 bp of the P-3-amplified sequence and was locatedwithin the intergenic region between rpl5 and nad6
(Fig. 2a). The last 25 bp of the common 711-bp sequence
was identical to the first 25 bp of rps10. The remaining
148 bp of the 859-bp band was not homologous to any
part of the intergenic region, whereas it exhibited 100%
homology with rps10 of cv. Désirée. This finding can be
explained by the primer location of ALM_5, which was

designed by Lössl et al. [15] to bind to rps10. Therefore,
the 859-bp band and the P-3 sequence shared a 686bp intergenic sequence plus the first 25-bp sequence ofrps10 in common; then, the 859-bp band continued thesequence of rps10, while the P-3 sequence was joined to
the 468-bp truncated nad6 sequence with a spacer region(868 to 886 bp in size) (Fig. 2a).
PCR amplification between nad6 and rps10 via
the primer pair nad6/ALM_5 or several other newly
designed primer pairs failed, indicating that nad6 is
not joined to rps10, with the exception of the 25-bp
rps10 remnant. Using the primer pairs rp15rps14outF/
nad6 (internal P-3) and rp15rps14outF/ALM_5 (forthe 859-bp band), we amplified sequences from
­TSCsto-carrying S. stoloniferum and its interspecifichybrids and ­TSCsto-noncarrying interspecific hybrids(Fig. 3). The primer pair rp15rps14outF/nad6 yieldedstrong bands exclusively from ­TSCsto-carrying plants,
while the primer pair rp15rps14outF/ALM_5 yielded
faint bands with 859 bp in size from ­TSCsto-carrying
plants and a > 2 kb band from one pollen-fertile clone.
These results indicated that the two primer sets amplified different molecules with different copy numbers.

Table 2 Polymorphisms in the region amplified by P-3 (rpl5-ψrps14–nad6) in S. stoloniferum and interspecific hybrids with ­TSCsto
cytoplasm
Speciesor hybrid­family1)

Polymorphism and its position in p
­ arentheses2)
Accession or pedigree

3)

Size (bp)3)

1 (1165/1166)

2 (1166) 3 (1247) 4 (1252) 5 (1575/1576)

CTT​

A

A

A

TCT​TCC​ACC​TCG​ACT​ A

2010

ATT​

C

C

T

TCT​TCC​ACC​TCG​ACT​ A

2010

S. stoloniferum PI 545726

TTT​

A

A

A

-

A

1995

S. stoloniferum PI 558455

-

A

C

T

-

A

1992

S. stoloniferum PI 161178, PI 275248, PI 310964, PI
545780, PI 545788, PI 558447, PI 558450,
PI 558451

A

C

T

-

G

1992

S. stoloniferum PI 255547, PI 498030, PI 498038, PI
558395, PI 558449
15H156 (1)19H81 (1)
19H69 (1)F1 (PI 255547 × 10H17)

F1 (PI 498038 × 10H17)

F1 (PI 558449 × Konafubuki)

S. stoloniferum PI 497994, PI 498035
19H71 (2)19H72 (3)19H73 (3)19H74 (3)19H75 (2)19H76 (3)19H77 (1)17H120 (6)17H121 (1)Alwara1)

6 (1807)

F1 (PI 497994 × 10H17)

F1 (PI 497994 × 10H17)
F1 (PI 497994 × 10H17)
F1 (PI 497994 × 10H17)
F1 (PI 498035 × 10H17)
F1 (PI 498035 × 10H17)

F1 (PI 498035 × Konafubuki)

F1 (PI 558450 × 10H17)

F1 (PI 558450 × Konafubuki)
4x cultivar

Number of hybrids within the family

2)

Polymorphic sites are shown in Fig. 2a. Their positions in cv. Alwara are indicated in terms of the number of base pairs from the 5’-end of the rpl5midR primer

3)

Insertion position

Sanetomo et al. BMC Plant Biology(2022) 22:302

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Fig. 3 Stoichiometric differences between P-3 and the 859-bp band. Electrophoresed gels of the PCR-amplified bands via (a) rpl5rps14outF/
nad6 and (b) rpl5rps14outF/ALM_5 (for the 859-bp-band) primer sets. 1, cv. Alwara; 2, 18H225; 3, 15H156; 4, 19H69; 5, 19H77; 6, 19H72-1; 7–9,
S. stoloniferum PI 498030, PI 545726 and PI 558455, respectively; 10, 17H117-9; 11, 17H131; 12, 10H6-1; 13, 17H125-1. A 1 kb Plus DNA ladder(Invitrogen™) was used as a size marker in the first lane. Full-length gels are presented in Supplementary Fig. S4. c–e Amplification differences
detected via real-time PCR. Relative quantities compared with those of aprt (adenine phosphoribosyl transferase, a housekeeping nuclear gene;
Nicot et al. [41]) were measured for rpl5-nad6 (c) and rpl5-rps10 (d), and their differences are shown as the relative quantities of rpl5-rps10 divided
by those of rpl5-nad6 (e). 1–5, S. stoloniferum PI 497994, PI 498038, PI 545780, PI 558450 and PI 558455, respectively; 6, 15H156; 7, 17H120-1; 8,
18H225; 9, 17H118-3; 10, 17H117-9; 11, 17H131; 12 and 13, S. verrucosum PI 275260 and PI 498061, respectively; 14, cv. Alwara; 15, cv. Désirée. The
samples with ­TSCsto are underlined

To confirm the copy number differences, real-time PCR
was performed in conjunction with the same forward
primer but with two different reverse primers: one
designed to target rps10 (rpl5-rps10 primer set) andthe other designed to target the spacer region toward
nad6 (rpl5-nad6 primer set). Relative quantities compared with those of the adenine phosphoribosyl transferase gene (aprt), a housekeeping gene encoded in the
nuclear genome [41], were measured. The relative quantities when rpl5-nad6 was used varied from 0.05 to 0.44
(Fig. 3c), while those when rpl5-rps10 was used varied
from 0.073 × ­10–5 to 0.74 × ­10–5 from T­ SCsto-carrying
plants (Fig. 3d). Thus, rpl5-rps10 amplified muchless mitochondrial DNA than rpl5-nad6 did (1 vs.
0.8 × ­105–8.8 × ­105, Fig. 3e). Mitochondrial DNA of
the pollen-fertile clones ­(TSCsto-noncarriers) was notamplified at all when these two primer sets were used.

Expression analysis of the intergenic region
between rpl5‑ψrps14 and nad6

To search for novel candidate genes associated with
T-CMS, a pollen transcriptome analysis was performed on the RC-I-specific intergenic region between
rpl5-ψrps14 and nad6. Total pollen RNA from two
S. stoloniferum accessions, both with ­TSCsto, wasextracted, and a 150 bp paired-end reads were generated via Illumina sequencing. A total of 17,624 reads
from S. stoloniferum PI 498035 (2010 bp in size forP-3) and a total of 12,593 reads from S. stoloniferum PI
558450 (1992 bp in size for P-3) were mapped to the413,590-bp contig 1 of cv. Alwara (Table S4). Two andsix reads of PI 498035 were mapped to rpl5 and nad6,
respectively, while no reads of PI 558450 were mappedto either gene. Three reads from PI 498035 (a, b, andc in Fig. 2a) and one read from PI 558450 (d) were

Sanetomo et al. BMC Plant Biology(2022) 22:302

mapped to the intergenic region between rpl5-ψrps14
and nad6 (Table S4). Reads b, c and d overlapped witheach other and occupied a 363-bp sequence in the lastpart of the intergenic region. One nucleotide substitution (A/G at 1807; Table 2) and a 15-bp duplication(between 1575 and 1576; Table 2) were confirmed in
the mapped reads. Read a (255 bp) was mapped to thehomologous region between the intergenic region andthe 859-bp band.
Polymorphisms in the cox2‑rps1 connecting region specificto RC‑II

The second recombinant type RC-II was found in contig
6 of 17H117-9 (pollen fertile). The central region of contig 6 consisted of genes of M01. However, both terminal sequences (each 46.6 kb in size) were symmetricallyarranged with genes of M01 and M02. The connecting
region consisted of rps1 of M02 and the cox2-partial geneof M01. A PCR primer set (P-5) (Table S2) was designed
to amplify the RC-II-specific region consisting of the last
98 bp of rps1, the intergenic region, and the first 24 bp ofcox2 (Fig. 4).
The same set of 38 accessions of S. stoloniferum and
39 interspecific hybrids were analyzed using P-5. Single 1715-bp bands were amplified from 18 accessionsof S. stoloniferum, of which five accessions also had theP-3 band. The 1715-bp band was amplified from six
interspecific hybrids derived from three accessions of S.
stoloniferum. These hybrids did not exhibit T-CMS [33],indicating that the 1715-bp band was not associated with
the T-CMS trait or with the cytoplasmic type (Table
S3). The 1715-bp band amplified from the 24 samples
was sequenced, the results of which revealed only one

Page 8 of 16

nucleotide substitution from G to C at a position 333 bp
from the top of the rps1_EO primer in only one accessionof S. stoloniferum (PI 498000) and its hybrid (Tables S3,
S5; Fig. S3).
Distribution of RC‑I and RC‑II in tuber‑bearing Solanum
species

The presence or absence of recombinant mitogenomes(RC-I and RC-II) was surveyed by the use of the P-3 andP-5 primer sets for 129 accessions of 27 mostly Mexican wild species in addition to the 38 accessions of S.
stoloniferum described above (Table S5).
The P-3 band was detected for S. verrucosum (3 of 10
accessions, Verrucosa Group), S. hjertingii (6 of 7 accessions, Longipedicellata Group) and five species of the
Iopetala/Acaulia Group, namely, S. guerreroense (1 of
2 accessions), S. hougasii (7 of 7 accessions), S. iopetalum (1 of 15 accessions), S. schenckii (4 of 7 accessions),
and S. demissum (1 of 23 accessions). With respect tothe remaining 106 accessions, clear P-3 bands were notdetected, although faint bands with the same size weresometimes detected, albeit inconsistently.
The P-5 band was detected for S. bulbocastanum (2 of
2 accessions, Bulbocastana Group), S. hjertingii (2 of 7
accessions, Longipedicellata Group), and S. iopetalum (4of 15 accessions, Iopetala Group). Only one accession of
S. hjertingii (PI 186559) yielded both P-3 and P-5 bands(Table S5).
Association of RC‑I with T‑CMS in S. verrucosum

Since RC-I, which was associated with T-CMS in
S. stoloniferum, was discovered in the diploid species S. verrucosum, plants from eight accessions of S.

Fig. 4 Schematic representation of the P-4 region in contig 6 of 17H117-9, amplified using the primer pair rps1_EO/cox2_partial_SO (1715 bp). The
dashed frames indicate symmetrically duplicated regions (46.6 kb)

Sanetomo et al. BMC Plant Biology(2022) 22:302

Page 9 of 16

verrucosum were pollinated with pollen from two or
three diploid clones (mostly S. phureja). Of these accessions, only PI 498061 had RC-I according to the P-3
marker assay results. A total of 92 interspecific hybrids,
with nine to 15 hybrids per accession, were examinedfor pollen shape and stainability. All but ten hybrids
exhibited high pollen stainabilities, with family means
ranging from 59.0% to 79.0% (Table 3). The ten hybrids
were all derived from PI 498061 and showed T-CMS.
Hence, the association of RC-I with a T-CMS trait wasverified in the S. verrucosum hybrids. The copy number
difference between molecules generating the P-3 bandand the 859-bp band was also confirmed via real-time
PCR, in which a 90,000-fold difference was found for S.
verrucosum PI 498061 (Fig. 3e).

Discussion
Mitogenome assembly

In this study, five mitogenomes were assembled via
4000 to 10,000 Nanopore long reads, resulting in two
to seven contigs for each clone, with total lengths
ranging from 463 to 535 kb. Recently, three individual molecules in each of 12 potato accessions fromdiverse taxa were reported by Achakkagari et al. [10].
Similarly, Varré et al. [9] reported three autonomous
molecules, namely, two circular (M02 and M03) and
one linear (M01) DNA molecules, for two S. tuberosum cultivars, and Cho et al. [11] reported five circular DNA molecules in S. tuberosum, in which three ofthem had shared sequences. In the present study, only
M03 was retained as a single molecule in all the clones,
whereas M01 and M02 were rearranged intensively
by inversions, translocations, and duplications andformed one to six contigs in each clone. Structural variability via repeat sequences is remarkably large in plantmitogenomes [8, 13]. The larger number of contigs

was constructed for 17H117-9 and 17H131, probably
because branching molecules often caused sequencetermination at the ends of these contigs [9, 13].
T‑CMS is associated with the intergenic region
between rpl5‑ψrps14 and rps10

We discovered a recombinant-type mitochondrial
molecule (RC-I) in T-CMS hybrids of S. stoloniferum
carrying ­TSCsto and their parental accessions. In
this recombinant-type molecule, a novel intergenic
sequence between rpl5-ψrps14 and nad6 was found.
rpl5-ψrps14 and nad6 are often located in different
molecules [9, 13]. Seven contigs assembled in the present study were terminated with rpl5-ψrps14, rps10, or
nad6. This implies that the noncoding spacer regions
around these genes were often branched or rearranged
and coexisted, resulting in the assembly of multiple smaller contigs or a large linear contig. Quiñones
et al. [37] reported alternative rearrangements downstream of rpl5-ψrps14, in which rps10 is present inspecies other than S. tuberosum, whereas ψcob is present only in S. tuberosum. The former and latter types
correspond to α- and β-type mitochondrial genomes,
respectively, in accordance with the classification of
Lössl et al. [34]. Translocation of ψcob downstreamof rpl5-ψrps14 was also found in the mitogenomes of
S. stenotomum, S. phureja, and S. bukasovii [13] andwas found to be rather frequent in tuber-bearing Solanum species [14]. An approximately 1.5-kb R4 repeat
overlaps the ψcob sequence [9, 13]. Thus, the region
between rpl5-ψrps14 and rps10 has been known as arecombination hot spot in Solanum species via the R4
repeat sequence [13–15, 34, 35, 42–44]. Therefore, themitogenome of T
­ SCsto is obviously one of the recombination variants detected via this recombination hot
spot.

Table 3 Stainability of pollen from interspecific hybrids resulting from crosses between S. verrucosum (female) and 2x clones (male)
Female accession

No. of male parents

No. of hybrids

Pollen stainability (%)

Pollen shape

AverageSD

PI 275260

3

14

73.3

17.60

PI 498061

3

10

2.81)

2.51

NormalT-CMS

PI 545745

2

10

79.0

19.30

Normal

PI 545746

2

9

73.7

15.83

Normal

PI 545747

2

9

67.2

13.90

Normal

PI 545810

3

15

67.0

7.63

Normal

PI 545811

2

10

62.7

22.78

NormalPI 558488

3

15

59.0

14.57

Normal1)

One of four pollen grains in tetrad pollen was stainedSanetomo et al. BMC Plant Biology(2022) 22:302

The 859‑bp band derived substoichiometricallyfrom a molecule that underwent fission

The 859-bp band was identified as a diagnostic marker
for ­TSCsto by Sanetomo and Nashiki [33]. The 859-bp
band showed complete homology in the first 711-bp
sequence with the RC-I-specific region between rpl5
and nad6 (the P-3 region). However, we revealed that
the P-3 region and the 859-bp band were amplified
from different molecules with different copy numbers.
The 108.3-kb region homologous to the M02 region in
the recombinant contig (RC-I) was highly conserved
among T-CMS clones and terminated with rpl5-ψrps14
on one end and with rps10 on the other end. Therefore,
we suggest that the 859-bp band was amplified from amolecule that resulted from fission, derived by the joining of both ends of the conserved region, resulting ina circular molecule (Fig. 2c). Because of the low copy
number, circular structure, and complete homology
between the circular molecule and the M02 region in
RC-I, the assembly algorithm used in this study wasprobably unable to detect this circular sequence as anindependent contig.
Although many DNA rearrangements and polymorphisms in mitochondrial DNA have been detected via
PCR in somatic hybrids [34, 35, 43], these two molecules,
namely, RC-I and its internal sequence-derived circularmolecule, have not been discovered. The two molecules
coexisted in cells with T­ SCsto, but the circular molecule
was 100,000 times rarer in the cells (Fig. 3e). Copy number variation, substoichiometric variation, and fusionand fission-type molecules occurring by recombination
are frequent in plant mitogenomes [45–48]. Thus, compared with the reference mitogenome of cv. Désirée,
RC-1 is likely a fusion-type molecule (relating to tetradsterility), and the circular molecule is a fission-type molecule, both of which are present in a heteroplasmic state.
Candidate gene for T‑CMS

New open reading frames (orfs) generated by recombination in the mitogenome are often associated with CMS.
Many of them have a chimeric structure with genes
encoded in the mitogenome [7, 49, 50]. Achakkagari et al.
[13] found orf210 in the intergenic region between rpl5ψrps14 and rps10 in a cultivated potato carrying W/αcytoplasm. In the intergenic region between rpl5-ψrps14
and nad6 in RC-I, a 105-bp sequence showed homology
with orf165 of a CMS hot pepper line [40]. We identified
four reads of pollen transcripts derived from this region,
suggesting the presence of a novel orf. However, thenumber of reads mapped to this region was too small toaccurately localize the orf. This occurred mainly because

Page 10 of 16

interspecific hybrids did not provide sufficient pollen
grain due to T-CMS, and thus, mature, normal pollen
from S. stoloniferum was used in this study.
The CMS-WA gene in rice, located in the same intergenic region found in this study, is expressed specificallyat the pollen mother cell (PMC) stage and induces CMS
by interacting with Cox11 to cause premature death oftapetum cells [51, 52]. The Arabidopsis quartert (qrt)mutant releases premature pollen in the form of tetrads [53–55], which is similar to the T-CMS phenotype
in our study. In this mutant, proper degradation of thecell wall of the PMC after meiosis and subsequent segregation of microspores fail [53, 56]. Shishova et al. [57]detected metabolic changes in anthers in T-CMS potato
varieties compared with normal pollen-producing varieties. Therefore, it can be assumed that the transcripts ofthe causal gene for CMS accumulate in the tapetal tissues
and PMCs and suppress pollen development. Reduced
accumulation of the transcripts allows the production of
normal pollen in wild species and fertility-restored lines
even if these plants have a gene causing CMS [58–63].
These findings are in accordance with our results in that
only a few transcripts were mapped in normal pollen
tissue of S. stoloniferum. To identify the causal gene, itis necessary to conduct a transcriptome analysis during
early development (encompassing PMCs to tetrad pollen)in the T-CMS clones.
Origin of ­TSCsto

Using P-3, we surveyed a large panel of accessions(mostly of Mexican wild species) for the presence of therecombinant-type molecule RC-I. RC-I was frequent
among Mexican tetraploid and hexaploid species and wasfound in three of ten accessions of the Mexican diploid
species S. verrucosum. The shared nature of cytoplasmic
factors among these species has also been reported forchloroplast DNA polymorphisms [64, 65], a mitochondrial DNA-derived amplified band “Band 1” [16], andthe 859-bp band [33]. Scotti et al. [14] classified mitogenomes in 53 genotypes of 30 cultivated and wild potato
species into 13 haplotypes of 7 subgroups according tomutations in the region containing rps10, cob, and rpl5ψrps14 and classified S. verrucosum, S. stoloniferum andS. demissum into the same group. These findings support
the idea that S. verrucosum is a cytoplasmic donor for
Mexican polyploid species [16, 33, 64, 65]. Therefore, it
was suggested that ­TSCsto originated from S. verrucosum[33] because the cytoplasm of S. verrucosum also causes
T-CMS [25, 27, 29].
We obtained interspecific hybrids from both P-3 target-carrying and P-3 target-noncarrying accessions of S.
verrucosum as female parents crossed with diploid potato

Sanetomo et al. BMC Plant Biology(2022) 22:302

plants. The T-CMS phenotype occurred only for the
hybrids generated from the P-3 target-carrying S. verrucosum accession, which strongly supports that the intergenic region of RC-I is responsible for T-CMS and that
­TSCsto originated from S. verrucosum.
RC‑II derived from S. bulbocastanum?

S. stoloniferum is an allotetraploid species with the
genome constitution of AABB [66–70]. S. verrucosum is
the A genome maternal progenitor of S. stoloniferum [16,64, 65, 70]. The B genome might be homologous to the
genomes of Mexican diploid species S. cardiophyllum, S.
ehrenbergii, S. jamesii, or S. bulbocastanum [67, 68, 70,71]. The P-5 band specific to RC-II was observed only in
S. bulbocastanum among the diploid species evaluated,
implying that, as a maternal progenitor to S. stoloniferum,
S. bulbocastanum contributed to the B genome, as suggested by the shared harboring of late blight resistancegenes [71, 72] and root-knot nematode resistance genes[73]. Since S. stoloniferum is a highly polymorphic species [32], different maternal progenitors (S. verrucosum
or S. bulbocastanum) might provide different nuclear
genomes (A or B) and form a superspecies of sorts, suchas S. stoloniferum. In fact, not all S. stoloniferum accessions have cytoplasm that causes T-CMS [33], and thesame was true for S. verrucosum (Table 3). However, chloroplast DNA analyses revealed clear differences between
S. stoloniferum and S. bulbocastanum [64, 65]. In the present study, one S. hjertingii and six S. stoloniferum accessions yielded both P-3 and P-5 bands, clearly indicatingthat the PCR bands alone cannot be used to deduce thecytoplasmic origin of those accessions. Whole-genome
sequences and structural variations of mitogenomes in S.
bulbocastanum are needed to resolve this.

Conclusion
We discovered that the recombinant mitogenome RC-I
was associated with T-CMS in interspecific hybrids of
S. stoloniferum and that the intergenic region between
rpl5-ψrps14 and nad6 was most likely a causal region
for T-CMS. We also found the same mitogenome RC-I
in a limited number of accessions of S. verrucosum,
which resulted in its hybrids exhibiting T-CMS. Since S.
stoloniferum has been widely used as a genetic resourcefor disease and pest resistance in potato breeding [74,75], ­TSCsto has already been introduced into potato varieties worldwide [15, 76–78], and materials with ­TSCsto
are increasing and gradually replacing pollen-producing
parents in cross-breeding [76, 77, 79]. The P-3 marker
facilitates the identification of ­TSCsto more accurately
than does the previously reported marker (the 859-bp
band [33]), which is helpful for control cross-breeding.

Page 11 of 16

However, T-CMS may be a useful trait for efficientseed production in diploid inbred-based hybrid breeding, providing that the functions of T-CMS and a pollen-fertility restoration mechanism are elucidated [23].
A dominant male fertility restorer (Rt) gene, locatedvery distal from the centromere, has been found for
CMS caused by S. tuberosum cytoplasm [31, 80]. In ourrecent study (unpublished data), some of the interspecifichybrids resulting from S. stoloniferum × S. tuberosum
backcrossed with the S. stoloniferum parent show pollen
stainability with acetocarmine, indicating the presenceof a pollen fertility restoration mechanism. However,the fertility and morphology of the pollen were highly
influenced by environmental conditions, and polyploidy
and aneuploidy hinder the identification of the restoring factor(s). Alternatively, T-CMS caused by RC-I in the
diploid species S. verrucosum could facilitate the exploration of the mechanism driving CMS and the discovery ofa pollen fertility restoration gene.

Methods
Plant materials and DNA samples

The plants used for whole-genome sequencing included
three ­F1 hybrid plants, namely, 15H156 (S. stoloniferum
PI 255547 × 10H17), 17H117-9 (S. stoloniferum PI
249929 × 10H17), and 17H131 (S. stoloniferum PI
498027 × 10H17); a ­BC1 plant, 18H255 [(S. stoloniferum
PI 558450 × 10H17) × 10H17]; and a tetraploid cultivar,
Alwara. Among them, 15H156, 18H225 and cv. Alwara
are T-CMS clones, while 17H117-9 and 17H131 arepollen-fertile clones [33]. All the plants were grown ina greenhouse. No specific permissions or licenses wererequired for our collections and experiments. All methods were done in accordance with national, and international guidelines for plant experiments.
Total DNA was extracted from 1 g of young leaves
using a Nucleobond HMW DNA kit (Macherey–Nagel,
Germany), with minor modifications: the lysing time was
extended to 3 h at 50 ℃. The extracted DNA was subjected to size selection using a Short Read Eliminator Kit(Circulomics, USA) and resuspended in 10 mM Tris–
HCl buffer (pH 8.0) for seven days at 4 ℃. The quality of
the DNA was assessed using a microspectrophotometer(NABI, MicroDigital, Korea). To detect mitochondrial
DNA polymorphisms and perform sequencing analysis,
DNA samples from 38 accessions of S. stoloniferum and
39 interspecific hybrids from S. stoloniferum × S. tuberosum, which were previously described in the study by
Sanetomo and Nashiki [33], were used. For a broad survey of mitochondrial DNA polymorphisms, DNA samples from 129 accessions of 27 Solanum species describedby Sanetomo and Hosaka [16] were used. For crossing
with S. verrucosum, eight breeding clones, mostly thoseSanetomo et al. BMC Plant Biology(2022) 22:302

derived from S. phureja (15H138-1, 15H140-2, 97H32-6,
98H20-5, S. phureja 1.22, W6522195-19, W872209-36,
and W872209-51), were used. Seeds of all accessions witha plant introduction (PI) number were obtained from the
US Potato Genebank at Sturgeon Bay, Wisconsin, USA,with appropriate permissions.
Library preparation and nanopore sequencing

For library preparation, high-molecular DNA was prepared for sequencing following the standard protocol ofa Ligation Sequencing Kit (SQK-LSK109, Oxford Nanopore, UK). The reagent mixture consisting of 2 μl of NEBNext FFPE Repair Mix, 3.5 μl of NEBNext FFPE Repair
Buffer, 3.5 μl of Ultra II End-prep Reaction Buffer, 3 μlof Ultra II End-Prep Enzyme Mix (NEBNext Companion Module for Oxford Nanopore Technologies Ligation Sequencing, NEB, UK), and 2 μg of DNA in 48 μl of
10 mM Tris–HCl buffer (pH 8.0) was incubated at 20 ℃
for 30 min and then at 65 ℃ for 30 min in a thermal cycler(GeneAMP ABI9700, Thermo Fisher Scientific, USA).
The DNA was cleaned with the same volume of AMPure
XP Beads (Beckman Coulter, USA), and the bead pellet was washed twice in 200 μl of 70% (w/w) ethanol in
nuclease-free water. The supernatant was removed completely, and the pellet was subsequently dried at room
temperature for 30 s. The bead pellet was resuspended
in 61 μl of nuclease-free water. One microliter of eluted
DNA was quantified using a QuantiFluor system (Promega, USA). For adapter ligation, 25 μl of LNB, 5 μl of
AMX, 10 μl of NEBNext Quick T4 Ligase, and 60 μl of
DNA were mixed together and incubated at 25 ℃ for 1 h.
The ligated DNA was purified with 40 μl of AMPure XP
Beads, and the bead pellet was washed twice with 250 μlof LFB by resuspension and pelleting on a magnet. Afterdrying at room temperature for 30 s, the bead pellet was
resuspended in 15 μl of EB buffer, and 1 μl of DNA sample was quantified using a QuantiFluor system. Finally,
37.5 μl of SQB, 25.5 μl of loading beads and the DNA
library contents were mixed and loaded into a MinION
flow cell R9.4 (FLO-MIN106SP and FLO-MIN106D). All
sequencing processes were controlled with MinKNOW
and run for 24 h, with the default parameters. Individual
samples with low output were sequenced several times
until enough outputs were obtained. The output data
were saved in Fast5 format.
Base calling, read preprocessing and de novo assembly

Fast5 files were base-called with Guppy ver. 3.3.3 (GPUand high-accuracy modes) on Ubuntu 16.04 LTS. Adapters were trimmed with Porechop (ver. 0.2.1, https://​github.​com/​rrwick/​Porec​hop). Low-quality reads (mean
QS < 7) and short reads (length < 1500 bp) were removed
using NanoFilt [81], and the preprocessed reads were

Page 12 of 16

analyzed by the same tool. To extract reads of the mitogenome, all the reads were mapped to the reference mitogenome sequences of cv. Désirée (MN104801, MN104802,
and MN104803 [9]) with Minimap2 (with the -x map-ont
option), and the mapped reads were extracted in BAM
format using SAMtools ver. 1.10 [82]. To reduce computation time, the extracted reads were randomly sampled
until enough coverage (50 × to 100 ×) was obtained using
SeqKit ver. 0.11.0 [83]. The sampled reads were inputtedfor de novo assembly using Flye ver. 2.7 with the meta
option [84], and contig sequences that did not show
homology with the reference mitogenome sequences
according to dot plot analysis using D-GENIES [85]were manually removed. Some collapsed contigs werejoined manually using Bandage [86]. Assembled outputs
were called for consensus three times with Racon ver.
1.4.3 [87] and polished with Pilon ver. 1.23 [88]; consensus calling and polishing were performed only for longreads. All computing was performed on a computer with
Ubuntu 16.04 LTS as the operating system, an Intel Corei5 8400 CPU, 64 GB of RAM (DDR4-2400) and a 1 TB of
SSD hard drive.
Annotation and homology searches

Annotation of the organelle genomes was performed
based on GeSeq [89]. For reference gene information, thedataset of three mitochondrial DNA molecules from cv.
Désirée [9] was used. The selected parameters were “Linear model” and “Plastid (land plants)” for the sequenced
source and “keep best annotation only” for the annotation revisions. Protein-coding DNA sequences (CDSs)
and rRNA (and not tRNA) were annotated with > 25%
protein search identity and > 50% rRNA and DNA search
identity, respectively. A homology search was performed
by BLAST analysis provided by the National Center for
Biotechnology Information (NCBI).
PCR amplification for the detection of mitochondrial
polymorphisms and for sequencing

PCR primer sets were designed to amplify several interand intragenic mitochondrial genes: cox2-partial gene,nad6, rpl5-ψrps14, rps1, rps4, and rps10 (Table S2). PCR
amplification was carried out in a 10 μl reaction solutionthat included 2 μl of DNA (5 ng/μl), 5 μl of 2 × Ampdirect® Plus (Shimadzu, Japan), 0.25 U of heat-activatedTaq DNA polymerase (LA Taq® Hot Start Polymerase,
Takara Bio, Inc., Kusatsu, Japan), and forward and reverse
primers (each at 0.3 μM). The thermal cycling profile was
as follows: 95 °C for 10 min, followed by an initial cycle of
94 ℃ for 30 s, 65 ℃ for 30 s, and 72 ℃ for 1 min; a reduction in the annealing temperature by 1 ℃ during each of
the next five cycles, followed by an annealing temperatureof 60 ℃ for the remaining 25 cycles; and a final extension

Sanetomo et al. BMC Plant Biology(2022) 22:302

at 72 ℃ for 5 min. The amplification products were electrophoresed on a 2% agarose gel with 1 × TAE buffer(40 mM Tris, 20 mM acetic acid, and 1 mM EDTA). The
PCR products were subjected to Sanger-sequencing by acommercial company (Takara Bio, Inc., Kusatsu, Japan).
Real‑time PCR for the detection of copy number variation

To detect copy number variation of mitochondrial DNA
molecules, real-time PCR was performed using a LightCycler 96 System (Roche, Switzerland). A ten microliter
solution that included 3 μl of DNA (5 ng/μl), 5 μl of FastStart Essential DNA Green Master Mix (Roche, Switzerland), and forward and reverse primers (each at 0.25 μM)
was used (Table S2). The thermal cycling profile was asfollows: 95 °C for 10 min; 45 cycles of 95 ℃ for 10 s, 60 ℃
for 10 s, and 72 ℃ for 10 s; and then 97 ℃ for 1 s for melting. The relative quantities were measured against thatof the adenine phosphoribosyl transferase gene (aprt), a
housekeeping gene encoded in the nuclear genome [41].
For each sample, the relative quantities of three replicates (with two tubes per reaction) were obtained andaveraged.
Transcriptome analysis

Total RNA from the pollen of S. stoloniferum PI 498035
and PI 558450 was extracted using an RNeasy® Plant
Mini Kit (QIAGEN, Germany). The RNA was treatedwith DNase (TURBO DNA-free™, Ambion, TX, USA)for 20 min at 37 °C. Transcriptome analysis was thenperformed by a commercial service provider (Rhelixa,
Japan). A library was prepared using a NEBNext® Poly(A)mRNA Magnetic Isolation Module (for PolyA selection) and a NEBNext Directional Ultra RNA Library
Prep Kit for Illumina® (for strand-specific libraries). A
150 bp paired-end run was performed on an Illumina
NovaSeq 6000 sequence platform. The reads were qualified using FastQC 0.11.8 [90] and MultiQC v1.8 [91]and then filtered using Trimmomatic 0.38 [92] with
“ILLUMINACLIP:TruSeq3-PE.fa:2:30:10 LEADING:20
TRAILING:20 SLIDINGWINDOW:4:15 MINLEN:36”
options. The trimmed reads were mapped to the mitochondrial genome sequences of cv. Alwara using HISAT2ver. 2.1.0 [93]. The reads mapped to the intergenic region
between rpl5-ψrps14 and nad6 were subsequently
extracted by SAMtools ver. 1.9 [82] and visualized by
IGV [94].
Abbreviations
CMS: Cytoplasmic male sterility; Mitogenome: Mitochondrial genomes; T-CMS:
Tetrad sterility-type cytoplasmic male sterility; Orf: Open Reading Frame; PCR:
Polymerase chain reaction; PMC: Pollen mother cell; RC: Recombinant contig.

Page 13 of 16

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s12870-​022-​03669-8.
Additional file 1: Table S1. Summary statistics of MinION sequencing. Table S2. PCR markers. Table S3. Characterization of interspecifichybrids generated from crosses between S. stoloniferum and S. tuberosum accessions. Table S4. Pollen transcript reads mapped to the region
between rpl5-ψrps14 and nad6. Table S5. Survey of P-3 and P-4 regions ina wide range of tuber-bearing Solanum species. Table S6. Mitogenome
sequence data are available under BioProject No. PRJDB12191.
Additional file 2: Figure S1. Dot-plot alignments of assembled contigs
vs. the reference mitogenome (MN104801 and MN104802) of cv. Désirée.
Figure S2. Sequence of the intergenic region between rpl5-ψrps14 andnad6 (P-3) in cv. Alwara. Figure S3. Sequence of the intergenic region
between rpl1 and cox2-partial (P-5) in cv. Alwara. Figure S4. Photographsof full-length electrophoresed gels used for Figure 3. Stoichiometric differences between P-3 and the 859-bp band via a rpl5rps14outF/nad6 andb rpl5rps14outF/ALM5 (for the 859-bp band) primer sets.
Additional file 3. Gene annotation data.
Acknowledgements
We thank Dr. K. Hosaka, Obihiro University of Agriculture and Veterinary Medicine, for improving the manuscript, and W. Takenaka for technical assistance.
We also thank Dr. Awie J. Hosaka, Rhelixa, Japan, for help in mapping pollentranscripts.
Authors’ contributions
AN and RS carried out the sequencing analysis and screened wild species. KA
carried out the DNA extraction, Nanopore sequencing, assembly and annotation. RS collected and analyzed all the data and wrote the manuscript. All theauthors have read and approved the final manuscript.
Funding
This research was supported in part by a JSPS Grant-in-Aid for Scientific
Research (C) (Grant Number 22K05570); by a grant from the JST Fusion Oriented
Research for Disruptive Science and Technology (Grant Number JPMJFR2006);
and by Calbee, Inc., the Hokkaido Potato Growers Association, Kewpie Corp.,
KENKO Mayonnaise Co., Ltd., and the Japan Snack Cereal Foods Association.
Availability of data and materials
All the data generated or analyzed during this study are included in this published article and its supplementary information files. Wild species accessions
with a plant introduction (PI) number are available from the US Potato Genebank at Sturgeon Bay, Wisconsin. The other clones and varieties are available
from the corresponding author. The genome sequence data that support thefindings of this study are available in GenBank of DDBJ at https://​www.​ddbj.​
nig.​ac.​jp/​ddbj/​index-e.​html under accession numbers LC649808–649830. The
associated BioProject number is PRJDB12191, and the BioSample numbers are
SAMD00400419–00400421 and SAMD00402078–00402079 (Table S6). RNA
sequence data is available in PRJDB13350, and the BioSample numbers are
SAMD00452379–SAMD00452382.

Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare that they have no conflict of interests.
Author details
1
Potato Germplasm Enhancement Laboratory, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080‑8555, Japan. 2 National

Sanetomo et al. BMC Plant Biology(2022) 22:302

Agriculture and Food Research Organization, Hokkaido Agricultural ResearchCenter, Memuro, Hokkaido 082‑0081, Japan. 3 Graduate School of Scienceand Technology, The University of Tsukuba, Tsukuba, Ibaraki 305‑8572, Japan.
Received: 23 April 2022 Accepted: 31 May 2022

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