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  • Research article
  • Open Access

The comparison of four mitochondrial genomes reveals cytoplasmic male sterility candidate genes in cotton

BMC Genomics201819:775

https://doi.org/10.1186/s12864-018-5122-y

  • Received: 12 July 2018
  • Accepted: 26 September 2018
  • Published:

Abstract

Background

The mitochondrial genomes of higher plants vary remarkably in size, structure and sequence content, as demonstrated by the accumulation and activity of repetitive DNA sequences. Incompatibility between mitochondrial genome and nuclear genome leads to non-functional male reproductive organs and results in cytoplasmic male sterility (CMS). CMS has been used to produce F1 hybrid seeds in a variety of plant species.

Results

Here we compared the mitochondrial genomes (mitogenomes) of Gossypium hirsutum sterile male lines CMS-2074A and CMS-2074S, as well as their restorer and maintainer lines. First, we noticed the mitogenome organization and sequences were conserved in these lines. Second, we discovered the mitogenomes of 2074A and 2074S underwent large-scale substitutions and rearrangements. Actually, there were five and six unique chimeric open reading frames (ORFs) in 2074A and 2074S, respectively, which were derived from the recombination between unique repetitive sequences and nearby functional genes. Third, we found out four chimeric ORFs that were differentially transcribed in sterile line (2074A) and fertile-restored line.

Conclusions

These four novel and recombinant ORFs are potential candidates that confer CMS character in 2074A. In addition, our observations suggest that CMS in cotton is associated with the accelerated rates of rearrangement, and that novel expression products are derived from recombinant ORFs.

Keywords

  • Mitochondrial genomes
  • Cytoplasmic male sterility
  • Chimeric ORFs
  • Comparative genomics
  • Transcriptomes
  • Gossypium

Background

Cytoplasmic male sterility (CMS), a phenomenon that the male reproductive structures fail to develop, is an important agronomical trait in higher plants. The CMS character is frequently used in crop breeding and commercial seed production to increase the yield of the crops such as rice, maize, rapeseed, and cotton [15]. CMS is maintained by the maintainer line which is similar to the CMS line in terms of the nuclear composition but is equipped with the male-fertile cytoplasm. Fertility is conferred by a third line which carries the nuclear restorer genes [68]. The CMS phenotype is closely associated with mutations in mitochondrial genomes (mitogenomes) [9, 10]. The different CMS phenotypes are the results of frequent recombination, gene shuffling and mutation [11, 12]. In CMS lines, mitogenomic sequences’ rearrangements produce chimeric genes which disrupt the normal physiological functions and cause male gametophyte abnormalities, such as pollen abortion [13]. Novel chimeric genes responsible for CMS were identified by evaluating the difference in mtDNA and transcriptional products among the following lines: CMS, maintainer, and restorer in maize [14], wheat [15], rice [16], pepper [17] and rapeseed [1820]. Unfortunately, there are few reports on mitochondrial genomes rearrangement and the role of CMS in cotton.

Next-generation sequencing technology (NGS) has been applied to plant chloroplast (cp) genomes, with over 1200 species sequenced [2125]. However, plant mtDNA has a large number of repeats sequences and rearrangements, thus limiting the use of NGS [2632]. Likewise, RNA sequencing has been broadly used to study plant transcriptome and mtDNA [3337]. However, the focuses of most studies were on mitochondrial global transcript levels. Comparative analysis of the mitochondrial transcriptomes of CMS, maintainer, F1 and restorer lines’ in the context of their nuclear genomes can provide insights into cytonuclear-related phenotypes, such as cytoplasmic male sterile [11, 38]. In this study, we performed a comparative analysis of the mtDNA of the CMS, restorer and maintainer lines from both Gossypium harknessii and G. hirsutum to determine candidate CMS factors. We also analyzed the expression patterns of uncharacterized ORFs, some of which are candidate genes for CMS. The results give some interesting clues about mitochondrial evolution and CMS generation, as well as generate a background for future studies on CMS molecular diversity and phenotypic variability in cotton.

Methods

Materials and mtDNA preparation

Line 2074A, an upland cotton cytoplasmic male sterile line with Gossypium harknessii Brandegee CMS-D2–2 cytoplasm, was from its original sterile line DES-HAMS277. Line 2074S, an upland cotton cytoplasmic male sterile line, was from G. hirsutum L. CMS-AD1. These two lines were genetically stable cotton sterile lines derived from 20 to 30 generations of backcross. Line 2074B, a cultivar of upland cotton ‘Sumian No. 20’, was the maintainer of these two cytoplasmic male sterile lines. The restorer E5903 is a nuclear restoring line with normal nuclear and normal male-fertile Gossypium harknessii Brandegee. 2074A, 2074S and E5903 cotton materials used in this study are developed in our own lab [39]. We breed these three cotton cultivars and the work started 20 years before. The mtDNA preparation was performed described previously [39].

Library construction and genome sequencing, assembly and sequence verification

The mitogenome Fosmid library was constructed according to the manufacturer’s protocols (MaxxPlaxTMLambda Packing Extract)/(CopyControlTM Fosmid Library Production Kits; Epicentre Technologies, Madison, WI). All these three mtDNAs Fosimd libraries have been constructed and screened with probes from sequences of conservative genes and scaffolds. From those libraries, 1000 clones were randomly selected and screened with 28 probes designed from sequences of mitochondrial genes. At last, 22, 26 and 21 positive clones were obtained from 2074A, E5903 and 2074S, from which 23 clones (seven for 2074A, nine for E5903, and seven for 2074S) were selected to cover larger repeats and sequenced the double-ends by shotgun strategy, with insert size of about 36.2 kb- 38.4 kb. Sequenced fragments were aligned using Blastn to determine the exactness of assembly [39].

The mtDNA samples were sequenced using Illumina strategy at BGI (Beijing Genomics Institute) and assembled primarily using SOAPdenovo [40]. The Illumina system produced 413–607 M usable reads in one run for genome assembly and about 700 × coverage with Solexa using paired-end (90 bp reads). Raw sequences were evaluated by two quality control tools, using the Trimmomatic [41] and FilterReads module in Kmernator (https://github.com/JGI-Bioinformatics/Kmernator) to remove potential undesirable artifacts, including adapters or low-quality or N bases or short sequences. The filtered reads Q30 > 85%. These filtered reads were a mixture of reads derived from chloroplast, mitochondrial and nuclear genomes; firstly, we removed the chloroplast and nuclear contaminant contigs through Blastn against nt/nr database (Additional file 1: Table S1). Through adjusting the software SOAPdenovo with the reasonable parameters (−s config_file -K 37 -R -D 1 [40]), we acquired 28–65 big contigs in 4 mitogenomes. Among them, the mitogenomic sequences of 2074B had been published and the sequence was deposited in GenBank database under the accession number: JX065074.1 [42]. In addition, known mitogenomic sequences from our previous studies, including G. hirsutum 2074B [42], G. barbadense [43], G. raimondii and G. arboreum [30], as well as eight other diploid and tetraploid species [31], were used to order/orient mitochondrial-type scaffolds.

Combined with the scaffolds’ information and one whole-genome backbone with positive clones, three procedures were adopted to finish the physics gaps. Firstly, we screened the library of the whole mitogenome according to the splicing sequence and the functional genes, constructed genome physical map and then sequenced the positive clones [39]. Secondly, according to the relationship of whole-genome physics map with the positive clones, we designed primers combination on the different scaffolds’ terminals, and used long-PCR to finish the gaps (Additional file 2: Table S2A). Thirdly, PCR amplification was performed based on primers pairs that consist of the terminal sequences of large repeats (Additional file 3: Table S2B). Finally, we assembled three circle mitogenomes (2074A, 2074S, E5903). To evaluate the quality and accuracy of these three mitogenomic sequences’ assemblies, pair-end reads were mapped onto their respective consensus sequences with BWA 0.7.10-r789 [44]. The resulting SAM files from BWA mapping were transformed into BAM files using samtools view program [45]. The BWA mapping results of these pair-end reads in BAM files were then used to calculate the depth of sequencing coverage using samtools depth program [45]. For three Gossypium species, the Illumina reads covered all parts of the genomes consistently, achieving an average sequencing depth of 214.3× in 2074A mitogenome (clean data, 413 M), 28.8× in 2074S mitogenome, 27.3× in E5903 mitogenome.

Analyses and annotations of mitogenomes and sequence data

Intersubspecific polymorphisms were firstly identified based on the MUMmer package (v3.06) [46]. The results were acquired using a custom-designed Perl script and were confirmed through careful visual inspection. We carried out analyses on repeat sequences using the Washington University (WU)-Blast, including forward, palindromic reverse, and complemented repeats with a minimal length of 20 bp. Cp-derived (chloroplast-derived) sequences were identified using BlastN search of mitogenomes against annotated cotton chloroplast genomes (Identity 90%, E-value 1e-5, and Length 30 bp). Nuclear-derived insertions were searched against the G. raimondii genome. The syntenic regions of mitogenomes between different cultivars were detected using Nucmer of the MUMmer package (v3.06) [47] with 50 bp exact minimal match. NCBI-BlastX and -BlastN searches of the genomes against databases of sequenced plant mitogenomes were performed to find protein-coding and structural RNA genes, respectively. tRNA genes were searched by tRNAscan-SE [48] and were identified by BlastN [47]. The annotated mitogenomes features, including gene coordinate and genome structures, in genomes were drawn by OGDRAW v1.1 [49] and R Project (https://www.R-project.org/).

We used YASS to analyze the genome complexity that was defined as the complete sequence information of a genome with only one copy of each duplicate (> 500 bp). We set parameters as follows: E-value < 1e-30 (with the score “+ 1” for one match and “-3” for one substitution); the rate of substitutions and insertions/deletions < 5% [50].

Analysis of candidate cytoplasmic male sterility genes

Based on the previous reports showing that CMS genes are chimeric [3, 7, 51, 52], a search for chimeric ORFs was conducted. Open reading frames (ORFs) were identified by ORFfinder (https://www.ncbi.nlm.nih.gov/orffinder/) and EMBOSS (6.3.1: getorf) [53]. All ORFs at least 300 bp in length were compared to the mitogenomes of the maintainer line 2074B and the restorer line E5903 using BlastN with an identity of 99% and an E-value cut off of 1 × 10− 5. ORFs containing at least 30 bp of an identified mitochondrial gene were characterized as chimeric, excluding any ORFs that overlap the genomic position of an identified gene. Transmembrane domains in each candidate ORF were predicted using TMHMM Server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM/).

Sequencing of the cotton mitochondrial transcriptome

The extracted mitochondrial RNA from the flower buds (3–5 mm size) in CMS line 2074A, its maintainer 2074B and fertile material F1 (2074A × E5903) were sequenced on an Illumina HiSeq2000 at BGI (Beijing Genomics Institute). Ribosomal RNAs were removed from the extracted mitochondrial RNA using Ribo-Zero (Epicentre, Madison, WI) and the mitochondrial RNA libraries were prepared using Illumina’s TruSeq RNAseq Sample Prep kit. Libraries were sequenced on one lane with 4 Gb clean reads/samples of an average length of 90 nt for paired-end. RNA sequence data quality was checked using FastQC to remove the adapters, low-quality, containing N bases and short sequences with reads Q30 > 85%. The reads were mapped to the assembled mitogenome of CMS line 2074A using bowtie2 [54] with the following parameters: -D 5 -R 1 -N 0 -L 25 -i S, 1, 2.00. Then, the resulting SAM files from bowtie2 mapping were transformed into BAM files using samtools view program [45]. The bowtie2 mapping results of these pair-end reads in BAM files were then used to calculate read count for each gene through HTSeq-count program [55]. Differentially expressed genes that showed up and down regulation between samples were defined based on the standards of cutoff: two-fold change and a p-value of less than 0.05.

Results and discussion

Structures and contents of CMS, maintainer, and restorer mitochondrial genomes

Cotton is the first species that the mitogenome is sequenced among the large numbers of malvales. We performed de novo sequencing of three mitogenomes lines: a) CMS-2074A, b) CMS-2074S, and c) E5903 (a restorer line). Lines 2074A and E5903 were derived from integrating the cytoplasm of diploid species G. harknessii (CMS-D2–2) into tetraploid G. hirsutum; while, 2074S was a result of alloplasmic G. hirsutum with G. hirsutum L. CMS-AD1-derived cytoplasm [39]. The mitogenomes of the three lines were highly conserved with the sequence identity more than 96%, indicating the preservation of the mitochondrial genome during cross-breeding (Table 1; Fig. 1). The mitogenomes of the three lines were 666,081 bp (E5903), 668,584 bp (2074A) and 668,464 bp (2074S), and there was about 3 kb difference detected (Table 1). These observations were close to the previous estimations based on restriction digestion patterns (690 kb – 710 kb) [56, 57]. Compared to the maintainer line 2074B, the above three lines (E5903, 2074A and 2074S) had more repeats. In four lines, the mitogenomic sequences belonging to the coding genes (including duplicated genes) and the plastid-derived sequences varied by less than 1% (Table 1, Additional file 4: Table S3). Both the proportions of nuclear-derived intergenic sequences and large repeats varied by 1–2%. Notably, the two CMS lines, 2074A and 2074S, contained two large inverse and direct repeats. Overall, the three mitogenomes, 2074A, 2074S, and E5903, had similar syntenic arrangements and were 87% identical in sequences’ similarity with the maintainer line, indicating general conservation among the varieties within species.
Table 1

Main features of the assembled Gossypium mitogenomes

Genome Characteristics

2074A

2074S

2074B

E5903

Genome size (bp)

668,464

668,584

621,884

666,081

GenBank ID

JX536494.1

JX944505.1

JX065074.1

JX944506.1

Circular chromosomes

1

1

1

1

Percentage G + C content (%)

44.97

44.98

44.98

44.95

Protein genes

37

37

36

37

tRNA genes

30

30

29

30

 Native

18

18

17

18

 Plastid-derived

12

12

12

12

 tRNAs with introns

3

3

3

3

rRNA genes

4a

4a

4a

4a

Genic content percent coverage of total genome

 Exonic

6.23

6.23

6.25

6.39

 Intronic-c

4.43

4.43

4.45

4.76

Intergenic content percent coverage

 Chloroplast-derived

1.43

1.44

1.35

1.37

 Nuclear-derived

8.44

8.83

7.11

8.29

Repeat content percent coverage of total genome

 Large repeats: > 1 kb(number)

11.78 (4)

11.74 (5)

9.44 (4)

11.32 (7)

 Small repeats: < 1 kb(number)

4.71 (475)

4.77 (476)

4.05 (465)

4.81 (470)

aPresent rrn26 has two copies

Fig. 1
Fig. 1

Linear maps of the four cotton mitogenomes. Known protein-coding genes, tRNA and rRNA genes, and gene fragments are shown on the line. Genes on the right side and left side of the line are transcribed direct and inverted, respectively. Colors indicate genes by function: Complex I (nad; yellow), Complex II (sdh; green), Complex III (cob; yellowish green), Complex IV (cox; light pink), Complex V (atp; olive-green), ribosomal proteins (brown), maturase (matR; orange), other genes (ccm and tRNA; purple), intron (white)

The main cycle (MC) molecules of two male sterile lines (2074A and 2074S) are 47 kb larger than that of the maintainer line (2074B). The reason is that they contain three large repeats, for examples, one is 10 kb larger than that of 2074B, which are similar as observed in the sterile line Ks3 that contains four repeats larger than 20 kb [41], and the sterile line TK18-MS that contains a pair of repeats of 86 kb in its MC molecule [58]. The intergenic regions of plant mtDNAs often contain retrotransposons from nuclear [25], chloroplast [59, 60] and other plant mitogenome [28]. 2074A and 2074S have more retrotransposons than 2074B and E5903, and they contain six unique regions with a total length of 31,694–36,741 bp. Furthermore, these sequences are novel and most are located in the intergenic regions, showing that they have a faster rate of evolution as similarly reported by Palmer et al., 2000 [61].

Gene evolution among CMS, restorer, and maintainer lines

Cotton mitogenomes typically contain 36 genes (Table 2; Fig. 1) whose content is largely conserved among species and across angiosperms, with few differences (for example, contrary to other sequenced plants, rpl2 in cotton lacks an intron [42, 43, 58]). As the case in the mitogenomes of other angiosperms, most of the genes encode proteins involved in ATP-generating electron transport: nine subunits of complex I (nad 1, nad 2, nad 3, nad 4, nad 4 L, nad 5, nad 6, nad 7 and nad 9), two subunits of complex II (sdh 3 and sdh 4), one subunit of complex III (cob), three subunits of complex IV (cox 1, cox 2 and cox 3), and five subunits of complex V (atp 1, atp 4, atp 6, atp 8 and atp 9); four genes involved in biogenesis of cytochrome c (ccm C, ccm B, ccm FC and ccm FN); 10 genes encode for ribosomal proteins (rpl 2, rpl 5, rpl 10, rpl 16, rps 3, rps 4, rps 7, rps 10, rps 12 and rps 14), while the numbers of the ribosomal proteins are usually variable among different species [58]. In addition, one gene (mttB) is involved in independent membrane targeting and translocation system, and one maturase gene (matR) is mapped within the 4th intron of nad1. 9 protein-encoding genes (ccmFC, cox2, rps10, rps3, nad1, nad2, nad4, nad5 and nad7) contain a total of 23 group II introns, among of which three are trans-spliced (nad 1, nad 2 and nad 5). As previously noted, the cotton mitogenomes presented here lack rps1, rps2, rps11, rps13, rps19 and sdh2, and only partial sequences of these genes were detected in cotton mitogenomes.
Table 2

Gene contents of Gossypium mitotypes

Product group

Gene

2074A

2074S

2074B

E5903

Product group

Gene

2074A

2074S

2074B

E5903

complex I

nad1

+

+

+

+

Ribosome

rps3

+ 2b

+ 2

+ 2

+/ψ-

nad2

+

+

+

+

 

rps4

+

+

+

+

nad3

+

+

+

+

 

rps7

+

+

+

+

nad4

+

+

+

+

 

rps10

+

+

+

+

nad4L

+

+

+

+

 

rps12

+

+

+

+

nad5

+

+

+

+

 

rps14

+

+

+

+

nad6

+

+

+

+

 

rpl2

+

+

+

+

nad7

+

+

+

+

 

rpl5

+

+

+

+

nad9

+

+

+

+

 

rpl10

+

+

+

+

complex II

sdh3

+

+

+

+

 

rpl16

+

+

+

+

sdh4

+

+

+

+

tRNA

trnC(GCA)-cp

+

+

+

+

complex III

Cob

+

+

+

+

 

trnD(GUC)-cp

+ 2

+ 2

+ 2

+ 2

complex IV

cox1

+

+

+

+

 

trnE(UUC)

+

+

+

+

cox2

+

+

+

+

 

trnF(GAA)

+ 2

+ 2

+ 2

+ 2

cox3

+

+

+

+

 

trnfM(CAU)-cp

+ 4

+ 4

+ 4

+ 4

complex V

atp1

+

+

+

+

 

trnG(GCC)

+

+

+

+

atp4

+

+

+

+

 

trnH(GUG)-cp

+

+

+

+

atp6

+

+

+

+

 

trnK(UUU)

+

+

+

+

atp8

+

+

+

+

 

trnM(CAU)

+ 2

+ 2

+ 1

+ 2

atp9

+

+

+

+

 

trnI(UAU)

+

+

+

+

Cytochrome C

ccmB

+

+

+

+

 

trnN(GUU)-cp

+

+

+

+

ccmC

+

+

+

+

 

trnP(UGG)

+ 3

+ 3

+ 3

+ 3

ccmFN

+

+

+

+

 

trnQ(UUG)

+

+

+

+

ccmFC

+ 2a

+ 2

+

+ 2

 

trnS(GCU)

+ 2

+ 2

+ 2

+ 2

Other gene

mttB

+

+

+

+

 

trnS(GGA)-cp

+

+

+

+

matR

+

+

+

+

 

trnS(UGA)

+

+

+

+

rRNA

rrn5

+

+

+

+

 

trnSup(UUA)

+

+

+

+

rrn18

+

+

+

+

 

trnV(GAC)

+

+

+

+

rrn26

+ 2

+ 2

+ 2

+ 2

 

trnW(CCA)-cp

+ 2

+ 2

+ 2

+ 2

      

trnY(GUA)

+

+

+

+

Note. −+, denotes present; −, denotes absent; aGene copy number is shown after +; brps3–2is a pseudo gene

As reported previously, several mitochondrial genes exist in repeat regions and in multiple copies (Additional file 5: Table S4). However, unlike the mitogenome of G. hirsutum maintainer line, those of 2074A, 2074S and E5903 contain duplicated trnM (CAU) and ccmFC in large repeats. In addition, congruent with prior results, rps3 is located at a repeat’s boundary and varied in structure among these four Gossypium mitogenomes. In cotton, rps3 contains a central domain (pfam00013) which has been lost in the incomplete duplicates (pseudogene rps3–2) of 2074A, 2074S, and 2074B.

A total of 47 SNPs exists within 21 protein-coding genes in four mitogenomes analyzed, and only 11 SNPs were synonymous mutations (1 in 2074A mitogenome and 10 in 2074B mitogenome, Table 3). Remarkably, the numbers of nonsynonymous mutations (36 SNPs) are over three times as that of synonymous mutations, and nonsynonymous mutations were nearly evenly distributed among the mitochondrial genomes (10, 10, 9, and 7 unique nonsynonymous SNPs in 2074A, E5903, 2074S and 2074B, respectively). Most of these SNPs represent transversions rather than transitions (29 versus 7), and many of them were found in ribosomal protein-coding genes, (i.e., rpl2, rpl5, rpl10, rpl16, rps3, rps4 and rps10; Table 3). As protein-coding genes are extraordinarily conserved and exhibit slow evolutionary rates, the abundance of non-synonymous changes reported here may represent CMS-related candidate genes, although this needs to be functionally verified in each case. Mitochondrial genes, rps3 and rpl2, separately containing 3 and 2 nonsynonymous changes between the mitogenomes of 2074A (and 2074S)/E5903 and 2074B, might represent suitable candidates.
Table 3

The protein variation in four Gossypium mitogenomes

Gene

Len

Var

IDY

Loc

2074A

2074S

E5903

2074B

NSM

SM

aa-Var

N-S

P-S

N-S

P-S

N-S

P-S

N-S

P-S

atp4

585

1

99.8

222

ttT

Phe

ttT

Phe

ttT

Phe

ttC

Phe

1

0

 

atp8

465

1

99.8

171

agA

Arg

agA

Arg

agA

Arg

agC

Ser

0

3

Ser-Arg

atp9

225

1

99.6

27

ggA

Gly

ggT

Gly

ggT

Gly

ggT

Gly

1

0

 

ccmB

621

1

99.8

11

cAt

His

cTt

Leu

cTt

Leu

cTt

Leu

0

1

Leu-His

ccmFC

1323

1

99.8

585

gtC

Val

gtC

Val

gtC

Val

gtG

Val

   

cox1

1593

3

99.8

415

Acc

Thr

Acc

Thr

Ccc

Pro

Acc

Thr

  

Thr-Pro

960

atA

Ile

atA

Ile

atA

Ile

atC

Ile

   

1428

atA

Ile

atA

Ile

atA

Ile

atC

Ile

2

1

 

cox2

783

1

99.9

481

Tta

Leu

Tta

Leu

Tta

Leu

Cta

Leu

1

0

 

cox3

798

4

99.6

157

Ctc

Leu

Atc

Ile

Atc

Ile

Ctc

Leu

  

Leu-Ile

294

ttT

Pro

ttT

Pro

ttT

Pro

ttG

Leu

  

Pro-Leu

295

Gct

Ala

Gct

Ala

Gct

Ala

Tct

Ser

0

4

Ala-Ser

matR

1968

1

99.9

1858

Aaa

Lys

Aaa

Lys

Aaa

Lys

Caa

Gln

0

3

Gln-Lys

nad2

1467

1

99.9

783

tcG

Ser

tcG

Ser

tcG

Ser

tcT

Ser

1

0

 

nad3

357

1

99.7

317

tCt

Ser

tCt

Ser

tCt

Ser

tTt

Phe

0

1

Ser-Phe

nad4

1488

3

99.8

33

gaT

Asp

gaT

Asp

gaT

Asp

gaC

Asp

   

240

atC

Ile

atC

Ile

atC

Ile

atA

Ile

   

242

aAt

Asn

aAt

Asn

aAt

Asn

aTT

Ile

2

1

Asn-Ile

nad7

1185

1

99.9

24

atC

Ile

atC

Ile

atC

Ile

atA

Ile

1

0

 

rpl2

1005

2

99.8

45

ttG

Leu

ttG

Leu

ttG

Leu

ttT

Phe

  

Phe-Leu

292

Ctc

Leu

Ctc

Leu

Ctc

Leu

Atc

Ile

0

6

Ile-Leu

rpl5

582

1

99.8

139

Caa

Gln

Caa

Gln

Caa

Gln

Aaa

Lys

0

3

Lys-Gln

rpl10

489

1

99.8

361

Aaa

Lys

Aaa

Lys

Aaa

Lys

Gaa

Glu

0

3

Glu-Lys

rpl16

435

1

99.5

270

gtC

Val

gtC

Val

gtC

Val

gtA

Val

1

0

 

rps3

1707

3

99.8

1670

aAg

Lys

aAg

Lys

aAg

Lys

aGg

Arg

  

Lys-Arg

1676

gGA

Gly

gGA

Gly

gGA

Gly

gAC

Asp

  

Gly-Asp

1678

Cgt

Arg

Cgt

Arg

Cgt

Arg

Ggt

Gly

0

3

Arg-Gly

rps4

1098

1

99.9

535

Caa

Gln

Caa

Gln

Caa

Gln

Aaa

Lys

0

3

 

rps10

333

1

99.4

311

gTC

Val

gAA

Glu

gAA

Glu

gAA

Glu

0

1

Glu-Val

sdh3

435

1

99.8

33

ttC

Phe

ttC

Phe

ttC

Phe

ttA

Leu

0

3

 

nonsynonymous mutation

31

  

10

 

9

 

10

 

7

total

10

36

17

Synonymous mutation

  

1

 

0

 

0

 

10

Note. –Len, length of gene CDS sequence; Var variant sites in fourmitogenomes, IDY identity of gene CDS sequences, Loc location of variant sites, N-S nucleotide sequence, P-S amino acid sequence, Boldface, variant nucleotide, Bold italic variant amino acids, NSM nonsynonymous mutation, SM synonymous mutation, aa-Var amino acidvariation, Boldface mark is mutated base and amino acid

In the four mitogenomes analyzed, we found many gene editing events, for example, ACG was edited into AUG as start codon in three genes (rps10, nad1 and nad4L), and AUU was modified into AUG in one gene (mttB). There were five cases where gene editing generated stop codon, thereinto, four cases were the conversion of TAG into CGA in rps10, ccmC, atp9 and ccmFC genes; however, TAG was converted to CAA in atp6 gene. of Evolutionary rates analysis (ka/ks or ω) revealed that the ratios of ka to ks of nine genes (rps12, matR, atp1, mttB, rps4, rrn18 and nad1) were greater than 1, which implied a positive selection. In addition, that of two genes (rpl5 and cox3) were less than 1, which implied a purifying selection. By contrast, the non-coding regions appeared to be rapidly diverged (Additional file 6: Table S5).

Repeated sequences and unique sequences

The plant mitogenomes harbor massive repeated sequences, and the genome sizes tend to increase the genomic coverage by large repeats [6265]. Our analysis revealed duplications were the main reason for the difference in size among the four lines. The duplicate lengths varied from 504 bp to 29 kb, which constituted 9.4–12.0% of the total genome lengths (Table 4). Two duplicated fragments 11,191 bp and 10,632 bp were present in the mitogenomes of all four lines. There was a common duplicate in three mitogenomes of 2074A, 2074S and E5903, but it was absent in 2074B. The mitogenomes of 2074A and 2074S were mostly identical, with one exception that a repeat sequence was present in 2074A but absent in 2074S. The mitogenome of 2074A is made up of a repeat sequence (29,277 bp), whereas that of 2074S consists of two repeat sequences (24,378 and 4621 bp) that are separated by a gap (Table 4). Total backbone DNA sequences represented a concatenation containing all basic fragments among all mitogenomes. When considering only one copy of each duplicated sequence, we found the genomic variations became small, especially from the same origin. The sizes of the backbone mtDNA sequences of 2074A and E5903 are same, and have a minimal difference with that of 2074S. Other repeats are smaller in size, and distribute distinctly and vary in copy number (Fig. 2).
Table 4

Length and percentage of duplicated fragments (up to 500 bp)

Genome

Genome length (bp)

Duplication length (bp)a

% of in genome

Minimal length (bp)b

Maximal. length (bp)b

Number of fragments

Genome length without duplication (bp) (Percentage)

2074A

668,464

80,545

12.0

504

29,277

7

587,919 (87.95%)c

2074S

668,584

80,269

12.0

505

27,666

8

588,315 (87.99%)

2074B

621,884

58,734

9.4

879

27,558

5

563,150 (90.56%)

E5903

666,082

78,161

11.7

504

21,563

11

587,921 (88.27%)

Note. –aAll duplicated copies less one; bLength of one copy; c% of backbone fragments in genome

Fig. 2
Fig. 2

The size distribution of repetitive content by the number of repeat pairs and total repeat length. The X position is repeat size category, which contains more than 10 kb, 1–10 kb, 0.5–1 kb, 101–500 bp, 41–100 bp, 31–40 bp, 21–30 bp. The Y positions are number of repeats pairs (primary axis) and proportion of total repeat length (secondary axis). The (a), (b), (c), (d) present 2074A, 2074S, 2074B, E5903 mitogenome, respectively

Intra-genomic recombination is an active phenomenon in the mitogenomes of plants [25]. Recombination frequency depends on the size of repeats, for example, large and direct repeats (> 1 kb) are associated with homologous recombination that will lead to the formation of sub-genomic molecules [66]. These four cotton mitogenomes exist as 4–7 larger repeats that produced subcircles. In 2074A, two pairs of subcircles are mediated by direct-repeat AR1, whereas one MC genomic circle may be produced by inverted-repeat AR4. However, 8–12 positive Fosmid clones covered all these large repeats and nine positive Fosmid clones covered all these repeats in E5903, which suggests that these four big repeats didn’t formed subcircles in 2074A and E5903 mitogenomes (Additional file 7: Figure S1). Importantly, every nodal point of large repeats is verified by long-PCR with special primers designed from their sequences (the PCR products were overlapped with repeats and non-repeats regions more than 300 bp, Additional file 2: Table S2A, Additional file 3: Table S2B).

Cytoplasmic male sterility is frequently associated with novel, chimeric, and often disruptive ORFs [3, 64]. In this study, we evaluated the mitogenomes of two CMS lines in the context of their maintainer and restorer lines for unique sequences that contain novel coding regions responsible for sterility in G. hirsutum. Six unique sequences (U1 to U6) were similar in two CMS lines, but were absent in the maintainer and restorer lines (Table 5). The total lengths of these regions were 31,694 bp in 2074A and 36,741 bp in 2074S, respectively (Table 5). Overall, these regions were unique with little similarities to known mitochondrial and nuclear sequences of other plants. These unique regions were frequently associated with repeats’ boundaries, which might indicate an origin from new sequence migration and recombination.
Table 5

The unique sequences in 2074A and 2074S compared with 2074B

No.

Locationa

Length(bp)

Joint of syntenic regions

Predicted ORFb

Identity sequencesd

U1

1–5156

5156

S1

Aorf1, Aorf2, Aorf3; Sorf1, Sorf2, Sorf3

2316–2885, 3107–3879 Carica papaya mitochondrion.

U2

16,918-17,305

388

S2-S3

 

236–379 Citrullus lanatus mitochondrion, Beta vulgaris subsp. 5 aponica genotype male-sterile E mitochondrion.

U3

143,667–151,556 (143,674-151,564)

7890

S6, S5-S7

Aorf7, Sorf7

2888–3734, 7191–7409, 6160–6348 Vigna 5 aponic mitochondrion, Malus x domestica mitochondrion, Beta vulgaris subsp. 5 aponica genotype male-sterile G mitochondrion.

U4

237,227- 238,728

(237,182–238,681)

1502

S10

 

893–1502, 413–720, Phoenix dactylifera mitochondrion, Nicotiana tabacum mitochondrion.

U5c

438,450–457,430 (443,399-457,334)

18,981

(13936)

S14, S13-S15

Aorf18, Aorf19, Aorf20, Aorf21, Aorf22; Sorf17, Sorf16, Sorf18, Sorf19, Sorf20, Sorf21

6486–7727, G. hirsutum clone MX008C17-jmb, 5270–5431, 5610–5890, 10,317–11,602, 12,127–12,482 Mimulusguttatus mitochondrion; 2286–2703 unknown chloroplastid sequence. 15,875–18,884 O. berteriana mtDNA for rps3 and rpl16.

U6

665,761 -668,584

(665,642-668,464)

2824

S22

Aorf29; Sorf30

565–857, 1918–2188, 879–1159, Phoenix dactylifera mitochondrion, Lotus 5 aponicas strain MG-20 mitochondrion.

Note. –a figures in brackets denote the sites in 2074A mitogenome; bthere are 5 ORFs predicted in U5; cU5 is 13936 bp in 2074A, and is longer 5148 bp at 3’end sequence in 2074S dthe identity is more than 80%, the figures denote the sites of alignment fragments

Mitochondrial genome syntenic evolution and organization

In addition, compared with 2074A, we analyzed the syntenic evolution of 4 cotton mitogenomes. We found 22 syntenic regions (named as S1 - S22), ranging from 2824 to 147,683 bp, which possessed at least 98% identity (Additional file 8: Figure S2). 2074B has lost syntenic segments S1 (U1), S6 (U3), S14 (U5), and S22 (U6). However, some segments are conserved in four mitogenomes, such as S5 - S6, S8 - S9, S10 - S13 and S15 - S20; the terminal sequences of S3, S4, S7, S13 and S21 are four large repeats (AR4, AR3, AR1, AR2, AR1, respectively), and the former sequence S20 was AR1 (as the difference of these larger repeats). The syntenic regions are broken, which suggests the repeat sequences are more dynamic and have undergone recombination in breeding process. S10 and S2 are broken by unique sequences U4 and U2, while other syntenic regions are more or less interrupted by insertion or deletion. These two cytoplasmic male sterile lines are more complex in nucleotide sequence composition, which suggests that male sterility may have been favored by faster rates of rearrangement and evolution, or CMS itself might have caused faster rearrangement and evolution.

CMS in two cytoplasmic male sterile lines of Gossypium hirsutum

CMS is a widespread phenomenon in plants and is associated with abnormal mitochondrial ORFs [7]. The occurrence of male sterility is an important feature in cotton breeding system. CMS is expected to be affected by mitochondrial gene(s), ORF content(s) and diversity during the emergence and selection of CMS specific mitochondrial genes. In other plants, several CMS-associated aberrant genes are located upstream or downstream of certain known genes and co-transcribed together [7, 67]. Since novel ORFs may be relevant to CMS, we analyzed all the predicted ORFs about their origin, conservation, function and expression. We compared all ORFs of 2074A and 2074S with that of the maintainer line 2074B, we observed 28 and 30 novel ORFs in 2074A and 2074S, respectively (Tables 6 and 7). The ORFs of 2074A were named as Aorf1 to Aorf28, and Aorf4 was duplicated in 2074A; while, those of 2074S were named as Sorf1 to Sorf30, and Sorf4 also was duplicated in 2074S. 11 of the ORFs are common in 2074A and 2074S. The length of polymorphisms in ORFs was frequently caused by frame shift mutations with several nucleotides’ insertions/deletions.
Table 6

Chimeric ORFs (> 300 bp) presented in 2074A mitogenomes

2074 AORF

Start

End

Strand

Length

(bp)

2074B

E5903

Tra-domc

Uni/R-seqd

Homologous sequencee

RNA-Seq Log2

2074B/2074A

F1-A/2074A

Aorf1

3872

4483

+

612

-a

*b

1

U1

No homologous sequence

−11.76

2.73

Aorf2

4461

3700

762

*

6

U1

583–759, 86%, papaya mtDNA

−12.58

2.62

Aorf3

5180

4878

303

*

0

U1

154–301, 96%, papaya mtDNA

− 14.54

2.52

Aorf4

27,402

26,845

558

Some

some

1

AR4(524)

45 bp, rps3(1–45)f; 47 bp sdh3

−0.22

2.92

atp8

28,431

27,967

463

*

*

   

−0.35

2.56

Aorf5

86,768

87,067

+

300

0

 

98%, other plant mtDNA

−11.92

3.33

Aorf6

143,074

142,757

318

Some

0

 

1–301, 95%, Phoenix ductylifera mtDNA

0.00

12.63

Aorf7

144,192

144,596

+

405

*

0

U3

No homologous sequence

0.00

9.96

Aorf8

149,251

148,952

300

*

0

U3

4–242, 94%, Beta vulgaris mtDNA

−13.47

2.25

rrn26

185,940

189,313

+

3374

*

*

   

1.26

3.17

Aorf9

189,332

189,688

+

357

Some

some

1

AR1(7125)

76 bp, nad7

−0.70

2.41

Aorf10

258,310

257,840

471

Some

some

1

up AR2 93 bp

4–424, 92%, Ricinus communis mtDNA

− 0.25

2.66

Aorf11

314,879

315,265

+

387

Some

some

0

 

43 bp, nad7

−1.00

2.63

Aorf12

323,440

323,838

+

399

Some

some

0

 

No homologous sequence

−12.06

2.01

rps14

324,431

324,751

+

321

*

*

   

−1.22

2.09

cob

326,094

327,256

+

1163

*

*

   

−0.91

3.37

Aorf13

335,286

334,948

339

Some

some

0

 

No homologous sequence

1.26

3.17

Aorf14

343,549

343,223

327

0

 

36 bp, cox2ex1

−1.25

3.38

rpl2

346,637

347,641

+

1005

*

*

   

−0.27

2.05

Aorf4–2

348,085

348,642

+

558

Some

some

1

AR4(524)

45 bp, rps3; 47 bp, sdh3

−0.22

2.92

Aorf15h

388,830

388,447

384

Some

some

1

 

No homologous sequence

−1.08

2.79

Aorf16

397,394

397,735

+

342

0

 

No homologous sequence

−1.64

3.41

Aorf17

415,813

415,265

549

Some

some

0

up AR2 23 bp

39–527, 94%, Citrullus lanatus mtDNA

−11.92

3.33

Aorf18

449,399

449,001

399

some

0

U5

123–393, 82%, tobacco mtDNA

−11.06

4.57

Aorf19

452,355

453,074

+

720

*

2

U5

No homologous sequence

0.00

15.49

Aorf20

452,473

452,781

+

309

*

1

U5

No homologous sequence

0.00

13.35

Aorf21

454,116

453,781

336

*

0

U5

93%, Mimulus guttatus mtDNA

0.00

14.48

Aorf22

454,900

454,451

450

*

0

U5

96%, Mimulus guttatus mtDNA

−10.88

3.10

Aorf23

465,398

465,751

+

354

Some

some

0

 

167–331, other plant mtDNA

0.09

2.15

Aorf24

490,816

491,292

+

477

2

 

64 bp, rps4

−2.19

3.93

Aorf25g

491,321

491,689

+

369

Some

*

0

 

20 bp, ccmFC

−3.15

4.83

Aorf26

508,562

507,753

810

Some

some

0

 

306–479, other plant mtDNA

1.45

5.21

cox1

631,928

633,518

+

1591

*

*

   

−0.32

1.86

Aorf28

633,740

634,606

+

867

Some

0

up AR1 1760 bp

56 bp, atp4(1–56)f

−1.47

3.02

cox3

634,937

635,734

+

798

*

*

   

−0.03

2.69

Aorf27

665,520

666,155

+

636

*

0

AR1, U6

No homologous sequence

−4.71

0.35

Note. –ano detected; *bhave this ORF; cTra-dom: transmembrane domain; dUni/R-seq: unique sequence or repeat sequence; eHomologous sequence contains the sequence of genes in cotton and mitochondrial sequences of other plants; f Aorf4 contain a fragment that is 1-45 bp of rps3, Aorf28 contain a fragment that was 1-56 bp of atp4, identity is 100%; gAorf25 is in upstream 70 bp of nad5ex4; hthe end of Aorf15 is longer 81 bp than Sorf14

Table 7

Chimeric ORFs (> 300 bp) presented in 2074S mitogenomes

2074S

Length (bp)

Tra-domc

Uni/Rep-seqd

2074B

E5903

Location

Homologous sequencee

Sorf25

660

-a

 

*b

down atp4 2192 bp

19 bp, ccmFN

Sorf16

381

1

U5

 

157 bp, rps3ex2

Sorf1

612

1

U1

*

 

No homologous sequence

Sorf26

810

 

partial

Partial

 

306–479, other plant mtDNA

Sorf21

450

U5

*

 

96%, Mimulus guttatus mtDNA

Sorf20

336

U5

*

 

93%, Mimulus guttatus mtDNA

Sorf17

399

U5

partial e

 

123–393, 82%, tobacco mtDNA

Sorf15

549

up SR2

partial

Partial

 

39–527, 94%, Citrullus lanatus mtDNA

Sorf7

405

U3

*

 

No homologous sequence

Sorf14

315

1

 

partial

Partial

 

No homologous sequence

Sorf13

327

 

 

36 bp, cox2ex1

Sorf12

339

 

partial

Partial

 

No homologous sequence

Sorf9

471

1

up SR2

partial

Partial

 

4–424, 92%, Ricinus communis mtDNA

Sorf6

318

 

partial

 

1–301, 95%, Phoenix ductylifera mtDNA

Sorf8

357

1

SR1

partial

Partial

down rrn26 19 bp

76 bp, nad7

Sorf4

558

1

SR4

partial

Partial

down atp8 565 bp

45 bp, rps3(1–45)f; 47 bp sdh3

Sorf4–2

558

1

SR4

partial

Partial

up rpl2 444 bp

45 bp, rps3; 47 bp, sdh3

Sorf3

303

U1

*

 

154–301, 96%, papaya mtDNA

Sorf2

762

6

U1

*

 

583–759, 86%, papaya mtDNA

Sorf10

387

 

partial

Partial

 

43 bp, nad7

Sorf11

399

 

partial

Partial

 

No homologous sequence

Sorf19

309

1

U5

*

 

No homologous sequence

Sorf18

720

2

U5

*

 

No homologous sequence

Sorf5

300

 

 

98%, other plant mtDNA

Sorf22

354

 

partial

Partial

 

167–331, other plant mtDNA

Sorf23

477

2

 

 

64 bp, rps4

Sorf24

369

 

partial

*

up nad5ex4 91bpg

20 bp,ccmFC

Sorf29

867

up SR1

partial

down cox1 222 bp, up cox3 331 bp

56 bp, atp4(1–56)f

Sorf30

636

SR1, U6

*

 

No homologous sequence

Sorf28

414

 

partial

Partial

 

No homologous sequence

Sorf27

951

3

 

partial

Partial

down rrn5 46 bp

No homologous sequence

Note.–ano detected; *bhave this ORF; cTra-dom: transmembrane domain; dUni/R-seq: unique sequence or repeat sequence; eHomologous sequence contains the sequence of genes in cotton and mitochondrial sequences of other plants. fThe similarity between 1–45bp in Sorf4 and 1–45bp in rps3, 1–56bp in Sorf29 and 1–56bp is 100%; g nad5ex4 is located at 91bp upstream of Sorf24

We categorized the specific ORFs into three basic groups: I) ORFs near the functional genes, which is transcribed in the same direction with adjacent positioned genes either up or down stream, and could be co-transcript relevant to CMS (Aorf4, Aorf25, Aorf27, Aorf28, Sorf4, Sorf8, Sorf14, Sorf27 and Sorf28); 2) Special ORFs in unique regions of sterile lines, which always have short-sequences homology to chloroplast or mitochondrial sequences of other plants; such as Aorf2, Aorf18, Sorf15, Sorf16 and Sorf2 that were found in unique sequences of two sterile lines. Mostly, they are similar to chloroplast or mitochondrial sequences of other plants, or have no homology sequences in NCBI-NR database. In the third group, the ORFs are comprised of homologous sequences of 2074B and unique sequences such as Aorf14, Sorf13 and Sorf14.

To further verify whether these ORFs were functionally associated with CMS, we profiled the expression of mitochondrial genes and ORF’s based on RNA-seq data of flower buds (3–5 mm in size) in CMS 2074A, maintainer 2074B, and the fertility-restored F1 (2074A × E5903). Among all the three lines, the expression of mitochondrial genes was highest in F1 and lowest in 2074B (Fig. 3, P < 0.05). The expression levels of shd3 and rpl10 genes were higher in 207A than in 2074B (Fig. 3, P < 0.05). Taking the sequences of 28 predicted ORFs in 2074A as a pool; we used Blastn to match all three-transcript data (2074A, 2074B and F1). As a result, 10 ORFs were expressed at high levels (10 fold) as compared to the similar sequence (with 1–3 gap) in 2074B; five ORFs were expressed at high levels as compared to the similar sequence in F1; the five ORFs were not expressed in 2074B (Additional file 9: Figure S3). Based on the first group principle, the ORFs near to functional genes, we found that four pairs of ORFs and their nearby genes (Aorf4 and atp8, Aorf9 and rrn26, Aorf4–2 and rpl2, Aorf28 and cox1/cox3) have same expression trend both in 2074B/2074A and F1/2074A, therefore, these four ORFs might be co-transcribed with functional genes and relevant to CMS.
Fig. 3
Fig. 3

Differential expression of mt genes in 2074A, 2074B and F1-A. Log2 transformations of the expression fold changes (2074B/2074A and F1-A/2074A) are represented by bars. Y axis denotes the levels of transformed expression fold changes

Furthermore, we analyzed 16 reported CMS-associated ORFs. We found that these ORFs (78–488 bp) are near to co-transcribed genes and form a bicistronic complex with many functional genes. In this context, six ORFs in CMS2074S (Sorf25, Sorf4, Sorf4–2, Sorf29, Sorf8 and Sorf27) and five ORFs in CMS2074A (Aorf12, Aorf4, Aorf4–2, Aorf28 and Aorf9) were close to functional genes within 565 bp, and six (Sorf4, Sorf29, Sorf8, Aorf4, Aorf28 and Aorf9) of them are the products of rearrangements by large repeats. Additionally, these ORFs have transmembrane domain (except Aorf28, Table 5, Fig. 4) and same expression trend with their nearby genes. More important, four ORFs (Aorf4, Aorf28, Aorf9 and Aorf4–2) and their functional genes (atp8, cox1, cox3, rrn26 and rpl2) might have higher expression in CMS-2074A compared to F1. Aorf4 (561 bp) is found at the downstream 565 bp of atp8. Besides, the first 45 bp of Aorf4 are derived from rps3, while other partial sequences are identical to sdh3 (the 5′-end of orfH79 has 84 bp homology to cox1) and have same expression trends with atp8 in 2074B/2074A (− 0.3) and F1-A/2074A (2.6~ 2.9). Aorf4–2 (561 bp) is found in the downstream 444 bp of rpl2 and have same expression trends with rpl2 in 2074B/2074A (− 0.2~ 0.5) and F1-A/2074A (2.9~ 3.2). Aorf28 (867 bp), located at the downstream 241 bp of cox1 and the upstream 311 bp of cox3 (331 bp in 2074S), shows 66% identity with Arabidopsis mitogenome and is close to AR1. In addition, the expression trend of Aorf28, cox1 and cox3 were same. Aorf9 (357 bp), located at the downstream 19 bp of rrn26, keeps same expression trends with rrn26 in 2074B/2074A (− 0.7) and F1-A/2074A (2.4); as well, Aorf9 also has 76 bp identity with nad7 and 89% identity with Ricinus mitogenome. These four ORFs show the characters of CMS-associated genes and are similar to other ORFs, such as T-urf13 of maize [14], S-orf355/orf77 [66], orf224 of rape [8, 6870], orf256 of wheat [15, 71], orf125 of radish [72], etc. All above chimeric ORFs from other plants are always near and co-transcribed with functional genes, which makes functional genes transcribe improperly and causes abortion [7376]. As to now, these results were only based on the genome and RNA-seq data, more experiments, including functional validation of overexpression or CRISPR/Cas9 these orfs, are needed to confirm the real CMS gene of upland cotton.
Fig. 4
Fig. 4

The probability of transmembrane domains of Aorf4, Aorf9, Aorf2 and Aorf28 gene products

Conclusions

The two almost identical male sterile lines, 2074A and 2074S, share high identity with the restore line E5903 but are different from their maintainer line 2074B, especially in non-coding regions. The cotton mtDNAs are 621,884–668,584 bp in length, and harbor 36 known protein-coding genes, three rRNAs (18S, 26S, and 5S rRNAs) as well as 18 different tRNAs. The rates of the coding genes (including duplicated genes) accounting for the total genomes’ length are almost similar, but the repeat sequences show a few differences. In addition, five genes (rps1, rps2, rps13, rps19 and sdh2) have been lost and 38 nonsynonymous mutations occurred in 21 protein-coding genes, though they are functionally irrelevant. Out of 28 ORFs in CMS 2074A, four ORFs (Aorf4, Aorf9, Aorf4–2 and Aorf28) are close to the functional genes and show similar characters to CMS-associated genes in other plants. These four ORFs may be the potential candidates conferring CMS in cotton.

Abbreviations

CMS: 

Cytoplasmic male sterility

cp: 

Chloroplast

G.: 

Gossypium

MC: 

Main cycle

mitogenome: 

Mitochondrial genome

mtDNA: 

Mitochondrial DNA

NGS: 

Next-generation sequencing technology

ORFs: 

Open reading frames

rRNAs: 

Ribosomal RNAs

tRNAs: 

Transfer RNAs

Declarations

Acknowledgments

We deeply thank Dr. Yi Huang (Institute of Oil Crops, Chinese Academy of Agricultural Sciences) and Corrinne E. Grover (Iowa State University) for their valuable comments and discussion on previous versions of the manuscript. We are also grateful to anonymous reviewers for their helpful suggestions and comments.

Funding

This work was supported by grants from the National Natural Science Foundation of China (31671741) and National Key R & D Program for Crop Breeding (2016YFD0100203). The funders did not have any role in the design, collection, analysis, and interpretation of data and in writing the manuscript.

Availability of data and materials

Complete mtDNA sequences were deposited in GenBank database (JX944505.1 for CMS-2074S, JX536494.1 for CMS-2074A, and JX944506.1 for E5903). Other data sets supporting the results of this article are included within the article and its additional files.

Authors’ contributions

SSL assembled the mitochondrial genome, annotated the mitochondrial genomes, performed the data analysis and prepared the original manuscript. ZWC performed the transcriptomes data analyses, attended discussion and revised MS. NZ critically revised MS and attended data re-interpreting discussion. YMW maintained the experimental platform and participated in the bench work. HSN attended the transcriptomes data analyses and discussion. JPH designed the experiments, provided research platform, and revised the manuscript. All authors approved the final manuscript.

Ethics approval and consent to participate

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Competing interests

The authors declare that they have no competing interests.

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Authors’ Affiliations

(1)
Laboratory of Cotton Genetics, Genomics and Breeding/Key Laboratory of Crop Heterosis and Utilization of Ministry of Education/Beijing Key Laboratory of Crop Genetic Improvement, College of Agronomy and Biotechnology, China Agricultural University, Beijing, 100193, China
(2)
Institute of Cash Crops, Hubei Academy of Agricultural Sciences, Wuhan, 430064, Hubei, China

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