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Article

Comparative Analysis of Mitochondrial Genomes between the B-Type Cytoplasmic Male Sterility Line and Its Maintainer Line in Wheat

by
Ying Li
1,2,3,4,5,
Qilu Song
1,2,3,4,5,
Jialin Guo
1,2,3,4,5,
Yulong Song
1,2,3,4,5,
Xinhong Chen
1,2,3,4,5,* and
Gaisheng Zhang
1,2,3,4,5,*
1
College of Agronomy, Northwest A & F University, Yangling 712100, China
2
National Yangling Agricultural Biotechnology & Breeding Center, Yangling 712100, China
3
Yangling Branch of State Wheat Improvement Centre, Yangling 712100, China
4
Wheat Breeding Engineering Research Center, Ministry of Education, Yangling 712100, China
5
Key Laboratory of Crop Heterosis of Shaanxi Province, Yangling 712100, China
*
Authors to whom correspondence should be addressed.
Agronomy 2022, 12(4), 851; https://doi.org/10.3390/agronomy12040851
Submission received: 21 February 2022 / Revised: 18 March 2022 / Accepted: 28 March 2022 / Published: 30 March 2022
Browse Figures
Versions Notes

Abstract

:
Cytoplasmic male sterility (CMS) is a complex phenomenon in plants, rendering them unable to produce functional pollen. In general, this is caused by an abnormal or dysfunctional mitochondrial genome. In wheat, however, the systematic structural characteristics of the mitochondrial genome from the CMS line, vis-à-vis its maintainer line, are rarely reported. Here, we identified the morphological characteristics, sequenced, assembled, and characterized the complete mitogenomes of the wheat B-type CMS line (B) and its maintainer line (YS9). The morphological results indicated that the B likely undergoes binucleate microspore abortion. The B and YS9 genomes were assembled into a typical circular molecule 452,794 and 452,453 bp in length, respectively, comprising 34 protein-coding genes (PCGs), 3 ribosomal RNA genes (rRNAs), and 16 transfer RNA genes (tRNAs). The codon usage analysis revealed leucine (Leu) and serine (Ser) as the most frequently used amino acid residues in the B and YS9 mitochondrial proteins. In particular, we uncovered a specific ORF2718, whose length of 501 bp was more 30 bp than that of the atp8 gene in the B genome, which perhaps could affect normal function of ATP8. Further, the existence of SNPs at the atp6 gene is probably associated with the CMS mechanism. This study suggests that sequencing and comparing the genomic features of the B and YS9 mitogenomes provides not only an important opportunity to conduct further genomic breeding studies, but also valuable information for future evolutionary and molecular studies of CMS in wheat.

1. Introduction

Wheat (Triticum aestivum L.) is one of the most widely planted crops in the world, with a production area of 216.03 million hectares in 2019, providing most of humanity’s food sources. With both its population and urbanization rapidly expanding, China’s production of wheat lags far behind the demand for it. In addition, the planting area for this crop is also shrinking and the rural labor force is in decline, leaving the prospect of future wheat production a matter of major concern [1,2]. In order to safeguard the supply of grain and ensure food security, it is imperative we improve the yield of wheat. Although enhancing disease resistance, stress resistance, and cultivation measures can raise the quality and yield of crops by a small margin, increasing the yield in a sustainable and efficient way is essential for meeting the growing global demand for wheat-based foods.
Hybrid vigor (or heterosis) is a common phenomenon in the biological world. Since the 1940s, hybrid maize (Zea mays L.) has been widely used in crop production and provided many remarkable benefits. This has spurred many researchers to study and apply heterosis in crop production on large scales. Currently, heterosis is widely used in rice (Oryza sativa L.), rape (Brassica campestris L.), cotton (Gossypium spp.), and other major plant crops, generating great social and economic benefits for the world [3,4]. In wheat, the phenomenon of heterosis was first reported in 1919. Since then, research and production practices have shown that wheat heterosis can be pronounced, not unlike that of corn, rice, and other crops. The yield of wheat, for example, can be increased by 3.5–15% through heterosis [5]. Therefore, the utilization of heterosis is an important direction and approach for achieving breakthroughs in wheat yield [6]. Yet, at present, applications of heterosis mainly rely on cytoplasmic male sterility in wheat.
Cytoplasmic male sterility (CMS) is a maternally inherited trait in which plants develop normally, but they fail to produce any functional pollen. The trait is widespread in flowering plants, with more than 140 species known to exhibit CMS [7]. These include maize, sunflower, sorghum, common beans, cotton, rice, pigeonpea, pepper, and wheat [8,9,10,11,12]. The CMS phenomenon provides a convenient model to study nuclear–cytoplasmic interactions, but its molecular mechanism is very complex [13,14]. In general, the mitochondrial genome serves as the carrier of CMS in plants [15].
Mitochondria (mt) are semi-autonomous organelles that are most likely derived from formerly free-living bacteria (α-proteobacteria) via endosymbiosis, which would explain why they have their own genome, the mtDNA [16,17,18]. With the rapid development in sequencing and genome assembly methods, more and more complete mitochondrial genomes have been assembled. By December 2021, 423 plant mitochondrial genomes had been collected in the NCBI. The plant mitochondrial genome is highly variable in size and extremely complex [19,20,21]. Its size ranges from the smallest (221 kb) for Brassica napus to the largest (11.3 Mb) for Silene conica [22,23]. Moreover, it also has numerous repetitive sequences, frequent gene recombinations or losses, and multiple RNA editing modifications [22,24,25,26,27,28].
Although plant mitochondrial genomes vary greatly in size, there are only about 20 additional genes in comparison with those of animals and yeasts [23]. For instance, Arabidopsis thaliana only has a 367 kb mtDNA that encodes 32 protein-coding genes (PCGs), 22 transfer RNA genes (tRNAs), and 3 ribosomal RNA genes (rRNAs) [23,29]. An explanation for this could be that the vast majority of mtDNA is composed of non-coding sequences from plastid and nuclear genomes and, in rare cases, from other plant species [30,31]. Moreover, plant mtDNAs are also shaped by multiple structural changes, including rearrangements and recombination events, which lead to the formation of novel open reading frames (ORFs) [13,32,33,34,35]. These ORFs are usually chimeric structures consisting of known mitochondrial genes and unknown repetitive sequences. According to previous studies, the accumulation of novel chimeric ORFs typically interferes with the functioning of the mitochondrion or is toxic for plant cells, which usually leads to the abnormal development of pollen grains, and ultimately CMS [13,36,37]. In wheat, a chimeric ORF was associated with CMS, and this orf256 gene region is composed of 261 bp of the cox1 gene region combined with a DNA fragment of unknown origin. The former part consists of 228 bp of the 5′ flanking regions and 33 bp of the encoded N-terminal region; hence, the first 11 amino acids encoded by orf256 are the same as those encoded by cox1 [38]. The molecular basis of CMS has been studied in rice. The chimeric orfH79 from CMS-HL is a variant of orf79 from CMS-BT that encodes proteins with a 5′ region similar to cox1 and a 3′ region of unknown origin [39,40]. For the rice RT98A CMS line, its orf113 has the same sequence as nad9 in the region from –151 to +11, while the rest is composed of unknown sequences [41].
CMS is a complex phenomenon of plant sterility that is caused by mutation, rearrangement, or recombination events in the mitochondrial genome. Availability of wheat’s mitochondrial genome (or mitogenome) is also important for practical applications, and the characterization of mitogenomes in CMS lines can facilitate the search and identification of events presumably responsible for the sterility affecting many agricultural plants [42,43,44,45,46]. Yet, due to the complexity of plant mitogenomes, to date, reporting on the systematic structural characteristics of wheat mitochondrial genomes has been restricted to those from T. aestivum, as well as T. aestivum cv. Chinese Yumai K-type cytoplasmic male sterility line and its maintainer line [47,48]. By contrast, possible molecular causes for the CMS in the wheat B-type CMS line remain under study; hence, its potential relationship must be explored. To investigate the CMS mechanism more systematically, here we sequenced and assembled the complete mitogenomes of the wheat B-type CMS line and its maintainer line. Furthermore, we determined and compared their complete nucleotide sequences and organization, and also clarified the relationship between the B-type CMS line and its maintainer line (YS9) based on analyses of phylogeny, synteny, repeated sequences, and single nucleotide polymorphisms (SNPs). The complete mitogenomes of the B and YS9 will provide crucial and timely information for the investigation of mitogenomic evolution, functional research of the CMS mechanism, and the screening of CMS-associated genes, while also providing an opportunity to carry out further important genomic breeding studies in wheat crops.

2. Materials and Methods

2.1. Plant Materials

The wheat B-type CMS line (B) and its maintainer line, Yanshi 9 Hao (YS9), were used in this study. Both lines have the same nuclear background. The cultivars were bred successively for more than 20 consecutive generations, so that their CMS lines were stable. Their seeds were disinfected with 5% sodium hypochlorite for 10 min, washed with distilled water, and soaked for 12 h at room temperature. The exposed seeds were spread on aseptic filter paper and cultured in the dark, at room temperature, for 7–10 days. Their fresh etiolated leaves were collected and frozen in liquid nitrogen and stored at −80 °C for later mitochondrial analysis. In addition, the wheat B and YS9 was grown in the experimental field (34°16′ N, 108°4′ E) of the Northwest A&F University, in Yangling, Shaanxi Province, China.

2.2. Phenotypic Characterization and Pollen Analysis of Anthers

Wheat anthers were visualized under a Motic K400 dissecting microscope (Preiser Scientific, Louisville, KY, USA) and digitally photographed using an Olympus SZX16 stereo microscope (Tokyo, Japan) at various stages of development. Different stages of pollen grain were identified by staining with 4′,6-diamidino-2-phenylindole (DAPI; Sigma-Aldrich, Oakville, ON, Canada) and iodine–potassium iodide (I2–KI). Samples were photographed using an ICc5 color, 503 mono high-resolution camera mounted on an Imager M2 fluorescence microscope (Imager M2, ZEISS, Oberkochen, Germany) and analyzed with Elements software (ZEISS, Oberkochen, Germany).

2.3. DNA Extraction, Library Construction, and Sequencing

Genomic DNA was extracted via the CTAB method [49]. Data processing, quality control/checking, and filtering were carried out before constructing the small DNA fragment library. High-throughput sequencing was conducted on the Illumina Hi-Seq XTen platform after the library was qualified, from which a total number of 77,679,566 paired-end reads (150 bp) were generated and used for mitochondrial genome assembly.

2.4. Mitochondrial Genome Assembly

The mitochondrial genome (GenBank number: EU534409) served as the reference sequence, onto which our original sequencing reads were mapped to obtain the preliminary assembly results. For some sites with poor conservation (i.e., weak matching), their flanking conserved region was selected as the starting reference sequence, and these sequences extended by the MITObim v1.9 tool [50]. These extension products were then aligned with the above preliminary assembly results by pairwise alignment, to modify the assembly results. Following the above assembly steps, we obtained the master circles of the B and YS9 in wheat and drew corresponding circular mitogenome maps with the online tool OGDraw (https://chlorobox.mpimp-golm.mpg.de/OGDraw.html, accessed on 18 April 2021) [51].

2.5. Annotation and Analysis of the Mitogenomes of the CMS Line and Its Maintainer Line

The mitogenomes were annotated mainly by homology alignments with the reference mitochondrial genome, with the parameter: evalue = 1e−10. We identified the tRNA genes, introns, and ORFs in the B and YS9 mitogenomes. The tRNA and rRNA genes were, respectively, identified by the tRNAscan-SE 2.0 web server (http://trna.ucsc.edu/tRNAscan-SE/, accessed on 21 April 2021) and rRNAmmer 1.2 tools [52,53]. The introns were detected with the RNAweasel tool [54]. For the potential ORFs >100 bp, they were predicted, according to the standard genetic code, by the ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder, accessed on 23 April 2021). The start and stop codons of protein coding genes (PCGs) were manually adjusted to fit the ORFs. Both the relative synonymous codon usage (RSCU) values and amino acid composition of PCGs were calculated in MEGA X software [55].

2.6. Alignment and Phylogenetic Analysis

The mitogenomes of five higher plants (T. aestivum (CS): AP008982, T. aestivum cv. Chinese Yumai Ks3: GU985444, T. aestivum cv. Chinese Yumai: EU534409, T. timopheevii: AP013106, and Aegilops speltoides: AP013107) were extracted from NCBI database (https://www.ncbi.nlm.nih.gov/genome/browse/, accessed on 6 May 2021). Next, MAFFT software was used to implement the multiple sequence alignment between those sequences and our B and YS9 mitochondrial sequences to obtain the consistent sequences [56]. Finally, the evolutionary divergence time was estimated using MCMCtreeR with these parameters: the replacement model: HKY85; evolution rate: rgene_gamma = 2 2000 1; variance of the evolution rate: sigma2_gamma = 1 1000 1; MCMC (Markov chain Monte Carlo) parameters: burnin = 2000, sampfreq = 10, nsample = 20,000 [57], and the fossil-based, time-calibrated phylogenetic tree was constructed.

2.7. Synteny Analysis of the CMS Line and Its Maintainer Line

Synteny of the mitochondrial genome is defined as the phenomenon distinguished by homologous genes and sequences of differing mitogenomes being arranged in the same order. The extent of synteny between two mitochondrial genomes could be used to measure the evolutionary distance and genetic relationship between the species. Using Mauve [58], we compared the CMS line and maintainer line, and their wide homologous regions and SNP sites were also determined. Then, using BLAST+ [59], the sequences of both lines were further compared under two set parameters (-evalue = 1e−10 and -num_alignments = 5), to refine their wide homologous regions, Next, Perl software was used to interrupt the comparison results, according to the gap, and these interrupted comparison results were imported into Circos software to draw the circos diagram [60].

2.8. Identification of Repeated Sequences

The vast majority of variance in the size of plant mitogenomes can be explained by differences in the sizes of their repeat sequences. The repeat sequences of the B and YS9 mitochondrial genomes were searched for using the REPuter tool (https://bibiserv.cebitec.uni-bielefeld.de/reputer, accessed on 18 May 2021) with these parameters: Hamming distance = 3; minimal repeat size = 30 [61]. Following that, simple sequence repeats (SSRs) or tandem repeats were found. Specifically, the SSRs were detected using a web-based microsatellite identification tool, MISA-web (https://webblast.ipk-gatersleben.de/misa/, accessed on 18 May 2021) [62], according to previously reported methods entailing a motif size of 1–6 nucleotides with thresholds of 10, 5, 4, 3, 3, and 3, respectively. The tandem repeats were identified using the online tool Tandem Repeats Finder v4.09 under its default settings (http://tandem.bu.edu/trf/trf.html, accessed on 18 May 2021) [63].

3. Results and Discussions

3.1. Observation of Phenotype and Pollen Defects during Anther Development of the B and YS9 Lines

Wheat anther development was divided into five stages—the tetrad, the early-uninucleate, the later-uninucleate, the binucleate, and the trinucleate—based on a previous classification of anther development [64]. To identify the morphological differences of anthers during their development, we compared the phenotype of B and YS9 anthers at all five stages (Figure 1). We found no marked difference between B and YS9 anthers from the tetrad stage through binucleate stage; however, the B anthers did not undergo anther dehiscence and failed to produce normal and fertile pollen at the trinuclear stage (Figure 1E,K,L). Moreover, at the binucleate and trinucleate stages, the anthers of B were slightly smaller and thinner than those of YS9 (Figure 1F,L).
To gain a more detailed understanding of the defects in B plant anthers and when they arise, we observed the microspore chromosome behavior at different developmental stages via DAPI staining (Figure 2A–L). These results revealed that its microsporocyte nuclei appeared rather distinct, having chromosomes that became highly contracted and which exhibited the DAPI stain brightly (Figure 2A,G). The microsporocytes of B and YS9 appeared to have developed normally and undergone meiosis to generate the tetrads of haploid microspores (Figure 2B,H). In the subsequent stage, the microspores of YS9 proceeded through two rounds of mitosis, to develop into mature tricellular pollen grains in going from the early-uninucleate to the trinucleate stage, and their chromosomes exhibited brighter DAPI staining (Figure 2B–F). By contrast, in the B-type CMS line, it was clearly apparent that the microspores were abnormal from the later-uninucleate through to the trinucleate stage; the nucleus failed to divide normally into two nuclei to produce mature fertile pollen grains; microspores were irregular in shape; and plasmolysis occurred (Figure 2J–L). More importantly, staining of pollen grains with iodine–potassium iodide (2% I2–KI) indicated that the pollen grains of YS9 underwent pronounced starch accumulation, whereas those of B could not be deeply stained and were almost entirely starch-deprived (Figure 2M–T). Further, the YS9 pollen grains displayed a smooth nearly round shape (Figure 2M–P), whereas the B pollen grains were severely malformed (Figure 2Q–T). These observations were consistent with the DAPI staining results and demonstrated that the anthers and pollen grains of B probably incurred their defective development during the transition from the later-uninucleate to the binucleate stage. Yet the B plant pistils showed normal development (Figure 1D,E) and were able to produce normal seeds when backcrossed with fertile pollen. Many early studies have shown that pollen abortion is key for inducing male sterility and that male sterility can manifest at different developmental stages [7]. Our findings thus suggest the B-type CMS line likely belongs to a binucleate microspore abortion type. Further research is needed, however, to gain an in-depth understanding of the processes affecting male sterility.

3.2. Comparative Analysis of Features and Gene Contents in the B and YS9 Mitogenomes

We assembled the complete mitogenomes of the B and YS9 into the typical circular molecule, these being 452,794 bp and 452,453 bp in length, respectively (Figure 3, Table 1). The genome of B was only 341 bp longer than that of YS9, and both were similar to the mitochondria of T. aestivum cv. Chinese Spring (CS, 452,528 bp), T. aestivum cv. Chinese Yumai (452,526 bp), Aegilops speltoides var. ligustica (476,091 bp), and T. timopheevii (443,419 bp) (Table 1). Because of its complex structure, it is difficult to assemble a complete mitochondrial genome. Therefore, the mitochondrial genome of the same species is typically used as the reference sequence in further splicing during the assembly process, which may be the reason why there is a small difference between the sequences of B and YS9. In fact, the mitogenome size can vary greatly in different cultivars of the same species; for example, that of T. aestivum cv. Chinese Yumai Ks3 is 647,559 bp, this being significantly larger than its corresponding maintainer line (Yumai), whose mitogenome is about 195 kb smaller.
Mitochondrial genes are relatively conserved in the same or similar species. We uncovered a total of 53 unique genes in the two mitochondrial genomes assembled, comprising 34 PCGs, 3 rRNAs, and 16 tRNAs (Table 2 and Supplementary Table S1). The total lengths of the PCGs accounted for 7.45% and 7.39% of the B and YS9 whole mitogenomes, respectively, being similar to CS (7.99%), Yumai Ks3 (6.70%), Yumai (7.39%), Aegilops speltoides (7.49%), and T. timopheevii (7.98%). Most PCGs had no introns; however, eight genes (ccmFC, cox2, nad1, nad2, nad4, nad5, nad7, and rps3) were found to contain one or more introns, of which three (cox2, nad2, and rps3) required trans-splicing to assemble fully-translatable mRNA.
The nucleotide composition of the B whole mitogenome was A: 27.9%, T: 22.8%, G: 22.2%, C: 22.1%. The overall GC content was 44.3%, the same as for YS9’s mitogenome. When compared with mitochondrial genomes of CS, Yumai Ks3, Yumai, T. timopheevii, and Aegilops speltoides, except for Aegilops speltoides, the results were basically the same. Nevertheless, we did uncover some discrepancies in the nucleotide composition between the B and YS9 protein-coding sequences (CDS), and their GC content was lower than that of their whole genome (Table 1). The GC skew was generally used to measure the excesses of G relative to C, which were given by (G − C)/(G + C) [65,66]. If G > C, the value of GC-skew is positive, otherwise it is negative. The data displayed that the value of GC-skew ranged from −0.0045 to 0.0189 in the mitochondrial genomes and protein-coding sequences of the B and YS9. Moreover, a majority of these values were positive, with only two being negative, thus indicating a generally higher relative content of G than C. In addition, the results showed that, in general, the GC-skew was more pronounced than AT-skew.

3.3. Prediction of the Open Reading Frames (ORFs)

Mounting evidence suggests CMS is caused by chimeric genes arising from mitochondrial genome rearrangement. In comparing the mitogenomes of CMS materials with those of normal fertile materials, researchers have found differences not only in the size of genomes, but also in the high degree of rearrangement between sequences, resulting in the production of some novel ORFs, with some candidate genes possibly related to CMS identified, such as orf224 and orf222 (Brassica campestris), orf79 (Oryza sativa), orf256 (T. aestivum), and so forth [39,67,68]. Therefore, it is very important to further study these specific ORFs. In our study, the ORF Finder tool predicted 4344 and 4332 ORFs whose length was greater than 100 bp in the B and YS9 mitochondrial genomes, respectively (Supplementary Table S2). Most of these ORFs were identical, however, with few differing in sequence length. Further, 89 and 83 specific ORFs existed separately in the B and YS9 lines, and could be explored as candidate genes for CMS. Interestingly and importantly, we identified the ORF4906 in the YS mitogenome, whose length was 471 bp, which was annotated as an ATP synthase subunit 8 gene. However, in the mitogenome of the B-CMS line, a specific ORF2718 with a length of 501 bp was found, harboring in the upstream region 30 bp more than the normal atp8 gene does. The protein encoded by the atp8 gene is a critical part of the F0 subunit of ATP synthase, the latter is a key enzyme of mitochondrial oxidative phosphorylation for which functional defects will lead to energy metabolism disorders [69,70]. Accordingly, we speculate that the specific ORF2718 may affect the normal function of ATP8, and this may be closely related to CMS. We plan to further study its transcription and function to explore the abortion mechanism of the B-type male sterility line.

3.4. Codon Usage and Relative Synonymous Codon Usage (RSCU) Analysis of PCGs

In the B and YS9 mitogenomes, most of the PCGs use ATG as the start codon, while nad1 and nad4L start with ACG, and matR and rps19-p start with AGA (Supplementary Table S1). Six types of stop codons were found in the PCGs: (1) TAA (12 genes: ccmFC, cox1, cox2, nad2, nad3, nad4L, nad9, rpl16, rpl5, rps4, rps7, and rps19-p in the B mitogenome; 11 genes: cox1, cox2, nad2, nad3, nad4L, nad9, rpl16, rpl5, rps4, rps7, and rps19-p in the YS9 mitogenome); (2) TAG (10 genes: atp4, ccmC, ccmFN, cob, matR, mttB, nad6, nad7, rps2, and rps3 in both B and YS9 mitogenomes); (3) TGA (12 genes: atp1, atp6-1, atp6-2, atp8-1, atp8-2, atp9, ccmB, cox3, nad4, rps1, rps12, and rp13 in both B and YS9 mitogenomes); (4) ATA (1 gene: nad5 in both B and YS9 mitogenomes); (5) CCG (1 gene: nad1 in both B and YS9 mitogenome); and (6) CGA (1 gene: ccmFC in the YS9 but not B mitogenome) (Supplementary Table S1). As evidenced by Figure 4 and Supplementary Table S3, the codon usage analysis revealed that leucine (Leu) and serine (Ser) were the most frequently used, while tryptophan (Trp) and cysteine (Cys) were the least used amino acid residues in the B and YS9 mitochondrial proteins. In comparing the composition of B with YS9, their distribution of amino acid residues across the mitochondrial proteins was very similar, except for two of them: Ala and Arg. Lastly, most of the amino acid residues were highly conserved in the seven higher plants.
The RSCU refers to the relative probability for a specific codon between synonymous codons encoding corresponding amino acids [71]. The RSCU analysis for the B and YS9 mitogenomes can be found in Supplementary Table S3, which shows the presence of all codons in the PCGs. Excluding the termination codons, the 34 PCGs in the B and YS9 mitogenomes consisted of a total of 11,247 and 11,149 codons, respectively. Additionally, according to the codon usage results, the RSCU values of NNA and NNT codons exceeded 0.9, except for Leu (CUA, 0.86) and Ile (AUA, 0.88) of B and only Leu (CUA, 0.79) of YS9, suggesting a strong As or Ts bias in the third codon position of B and YS9 mitochondrial PCGs. This was a very common phenomenon, in that we observed it in all the studied plant mitogenomes.

3.5. Phylogeny and Synteny Analysis during Evolution

Given the burgeoning advances in sequencing technology and assembly methods, an increasing number of complete plant mitogenomes have been assembled, providing an promising opportunity for new phylogenetic analyses that use mitogenomes. Here, to determine the evolutionary relationship of the B and YS9 lines of wheat, we downloaded the mitogenomes of the other five higher plants from the NCBI database to construct a phylogenetic tree for them (Figure 5). For the most part, the CMS line and its maintainer line could be separated. The Mauve homologous analysis indicated the B and YS9 mitogenome had excellent collinearity (Figure 6), and there were many identical or similar sequences shared between the two genomes. Using BLAST+, the sequences of both B and YS9 were compared next; these results corroborated the Mauve analysis (Figure 7A). We then imported these comparison results into Circos software to draw the circos diagram, for which the similarity sequences were standardized according to the length of all alignment regions, and then connected by different colors (red, orange, green, and blue). As the two genome sequences were basically the same, most of the same or similar sequences corresponded to large fragments (in red and orange), and, conversely, the small blank areas denote regions with low or no similarity between the two lines (Figure 7B). From the phylogenetic and synteny analyses of mitochondrial genome sequence, evidently there was excellent homology and a linear relationship in mitogenomes between the isonuclear alloplasmic male sterility line and the maintainer line. The rate of mitochondrial recombination and variation is typically very fast in plants, and the difference between related species may be substantial [34]. We speculate that this might be due to hybridization between different generations that is effective at reducing the mutation rate of the mitochondrial genome.

3.6. Comparison of Repeated Sequences in the Mitochondrial Genomes

A differential mitogenome structure between the same species and different genera in plants could be due to the rearrangement of their mitochondrial genomes during evolution. The vast majority of variance in genome size of plant mitogenomes can be explained by differences in the sizes of repeat sequences, such as SSRs and tandem repeats. Plant mitochondrial genomes are well known for their abundant repetitive sequences. SSRs are DNA tracts of repeated motifs of one to six bases that are useful molecular markers in studying genetic diversity and for species identification [72]. Here, a total of 107 and 106 SSRs were identified in the B and YS9 mitogenomes, respectively, including mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide repeats (Table 3 and Supplementary Table S4). In both mitogenomes, the tetranucleotide repeats (p4) were the most abundant motif type, and the mononucleotide repeats of A/T were found to be more prevalent than other repeat types, while di-, tri-, penta-, and hexa-nucleotide repeats were fewer in number. Moreover, most of the repeats were observed in the intronic or intergenic regions, with only a small portion positioned in the exonic regions of genes (atp8-1, atp8-2, ccmFN, cox2, matR, nad1, nad2, nad4, nad7, rps2, and rps3). As presented in Supplementary Table S5, tandem repeats with lengths spanning 6 bp to 70 bp were also detected in the B and YS9 mitogenomes, with the former having two more tandem repeats than the latter. Among the tandem repeats, only four were localized in a coding region (atp6-1, atp6-2, rps2, and rps4), while the rest were situated in intergenic spacers.
Besides the SSRs and tandem repeats, we also used online software to annotate mitochondrial repetitive sequences of four types—forward (F), reverse (R), complex (C), and palindromic (P)—and the same number of repeats (50 pairs) with lengths > 30 bp were also identified in both mitogenomes (Supplementary Table S6). These results uncovered 16 pairs of repeats >1000 bp in the mitochondrial genome of YS9, the largest of which was 6500 bp, with the other 34 pairs between 107 bp and 877 bp in size. Further, 30 of the 50 pairs were forward (F) and 20 were palindromic (P). Compared with the YS9, the repeat sequence of B was longer, ranging from 513 bp to 5364 bp, for which 28 pairs surpassed 1000 bp; 37 pairs were forward (F); and 13 were palindromic (P). The difference in the number of long and short repeats may be the main reason for the difference in mitochondrial genome size between B and YS9.
Given the myriad repetitive sequences in the plant mitochondrial genome, recombination among them can affect the functions and biological characteristics of mitochondria. More often than not, the number and total length of repetitive sequences in the male sterility lines are greater than those of fertile lines [68,73]. Although the genome of the B-type CMS line was only 341 bp longer than its maintainer line, the results of this study are consistent with previous results. Meanwhile, a considerable number of SSRs, tandem repeats, and dispersed repeats were detected, which could be further studied to excavate mitochondrial-specific markers and may prove to be of great significance in species origin, evolution, classification, and germplasm analysis, as well as in the study and elucidation of the mechanism responsible for male sterility in wheat.

3.7. Identification of SNP Sites in the PCGs

When analyzing the conserved mitochondrial protein coding genes of the B and YS9, we identified 67 SNP sites, distributed in 19 PCGs (atp1, atp4, atp6-1, atp6-2, ccmFN, cox3, nad1, nad2, nad3, nad4, nad5, nad6, nad7, rps1, rps13, rps2, rps3, and rps4). Among them, the tally of SNPs in the nad6 gene region was highest, accounting for 38.81% of the total SNPs, which may be related to fertility. Whereas the number of SNPs of nad4, atp6-1, and atp6-2 were 7, 5, and 4, accounting for 10.45%, 7.46%, and 5.97%, respectively; just one to three SNPs were found in the remaining gene regions (Supplementary Table S7). The amino acids encoded by the corresponding codons were further compared before and after the SNP site mutations, and the mutation types and proportions of SNP sites affected were statistically analyzed. These results showed that 22 (32.84%) synonymous events had occurred that did not change the type of amino acid, but 45 (67.16%) non-synonymous events could have altered the residue. We also found 16 transitions and 51 transversions in the 67 SNPs. ATP synthase plays an important role in providing energy for plant growth and development, for which anthers need more energy than do vegetative organs, and atp6 is a critical gene encoding an ATP synthase subunit, so it reasonable to expect that variation in the nucleotides and corresponding protein structure of atp6 may affect the fertility of plants [74]. Whether the SNPs of the nad6 and atp6 genes induce pollen abortion needs further experimental verification in the B-CMS and maintainer lines. At the same time, these SNPs may also serve as valuable markers to distinguish among wheat lines harboring different cytoplasm.

4. Conclusions

This project is the first to have sequenced, assembled, and characterized the complete mitogenomes of the B-type CMS line and its maintainer line, YS9. Our comprehensive analyses of their mitochondrial genomes uncovered structural variations, including ORFs, repeated sequences and SNP sites in protein-coding genes, which might be related to CMS. More specifically, the CMS-specific ORF2718 and the existence of SNPs at the atp6 gene may be closely associated with the CMS mechanism. Our data suggest that sequencing the B and YS9 mitogenomes and comparing their genomic features, offers a promising avenue for pursuing further genomic breeding studies, while also providing valuable information for future evolutionary and molecular studies of CMS in wheat.

Supplementary Materials

The following are available online at https://www.mdpi.com/article/10.3390/agronomy12040851/s1. Supplementary Table S1: Mitochondrial genome of the B-type CMS line and the maintainer line, YS9: Arrangement and annotation; Supplementary Table S2: The predictions of the open reading frames (ORFs) of the B and YS9; Supplementary Table S3: The relative synonymous codon usage (RSCU) of PCGs in the mitochondrial genome of the seven higher plants; Supplementary Table S4: The location information of SSRs in the mitochondrial genome of B and YS9; Supplementary Table S5: Distribution of tandem repeats in the mitochondrial genome of B and YS9; Supplementary Table S6: The information of dispersed repeats within two mitogenomes of the B and YS9; Supplementary Table S7: Identification of SNP sites in PCGs of the B and YS9.

Author Contributions

G.Z. and X.C. supervised the experiments; Y.L. performed most of the experiments, analyzed the data, prepared the figures and tables, and wrote the manuscript; Q.S., J.G. and Y.S. helped revise the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by the National Support Program of China (No. 2015BAD27B01), the Technological Innovation and Over Planning Projects of Shaanxi Province (No. 2014KTZB02-01-02) and the National Natural Science Foundation of China (31771785).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Data is contained within the article or Supplementary Materials.

Acknowledgments

We thank Charlesworth for its linguistic assistance during the preparation of this manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. Phenotypes of wheat anthers at five stages of development in the B-type CMS line (A–E) and its maintainer line (G–K). (A,G) Tetrad stage; (B,H) early-uninucleate stage; (C,I) later-uninucleate stage; (D,J) binucleate stage; (E,K) trinucleate stage; (F) binucleate stage, where ‘a’ is the single B anther and ‘b’ the single YS9 anther; and (L) trinucleate stage, where ‘c’ is the single YS9 anther, and ‘d’ the single B anther. Scale bars are 2 mm (2000 µm).
Figure 1. Phenotypes of wheat anthers at five stages of development in the B-type CMS line (A–E) and its maintainer line (G–K). (A,G) Tetrad stage; (B,H) early-uninucleate stage; (C,I) later-uninucleate stage; (D,J) binucleate stage; (E,K) trinucleate stage; (F) binucleate stage, where ‘a’ is the single B anther and ‘b’ the single YS9 anther; and (L) trinucleate stage, where ‘c’ is the single YS9 anther, and ‘d’ the single B anther. Scale bars are 2 mm (2000 µm).
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Figure 2. DAPI- and I2–KI-stained samples showing microspore development of the wheat YS9 and B lines. Different stages of microspore development in the YS9 (A–F,M–P) and the corresponding stages of the B (G–L,Q–T) were compared. (A,G) Dyad stage; (B,H) tetrad stage; (C,I,M,Q) early-uninucleate stage; (D,J,N,R) later-uninucleate stage; (E,K,O,S) binucleate stage; and (F,L,P,T) trinucleate stage. Scale bars are 50 µm.
Figure 2. DAPI- and I2–KI-stained samples showing microspore development of the wheat YS9 and B lines. Different stages of microspore development in the YS9 (A–F,M–P) and the corresponding stages of the B (G–L,Q–T) were compared. (A,G) Dyad stage; (B,H) tetrad stage; (C,I,M,Q) early-uninucleate stage; (D,J,N,R) later-uninucleate stage; (E,K,O,S) binucleate stage; and (F,L,P,T) trinucleate stage. Scale bars are 50 µm.
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Figure 3. The two circular mitochondrial genomes of the wheat CMS line and its maintainer line, (A) the maintainer line YS9, (B) the B-type CMS line. Genes shown on the inside of the circle are transcribed clockwise whereas genes on the outside are transcribed counter-clockwise; different functional genes are indicated by different colors. GC content is conveyed on the inner circle by dark gray shading.
Figure 3. The two circular mitochondrial genomes of the wheat CMS line and its maintainer line, (A) the maintainer line YS9, (B) the B-type CMS line. Genes shown on the inside of the circle are transcribed clockwise whereas genes on the outside are transcribed counter-clockwise; different functional genes are indicated by different colors. GC content is conveyed on the inner circle by dark gray shading.
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Figure 4. Codon usage patterns of B and YS9 wheat mitogenomes compared with those of the other five higher plants. The ratio of each amino acid residue to the whole mitochondrial proteins is shown as a percentage on the y-axis.
Figure 4. Codon usage patterns of B and YS9 wheat mitogenomes compared with those of the other five higher plants. The ratio of each amino acid residue to the whole mitochondrial proteins is shown as a percentage on the y-axis.
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Figure 5. The constructed phylogenetic tree was based on the mitogenomes of seven higher plants (Triticum aestivum: AP008982; T. aestivum cv. Chinese Yumai Ks3: GU985444; T. aestivum cv. Chinese Yumai: EU534409; T. timopheevii: AP013106; and Aegilops speltoides: AP013107). The numbers represent the evolutionary divergence time of each species.
Figure 5. The constructed phylogenetic tree was based on the mitogenomes of seven higher plants (Triticum aestivum: AP008982; T. aestivum cv. Chinese Yumai Ks3: GU985444; T. aestivum cv. Chinese Yumai: EU534409; T. timopheevii: AP013106; and Aegilops speltoides: AP013107). The numbers represent the evolutionary divergence time of each species.
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Figure 6. Mauve linear comparison of four mitogenomes: the B-type CMS line and its YS9 maintainer line, and Yumai Ks3 and Yumai (the K-type CMS line and its maintainer line).
Figure 6. Mauve linear comparison of four mitogenomes: the B-type CMS line and its YS9 maintainer line, and Yumai Ks3 and Yumai (the K-type CMS line and its maintainer line).
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Figure 7. A similarity comparison of the two respective mitogenomes in the B (the B-type CMS line) and YS9 (its maintainer line) wheat. (A) Result for the comparison using BLAST+; (B) comparison results were imported into Circos software to draw the circos diagram, whose colors represent different comparison lengths of the sequences: red, quartile to max value; orange, median to upper quartile; green, lower quartile to median; blue, min to lower quartile.
Figure 7. A similarity comparison of the two respective mitogenomes in the B (the B-type CMS line) and YS9 (its maintainer line) wheat. (A) Result for the comparison using BLAST+; (B) comparison results were imported into Circos software to draw the circos diagram, whose colors represent different comparison lengths of the sequences: red, quartile to max value; orange, median to upper quartile; green, lower quartile to median; blue, min to lower quartile.
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Table
Table 1. Genomic features of B and YS9 mitogenome.
Table 1. Genomic features of B and YS9 mitogenome.
SpeciesBYS9CSYumai Ks3YumaiAegilops_speltoidesTriticum_timopheevii
GenomeSize (bp)452,794452,453452,528647,559452,526476,091443,419
GC content (%)A27.927.927.927.827.927.727.7
T27.827.827.827.927.827.927.9
G22.222.222.222.222.222.122.1
C22.122.122.122.122.122.322.2
G + C44.344.344.344.344.344.444.3
AT-skew0.00180.00180.0018−0.00180.0018−0.0036−0.0036
GC-skew0.00230.00230.00230.00230.0023−0.0045−0.0023
CDSSize (bp)33,74133,44736,17143,40733,44735,67435,403
GC content (%)A26.527.026.826.526.626.226.4
T30.930.530.831.130.931.030.8
G21.621.621.621.521.521.721.7
C21.020.920.820.921.021.121.1
G + C42.642.542.442.442.542.842.8
AT-skew−0.0767−0.0609−0.0694−0.0799−0.0748−0.0839−0.0769
GC-skew0.01410.01650.01890.01420.01180.01400.0140
Table
Table 2. Genomic contents of B and YS9 mitogenome.
Table 2. Genomic contents of B and YS9 mitogenome.
Group of GenesGene Name
Complex I (NADH dehydrogenase)nad1 *, nad2 ***, nad3, nad4 ***, nad4L, nad5 **, nad6, nad7 ****, nad9
Complex III (ubichinol cytochrome c reductase)cob
Complex IV (cytochrome c oxidase)cox1, cox2 *, cox3
Complex V (ATP synthase)atp1, atp4, atp6(x2), atp8(x2), atp9
Ribosomal proteins (SSU)rps1, rps2, rps3 ***, rps4, rps7, rps12, rps13, rps19
Ribosomal proteins (LSU)rpl5, rpl16
MaturasesmatR
Transfer RNAs (tRNAs)trnA, trnC, trnD(x2), trnE, trnF, trnfM(x3), trnH, trnI, trnK(x3), trnM, trnN, trnP(x2), trnQ(x3), trnS(x3), trnW, trnY
Ribosomal RNAs (rRNAs)rrn5(x3), rrn18(x3), rrn26(x3)
Other genesccmB, ccmC, ccmFC *, ccmFN, mttB
Note: * The asterisks denotes the number of introns (**, ***, **** is for two, three, four introns, respectively); the numbers in parentheses represent the number of copies corresponding to the multi-copy genes.
Table
Table 3. Frequency of identified SSR motifs in YS9 and B mitogenome.
Table 3. Frequency of identified SSR motifs in YS9 and B mitogenome.
Motif TypeNumber of RepeatsTotalProportion (%)
34567810111213141517
The Maintainer line (YS9)
Monomer (p1)000000123211102018.87
Dimer (p2)001181000000002018.87
Trimer (p3)015100000000001615.09
Tetramer (p4)332000000000003533.02
Pentamer (p5)110000000000001110.38
Hexamer (p6)310000000000043.77
Total47181281012321110106100
the CMS line (B-type)
Monomer (p1)000000102411112018.69
Dimer (p2)001171200000002119.63
Trimer (p3)015100000000001614.95
Tetramer (p4)332000000000003532.71
Pentamer (p5)110000000000001110.28
Hexamer (p6)310000000000043.74
Total47181271210241111107100
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Li, Y.; Song, Q.; Guo, J.; Song, Y.; Chen, X.; Zhang, G. Comparative Analysis of Mitochondrial Genomes between the B-Type Cytoplasmic Male Sterility Line and Its Maintainer Line in Wheat. Agronomy 2022, 12, 851. https://doi.org/10.3390/agronomy12040851

AMA Style

Li Y, Song Q, Guo J, Song Y, Chen X, Zhang G. Comparative Analysis of Mitochondrial Genomes between the B-Type Cytoplasmic Male Sterility Line and Its Maintainer Line in Wheat. Agronomy. 2022; 12(4):851. https://doi.org/10.3390/agronomy12040851

Chicago/Turabian Style

Li, Ying, Qilu Song, Jialin Guo, Yulong Song, Xinhong Chen, and Gaisheng Zhang. 2022. "Comparative Analysis of Mitochondrial Genomes between the B-Type Cytoplasmic Male Sterility Line and Its Maintainer Line in Wheat" Agronomy 12, no. 4: 851. https://doi.org/10.3390/agronomy12040851

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