Comparative analysis of mitochondrial genomes between the hau cytoplasmic male sterility (CMS) line and its iso-nuclear maintainer line in Brassica juncea to reveal the origin of the CMS-associated gene orf288
© Heng et al.; licensee BioMed Central Ltd. 2014
Received: 12 October 2013
Accepted: 23 April 2014
Published: 30 April 2014
Cytoplasmic male sterility (CMS) is not only important for exploiting heterosis in crop plants, but also as a model for investigating nuclear-cytoplasmic interaction. CMS may be caused by mutations, rearrangement or recombination in the mitochondrial genome. Understanding the mitochondrial genome is often the first and key step in unraveling the molecular and genetic basis of CMS in plants. Comparative analysis of the mitochondrial genome of the hau CMS line and its maintainer line in B. juneca (Brassica juncea) may help show the origin of the CMS-associated gene orf288.
Through next-generation sequencing, the B. juncea hau CMS mitochondrial genome was assembled into a single, circular-mapping molecule that is 247,903 bp in size and 45.08% in GC content. In addition to the CMS associated gene orf288, the genome contains 35 protein-encoding genes, 3 rRNAs, 25 tRNA genes and 29 ORFs of unknown function. The mitochondrial genome sizes of the maintainer line and another normal type line “J163-4” are both 219,863 bp and with GC content at 45.23%. The maintainer line has 36 genes with protein products, 3 rRNAs, 22 tRNA genes and 31 unidentified ORFs. Comparative analysis the mitochondrial genomes of the hau CMS line and its maintainer line allowed us to develop specific markers to separate the two lines at the seedling stage. We also confirmed that different mitotypes coexist substoichiometrically in hau CMS lines and its maintainer lines in B. juncea. The number of repeats larger than 100 bp in the hau CMS line (16 repeats) are nearly twice of those found in the maintainer line (9 repeats). Phylogenetic analysis of the CMS-associated gene orf288 and four other homologous sequences in Brassicaceae show that orf288 was clearly different from orf263 in Brassica tournefortii despite of strong similarity.
The hau CMS mitochondrial genome was highly rearranged when compared with its iso-nuclear maintainer line mitochondrial genome. This study may be useful for studying the mechanism of natural CMS in B. juncea, performing comparative analysis on sequenced mitochondrial genomes in Brassicas, and uncovering the origin of the hau CMS mitotype and structural and evolutionary differences between different mitotypes.
KeywordsBrassica juncea Mitochondrial Cytoplasmic male sterility orf288 Mitotype
Cytoplasmic male sterility is a phenotypic trait that is widespread among plants and results in the inability of the plant to produce viable pollen . Numerous studies have shown that cytoplasmic male sterility in plants is associated with aberrant recombination in the mitochondrial genome, which results in the production of chimeric ORFs that are expressed as novel polypeptides . Since the first plant mitochondrial genome sequencing in Arabidopsis, a large number of mitochondrial genomes have been sequenced in angiosperm plants [4–10], especially in those that contain CMS cytoplasm. CMS-associated mitochondrial genome of crop species reported to date include Beta vulgaris, Oryza sativa[12–14], Triticumae stivum, Zea mays, Brassica napus[17, 18], Raphanus sativus[19, 20]. In this study, the mitochondrial genome of hau CMS line, its maintainer line and the normal type line “J163-4” were fully sequenced and assembled into a master circle. As in other higher plants, all three sequenced mitochondrial genomes had large sizes and distinctive features, including slow evolutionary rates, rapid rearrangement, frequent insertion, complex multipartite structures, specific modes of gene expression, cis- and trans-splicing, RNA editing and the use of universal genetic code . Comparative analysis of the CMS line and its iso-nuclear maintainer line may help us verify the CMS-associated gene in hau CMS line, and contribute to a better understanding of the plant mitochondrial genome in Brassicas.
The male sterile hau CMS line (00-6-102A) emerged as a spontaneous male sterile mutant in B. juncea. The anthers in the hau CMS plants are replaced by thickened petal-like structures, and hau CMS sterility starts at the stamen primordium polarization stage, much earlier than the other four CMS systems used in Brassicas (pol, ogu, nap, and tour) . A novel chimeric gene named orf288 was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Subcellular localization analysis showed that this CMS-associated gene was translated in the mitochondria of male-sterile plants. Transgenic result also showed that ORF288 is associated with the male sterility of hau CMS in Brassica juncea.
In this study, we sequenced the complete mitochondrial genomes of hau CMS line, its iso-nuclear maintainer line and the normal type line “J163-4” in B. juneca using Roche/454 pyro-sequencing technology. Comparative analysis of the hau CMS mitochondrial genome further confirmed that orf288 was a cytoplasmic male sterility-associated gene in B. juneca. The sequenced mitochondrial genomes may help us identify the mechanism of natural CMS and uncover the origin and structure of the hau CMS mitotype as well as understand evolutionary differences between the different mitotypes in B. juncea. Our data give new insight into the evolution of the Brassicas mitochondrial genome.
The mitochondrial genomes of the hauCMS line and its maintainer line
Summary features of mitochondrial genome contents in the hau CMS mitotype and its maintainer mitotype
Genome size (bp)
GC content (%)
Coding sequence (%)
Protein coding genes
Repeats in the hauCMS line and its maintainer line
The specific ORFs of different mitochondrial genomes
Mitotype-specific ORFs and protein coding genes between the hau CMS line and its maintainer line
Similarity of predicted protein
hau mitotype specific
nad3 and rps12 genes, partial sequence [Brassica napus]
Between syntenic regions 12 and 10
ADN44176.1 photosystem 1 subunit A, partial (chloroplast)
In the syntenic region 12
CAA58667.1 orf263 (mitochondrion) [Brassica tournefortii]
Between syntenic regions 6 and 13
Normal mitotype specific
YP_717154.1 hypothetical protein BrnapMp057 [Brassica napus]
Between syntenic regions 5 and 6
YP_004927826.1 orf293 (mitochondrion) [Brassica rapa subsp. campestris]
The edge of syntenic region 12
NP_085579.1 ribosomal protein S7 [Arabidopsis thaliana]
Between syntenic regions 6 and 7
The hau CMS-associated gene orf288 in Brassica juncea
Comparative analysis of the mitochondrial genome of the hauCMS line with its maintainer line
Over the past two decades, our knowledge of the organization and evolution of mitochondrial genomes has been rapidly expanding as a large number of fully sequenced mitochondrial genomes have been published along with their structure, expression and evolutionary profiles. In light of the pivotal role of the CMS line in crop breeding and its convenience for studying the cytoplasmic and nuclear interaction, we compared the mitochondrial genomes of the hau CMS line and its iso-nuclear maintainer line to investigate the origin of the hau CMS mitotype and to dissect the structural and evolutionary differences between the different mitotypes. Mitochondrial genome of the hau CMS line (247,903 bp) was larger than its maintainer line (219,863 bp) and the repeats (>100 bp) appeared noticeably more frequent than those in its maintainer line. The size of plant mitochondrial genome was relevant to the repeats it contained, and the repeats in the mitochondrial genomes also revealed the structural dynamics of the mitochondrial genome in plant development via intramolecular and intermolecular recombination. In this study, we report that small repeats (<50 bp) might contribute to larger repeats (>100 bp) in the sequenced hau CMS mitochondrial genome, but the emergence of these small repeats and their function in the hau CMS line and its maintainer line mitochondrial genomes requires further study.
As reported in rice , maize , pearl millet , wheat , B. napus and cybrids in Brassicaceae, our results also confirmed that substoichiometrically different mitotypes coexist in mitochondrial genomes of the hau CMS line and its maintainer line in B. juncea. The ratio of the alternative genomes may be variable, but in plants, the usually prevalent main genome is accompanied by sublimons—substoichiometric mitochondrial DNA (mtDNA) molecules . In plants, the relative copy number of recombination-derived sub-genomic DNA molecules within mitochondria is controlled by nuclear genes and a genomic shifting process can result in their differential copy number suppression to nearly undetectable levels . Most of the mitotype-specific ORFs in one mitochondrial genome present at a substoichiometric level in the other genome. At 25 cycles of PCR amplification, only templates carrying specific ORFs were able to produce a detectable PCR product in the hau CMS line and its maintainer line, and when the cycles were up to 30 to 35, all primer pairs (P1-P6) used were able to amplify specific ORFs in hau CMS line and its maintainer line, but the pattern of amplification differed in the 2 lines. The substoichiometric amount of specific ORFs from one genome to the other is thus sufficient to be detected by PCR amplification but not enough to have been picked up by the coverage depth of the 454 sequencing that was performed . Although substoichiometrically different mitotypes coexist in mitochondrial genomes of different plants, the molecular mechanism of such coexistence in different mitotypes requires further study. The coexistence of different mitotypes may play a prominent role in the coordination of nuclear and mitochondrial interaction and also make valuable contributions to the hybrid vigor in different crop plants.
Heterosis plays an irreplaceable role in China’s high-yield crop production, and male sterility, which is a prerequisite for the mass production of hybrid seeds, acts as a key factor . Cytoplasmic male sterility (CMS) in plant, which is determined by the mitochondrial genome is associated with a pollen sterility phenotype and caused by mitochondrial genome mutation. Identification of a CMS-associated gene and uncovering the mechanism of this trait may facilitate plant breeding. With the sequenced mitochondrial genome, more novel mitochondrial genome types and molecular markers for cytoplasm classifications will be identified. Recently, Chang et al.  sequenced the mitotypes of cam (B. rape), ole (B. oleracea), jun (B. juncea) and car (B. carinata) and analyzed them together with previously sequenced mitotypes of B. napus (pol and nap) to show the evolutionary mechanism of mitochondrial genome formation in Brassica. Molecular markers such as RFLPs, AFLPs, SCARs, and SSRs were used to distinguish the CMS line from its maintainer line in the Brassicas. Based on the sequenced mitochondrial genome, specific SCAR markers (the hau CMS line specific primers combination P1, P2 and P3 and its iso-nuclear maintainer line specific primers combination P4, P5 and P6) were also developed to separate the hau CMS line from its maintainer line at the seedling stage.
The origin and emergence of the CMS-associated gene orf288 in B. juncea
A large number of CMS-associated genes have been found in crop species, but the origin and precise mechanism of CMS remains elusive. In different CMS systems, the CMS-associated genes show little or no structural relationship. Often the CMS-associated genes or loci are located close to an atp gene or contain parts of a gene encoding an ATPase subunit and are co-transcribed with flanking mt-genes . Although the CMS phenotype also occurs at different stages during reproductive development, they were the root cause of male sterility. In our study, the CMS-associated gene orf288 located downstream of atp6 and is co-transcribed. The transgenic result further verified that orf288 is associated with the male sterility of hau CMS in Brassica juncea. As a chimeric gene, orf288 is composed partially of nad5 and orf293 in B. juncea, and there were 3 large repeats larger than 100 bp located downstream of orf288. These repeats may be related to the formation of the CMS-associated gene. Although it has relatively high similarity with orf263 in alloplasmic male sterile Brassica tournefortii at the nucleotide level, the restorer lines for tour CMS systems were found to be ineffective for restoring fertility in the hau CMS line. This may suggest that they were different from each other . A comparative analysis of the mitochondrial genome of the hau CMS line and its maintainer line further confirmed that orf288 was CMS-associated gene in hau CMS line in B. juncea.
Voluminous evidence suggests that mitochondrial gene expression can affect the function of the nuclear gene products that control floral development. In Honglian cytoplasmic male sterile rice, the CMS-associated gene orfH79 impaired mitochondrial function via interaction with P61 (a subunit of electron transport chain complex III), and resulted in an energy production dysfunction and oxidative stress in mitochondria, which may work as retrograde signals leading to abnormal pollen development . In Wild Abortive CMS rice, WA352 accumulates preferentially in the tapetum of anthers, where it inhibits COX11 function in peroxide metabolism and triggers premature tapetal programmed cell death and consequent pollen abortion. These CMS models provided a mechanistic link between the gain of function of a newly identified mitochondrial CMS gene product and the loss of activity of the essential nuclear-encoded mitochondrial protein through their detrimental interaction . It is thus likely that different recombinations in different plant mitochondrials gave rise to different chimeric genes that caused male sterility through interaction with genes in the anther development pathways and eventually caused male sterility in different CMS systems. This hypothesis might explain why dissimilar CMS genes in different plants all caused similar phenotypic male sterility. The nature of different CMS-associated genes that interact with the anther development pathways still needs to be studied further.
The hau CMS mitochondrial genome was highly rearranged as was reported for mitochondrial genomes in CMS lines of other crops. The chimeric CMS-associated gene orf288 was composed of 94 bp partial sequences of nad5 (a subunit of complex I in the electron transport respiratory chain system) and 749 bp sequences that were highly similar to orf293 in its maintainer line. Three large repeats downstream of orf288 may be related to the formation of the CMS-associated gene in the hau CMS line. These findings may help us to identify the mechanism of natural CMS in B. juncea and to uncover the origin of the hau CMS mitotype and the structural and evolutionary differences between different mitotypes.
The hau CMS line (00-6-102A) used in this study was originally discovered as a spontaneous male-sterile mutant in B. juncea in the experimental field at Huazhong Agricultural University in 1999. The maintainer line (00-6-102B) was iso-nuclear to the hau CMS line in B. juncea. A cultivar trilocular line, J163-4, in B. juncea with fertility anther was also used as control in our study . The anthers in the hau CMS plants are replaced by thickened petal-like structures and the sterility of the hau CMS initiates at the stamen primordium polarization stage . Seeds from the hau CMS line, its iso–nuclear maintainer line and the normal type line “J163-4” were harvested in an experimental field at Huazhong Agricultural University in 2011.
Isolation of mitochondrial DNA and total RNA
Discontinuous Percoll gradient centrifugation was used to separate highly purified mitochondria from 7-day-old etiolated seedlings (Additional file 7) for hau CMS line, its maintainer line and the normal type line “J163-4” from B. juncea. A 100 g sample of each seedling-stage hypocotyls from the 3 different lines were homogenized in 200 ml homogenization medium (0.4 M mannitol, 5 mM EDTA, 8 mM cysteine, 10 mM tricine, 1% BSA, 1% polyvinyl-pyrrolidone, pH 7.8). The homogenate was filtered using four pieces of Miracloth and centrifuged at 1000 g for 5 minutes. The resulting supernatants were centrifuged at 18,000 g for 15 minutes and the pellet was re-suspended in wash buffer (0.4 M mannitol, 1 mM EDTA, 10 mM MOPS-KOH and 1% BSA) to repeat the above procedure at 1000 g for 5 minutes and 18,000 g for 15 minutes. The reaction was terminated with the addition of 20 mM EDTA. The pellet was resuspended in wash buffer and layered onto a step gradient consisting of 15%, 20%, 28% and 40% Percoll in 0.4 mM mannitol, 1% BSA and 10 mM MOPS-KOH. Purified mitochondrial were removed from the 20% and 28% interphase (Additional file 7). The pellet was resuspended in wash buffer without EDTA and 25 mg/ml DNase (Roche 104159) was added at room temperature for 1-3 h. Finally, samples were centrifuged at 18,000 g for 20 minutes and resuspended in the lysis buffer (50 mM Tris–HCl, 10 mM EDTA, 1% SDS, and 200 mg/ml proteinase K (Sigma) at room temperature for 3 h . The CTAB method was used to obtain the purified mitochondrial DNA . Total RNA was isolated from flower buds, fresh leaves, roots, and hypocotyls using Trizol (Invitrogen) according to the manufacturer’s protocol.
The sequencing strategy
The complete mitochondrial genomes of the hau CMS line, its maintainer line and the normal type line in B. juneca were sequenced using the Roche 454 FLX + pyro-sequencing technology. The sequencing service was provided by Personal Biotechnology (Shanghai, China). Nucleotide sequences of 13,130,330 bp, 43,164,917 bp and 15,240,642 bp in total were obtained from the 00-6-102A, 00-6-102B and J163-4 lines, respectively. The average sequence depth was 52, 196 and 69. The sequence was assembled to 7, 3 and 4 contigs in the hau CMS line, its maintainer lines and the normal type line “J163-4”. The genomic PCR products sequence between contigs was obtained by Sanger sequencing.
ORF Finder, BLASTX, BLASTN, and tRNA-SE were used to identify mitochondrial genes, rRNA, and tRNA. Artemis software , which allowed the use of a threshold to identify ORFs, was used to identify ORFs whose function was unknown. Both of these mitochondrial genome sequences were assembled using the Seqman software (DNAStar). Bl2seq (http://www.ncbi.nlm.nih.gov/) was used to do sequence alignment to find the syntenic region in the sequenced mitochondrial genomes. ClustalW2 and MEGA 4 were used for a phylogenetic analysis of CMS associated gene orf288 in Brassicaceae. Circos was used to visualize data and information of the mitochondrial genome of the hau CMS line and its maintainer line . Progressive Mauve was used for multiple alignment among the 8 sequenced mitochondrial genomes in Brassicas. The mitochondrial genome sequences of the hau CMS line and its maintainer line have been deposited to the GenBank Database under accession numbers: KF736092 and KF736093.
The authors are grateful to thank the two anonymous reviewers for their helpful comments and Dr. Jitao Zou from National Research Council Canada, Saskatoon and Mr. Wenxiang Jia for critically reading the manuscript and Xiao Lin for technical support. This study was supported by the National Natural Science Foundation of China (NSFC grant number 31271761), the Doctoral Fund of Ministry of Education of China (grant number 20120146110011) and the Fundamental Research Funds for the Central Universities (grant number 2662014PY060).
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