Analysis of genetic diversity in cytoplasmic male sterility,and association of mitochondrial genes with petaloid-type cytoplasmic male sterility in tuber mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tumida</Emphasis> Tsen et Lee)

In our previous study, we bred a stable cytoplasmic male sterility (CMS) line of tuber mustard by using distant hybridization and subsequent backcrosses. In this CMS plants, all floral organs are normal except the anthers, which are transformed into petals or tubular structures. Recently, 2 mitochondrial genes—atpA and orf220—that are distinctively present in the CMS line of tuber mustard were cloned and partially characterized. In our study of genetic diversity analysis of CMS, 7 species of Brassica and Raphanus crops, which included 5 CMS lines and their respective maintainer lines, were used to compare the constitution of protein-coding genes in the mitochondrial genomes. In 4 of the 43 mitochondrial genes, namely, atpA, orf220, orf256, and orf305/orf324, polymorphisms were detected among the tuber mustard CMS line and its maintainer line. The results of a cluster analysis indicate that petaloid CMS phenotype of tuber mustard is a novel CMS type and is nearer to the nap CMS in Brassica napus at the phylogenetic level. The results of individual amplifications of these genes indicate the presence of 4 sequence-characterized amplified region (SCAR) markers, which enable rapid and reliable identification of this CMS. Expressions of the orf220 and orf256 genes were detected only in the CMS line, while expression of the orf305 gene was detected in the maintainer line. The different expression patterns of different mitochondrial-specific marker genes indicate that the quantity of mitochondrial proteins is differentially regulated during organ/tissue development in tuber mustard. The results of this study suggest that the above mentioned 4 mitochondrial genes are associated with the petaloid CMS phenotype in tuber mustard.     

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

Background

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.

Results

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.

Conclusion

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.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-322) contains supplementary material, which is available to authorized users.     

Mitotype-specific sequences related to cytoplasmic male sterility in Oryza species

Plant mitochondrial genomes contain a large number of mitotype-specific sequences (MSS) which establish a mitochondrial genome structure distinct from other mitotypes. In rice, nine mitochondrial genomes have been sequenced, which provides us with the possibility of characterizing the MSS of rice and probing their relationship to cytoplasmic male sterility (CMS) in rice. We therefore analyzed the mitochondrial genomes of CW-CMS, LD-CMS, WA-CMS, N and Nipponbare lines, and found 57 MSS with sizes ranging from 102 to 5,745 bp, and with an aggregate length of 92.4 kb. The MSS account for more than 14.5 % of the rice mitochondrial genome and are a significant contributing factor in the variation of mitochondrial genome sizes. Of the MSS tested, 34 MSS exhibited polymorphism among rice lines, and 14 MSS were further confirmed as being specific to CMS. This includes nine MSS specific to sporophytic CMS, three specific to gametophytic CMS, and two shared by all types of CMS. Interestingly, except for CMS genes orf(H)79 and orf352 which are partly or fully overlapping with some MSS fragments, there are ten more open reading frames of unknown function that were detected in CMS-specific MSS, hinting at their possible roles in plant CMS. These novel findings provide us with potential new molecular tools to direct the breeding of CMS lines in hybrid rice breeding programs.     

Nucleo-cytoplasmic interactions affect RNA editing of cox2, atp6 and atp9 in alloplasmic male-sterile rice (Oryza sativa L.) lines

RNA editing plays an important role in the regulation of mitochondrial gene expression in flowering plants. In this study, we examined RNA editing of the mitochondrial genes cox2, atp6 and atp9 in five isonuclear alloplasmic male-sterile lines (IAMSLs) of rice to investigate whether different cytoplasmic types affect RNA editing. Although many editing sites were conserved among the three genes, we found that the editing efficiency of certain sites was significantly different between different IAMSLs or between IAMSLs and their corresponding cytoplasmic donor CMS lines. Furthermore, several editing sites were found to be either present or absent in certain IAMSLs and their corresponding CMS lines. These results indicate that nuclear loci, as well as unknown editing factors within the mitochondria of different cytoplasmic types, may be involved in RNA editing, and they suggest that RNA editing in plant mitochondria is affected by nucleo-cytoplasmic interactions.     

A comparative study of the atp9 gene between a cytoplasmic male sterile line and its maintainer line and further development of a molecular marker specific for male sterile cytoplasm in kenaf (Hibiscus cannabinus L.)

mtDNA was isolated from cytoplasmic male sterility (CMS) line P3A and its maintainer P3B of kenaf (Hibiscus cannabinus L.). The atp9 gene and its two flanking sequences were obtained using homology cloning and high-efficiency thermal asymmetric interlaced PCR methods. The coding sequences showed only two base pairs difference between the CMS and its maintainer, and shared a homology of over 87 % with atp9 genes from other species in GenBank. However, when comparing the flanking sequences, a 47-bp deletion was characterized at the 3′ flanking sequence of atp9 in the CMS line. Quantitative PCR analysis indicated that the expression level of atp9 in the CMS line was 0.937-fold that of its maintainer. Furthermore, the respiratory rate of anthers in the CMS line was markedly lower than that of its maintainer. The results indicated that the 47-bp deletion at the 3′ flanking sequence of atp9 and/or down-regulated expression of the atp9 gene in the CMS line might be closely related to CMS in kenaf. To confirm whether the 47-bp deletion was specific to cytoplasm of male sterile lines, another 21 varieties were used for further analysis. The results showed that the 47-bp deletion was specific to male sterile cytoplasm (MSC) of kenaf. Based on these, a specific molecular marker was developed to distinguish the MSC from male fertile cytoplasm of kenaf.     

Analysis of chloroplast differences in leaves of rice isonuclear alloplasmic lines

The chloroplast being an important organelle of plant cells could possibly be associated with plant cytoplasmic male sterility (CMS). To better understand the correlation between (CMS) and chloroplast, we presented a comprehensive analysis based on the changes of photosynthetic parameters, chloroplasts ultrastructure, soluble sugar and starch content, the relative expression of sugar and starch metabolism genes, and chloroplast genome in rice isonuclear alloplasmic CMS lines at the flowering stage. Leaf gas exchange parameters did not affect by CMS lines (M2BS and M2A), although intercellular CO₂ concentration (C _i) was influenced in both M2BS and M2A. Ultrastructural observation results indicated that many starch granules were observed in the chloroplast of CMS lines, especially bigger size in M2BS, while few ones in M2B. Only the chloroplasts of M2A contained some additional number of lipoids compared with those of the other two lines (M2B and M2BS). Soluble sugar and starch contents in CMS lines (M2BS and M2A) were significantly higher than those in maintainer line (M2B) (p?<?0.01). The relative expression of sugar and starch metabolism genes indicated the imbalance of starch and sugar synthesis and decomposition may lead to accumulation of starch granules and demonstrated the presence of cytoplasmic effects. Moreover, chloroplast genome sequencing results showed similarity in both CMS lines, which revealed different single nucleotide polymorphisms (SNPs) and insertion/deletion (InDels) models compared with their maintainer line. Those models were located in psbD, rpoC2, rpl33, psbB, ndhA, ndhH, and intergenic regions. These findings, aligned with the possible association of CMS characteristics with cpDNA and genetically close relationship among both CMS lines, may contribute for future research.     

Development of a SCAR marker for early identification of S-cytoplasm based on mitochondrial SRAP analysis in pepper (Capsicum annuum L.)

Molecular markers, coxII SCAR, atp6-2 SCAR and accD-U, have been used for marker-assisted selection of cytoplasmic male sterility (CMS) in pepper. However, the presence of these markers at the sub-stoichiometric level in maintainer lines affects the reliable selection of male sterile (S-) cytoplasm. This study aimed to develop a new CMS-specific molecular marker, SCAR₁₃₀, for reliable identification of S-cytoplasm in pepper, while the new and three previous molecular markers were used to determine the cytoplasm types of pepper lines. Based on mitochondrial genome sequence related amplified polymorphism (SRAP) analysis of the CMS lines and the maintainer lines, SCAR₁₃₀ was developed from a 10-bp deletion at the SRAP primer binding site in the CMS line (130 bp) compared with that in the maintainer line (140 bp). S-cytoplasm could be unambiguously selected from the pepper lines by the different length of the marker bands. Application of the four molecular markers to various pepper lines revealed that SCAR₁₃₀ is more reliable than the other three previous markers, orf507, ψatp6-2 and accD-U. Homology alignment with BLAST showed that the marker was located between trnE and trnS in the Nicotiana tabacum mitochondrial genome. Furthermore, expression of the marker-linked gene was significantly higher at the pollen abortive stage in the CMS line (HW203A) than in the maintainer line, which indicated that the marker was closely related to male sterility. Hence, factors other than orf507 and ψatp6-2 may exist for the regulation of male sterility in pepper.     

Alterations of RNA Editing for the Mitochondrial ATP9 Gene in a New orf220-type Cytoplasmic Male-sterile Line of Stem Mustard （Brassica juncea var. tumida）

RNA editing for the mitochondrlal ATP9 gene of encoding regions has been observed in both cytoplasmic malesterile and maintainer lines of stem mustard, where its editing capacity varied spatially and temporally in the cytoplasmic male sterility （CMS） line. There were four RNA editing sites for the mitochondrial ATP9 gene according to Its normal editing sites in mustard, of which three sites occurred as C-to-U changes and one as a U-to-C change. As a result, the hydrophobicity of deduced ATP9 protein was reduced due to the conversions at its 17th, 45th and 64th positions. Meanwhile, the conservation of deduced ATP9 protein was enhanced by changes at the 56th position. Loss of a specific editing site for ATP9 was observed in juvenile roots, senile roots, senile leaves and floret buds of the CMS line. Comparatively, complete RNA editing for ATP9 gene was retained in juvenile roots, juvenile leaves and floret buds of its maintainer line; however, the loss of a specific editing site for ATP9 gene occurred at senile roots and senile leaves in its maintainer line. These observations allow us to produce a hypothesis that the dysfunction of a specific mitochondrial gene arising from RNA editing could probably be a factor triggering CMS and organ senescence through unknown cross-talk pathways during development.     

Identification of the predominant nonrestoring allele for Owen-type cytoplasmic male sterility in sugar beet (Beta vulgaris L.): development of molecular markers for the maintainer genotype

Hybrid seed production in sugar beet relies on cytoplasmic male sterility (CMS). As time-consuming and laborious test crosses with a CMS tester are necessary to identify maintainer lines, development of a marker-assisted selection method for the rf gene (the nonrestoring allele of restorer-of-fertility locus) is highly desirable for sugar-beet breeding. To develop such a method, we investigated genetic variation at the Rf1 locus, one of two Rf loci known in sugar beet. After HindIII-digestion, genomic DNAs from beet plants known to have a restoring Rf1 allele yielded a range of hybridization patterns on agarose gels, indicating that Rf1 is a multi-allelic locus. However, the hybridization patterns of 22 of 23 maintainer lines were indistinguishable. The nucleotide sequences of the rf1 coding regions of these 22 maintainer lines were found to be identical, confirming that the lines had the same rf1 allele. Two PCR markers were developed that targeted a downstream intergenic sequence and an intron of Rf1. The electrophoretic patterns of both markers indicated multiple Rf1 alleles, one of which, named the dd(L) type, was associated with the maintainer genotype. To test the validity of marker-assisted selection, 147 sugar beet plants were genotyped using these markers. Additionally, the 147 sugar beet plants were crossed with CMS plants to determine whether they possessed the maintainer genotype. Analysis of 5038 F1 offspring showed that 53 % of the dd(L) plants, but none of the plants with other alleles, had the maintainer genotype. Thus, selection for the dd(L) type considerably enriched the proportion of plants with the maintainer genotype.     

Mitochondrial DNA genetic polymorphism in thirteen rice cytoplasmic male sterile lines

Key message

Thirteen rice CMS lines derived from different cytoplasms were classified into eight groups by PCR amplification on mtDNA. The orf79 gene, which causes Boro II CMS, possibly results in Dian1-CMS.

Abstract

Thirteen rice cytoplasmic male sterile (CMS) lines derived from different cytoplasms are widely used for hybrid rice breeding. Based on 27 loci on mitochondrial DNA, including single nucleotide polymorphisms and segmental sequence variations between typical indica and japonica as well as high-polymorphism segmental sequence variations and single nucleotide polymorphisms among rice CMS lines, the 13 rice CMS lines were classified into eight groups: (I) wild-abortive CMS, Indonesian Shuitiangu CMS, K-CMS, Gang CMS, D-CMS and dwarf abortive CMS; (II) Maxie-CMS; (III) Honglian CMS; (IV) Boro II CMS; (V) Dian1-CMS; (VI) Liao-CMS; (VII) Lead CMS; and (VIII) Chinese wild rice CMS. According to their pollen abortion phenotypes, groups I and II (including 7 CMS lines) were classified as sporophytic CMS lines, the cytoplasmic genetic relationships among which were very close. They could have originated from similar, or even the same, cytoplasm donors. Groups III–VIII (including 6 CMS lines) were categorized as gametophytic CMS lines, the cytoplasms of which differed from one another, with some having relatively far genetic relationships. Dian1-CMS was found to harbor the orf79 gene, which causes Boro II CMS, whereas Liao-CMS had an orf79 structure that does not result in Lead CMS. Therefore, we speculated that orf79 is associated with Dian1-CMS but not with Liao-CMS. The atp6–orf79 structure related to sterility was also found to experience multiple evolutionary turnovers. All sporophytic CMS lines were indica-like. Except the Honglian CMS line, which was indica-like, all gametophytic CMS lines were japonica-like.     

Reduced expression of CTR1 gene modulated by mitochondria causes enhanced ethylene response in cytoplasmic male-sterile Brassica juncea

We studied how mitochondria affect ethylene response via modulation of CTR1 expression in cytoplasmic male-sterile (CMS) Brassica juncea. The expression of CTR1 gene was reduced in CMS compared with male-fertile (MF) lines. We observed that hypocotyl and root lengths were shorter than in the MF line during germination in the dark. An enhanced ethylene response was observed in CMS plants as shown by the CMS and maintainer line phenotypes treated with 1-aminocyclopropane-1-carboxylic acid. The phenotype in CMS plants could be recovered to the maintainer line when treated with Ag(+) . One ethylene response gene, plant defensin gene, was detected to be induced in CMS. The behavior of this phenotype could be mimicked by treating the maintainer line with antimycin A that disturbs mitochondrial function, which showed reduced length of hypocotyl and roots, and resulted in similar expression patterns of ethylene-related genes as in CMS. The reduced length of hypocotyl and roots could be recovered to the maintainer line by treatment with gibberellic acid (GA(3) ). In addition, the GA(3) content was reduced in CMS plants and in the MF line treated with antimycin A. Ethylene treatment markedly affects GA(3) content; however, GA(3) did not significantly affect ethylene-related gene expression in regards to regulation of hypocotyl and root length, which suggests that ethylene acts upstream via gibberellin to regulate hypocotyls and root development. Taken together, our results suggest a link between mitochondrial modulation of the ethylene and gibberellin pathway that regulates the development of hypocotyl and roots.