Pol CMS Research Articles

High-generation near-isogenic lines combined with multi-omics to study the mechanism of polima cytoplasmic male sterility

BackgroundCytoplasmic male sterility (CMS), which naturally exists in higher plants, is a useful mechanism for analyzing nuclear and mitochondrial genome functions and identifying the role of mitochondrial genes in the plant growth and development. Polima (pol) CMS is the most universally valued male sterility type in oil-seed rape. Previous studies have described the pol CMS restorer gene Rfp and the sterility-inducing gene orf224 in oil-seed rape, located in mitochondria. However, the mechanism of fertility restoration and infertility remains unknown. Moreover, it is still unknown how the fecundity restorer gene interferes with the sterility gene, provokes the sterility gene to lose its function, and leads to fertility restoration.ResultIn this study, we used multi-omics joint analysis to discover candidate genes that interact with the sterility gene orf224 and the restorer gene Rfp of pol CMS to provide theoretical support for the occurrence and restoration mechanisms of sterility. Via multi-omics analysis, we screened 24 differential genes encoding proteins related to RNA editing, respiratory electron transport chain, anther development, energy transport, tapetum development, and oxidative phosphorylation. Using a yeast two-hybrid assay, we obtained a total of seven Rfp interaction proteins, with orf224 protein covering five interaction proteins.ConclusionsWe propose that Rfp and its interacting protein cleave the transcript of atp6/orf224, causing the infertility gene to lose its function and restore fertility. When Rfp is not cleaved, orf224 poisons the tapetum cells and anther development-related proteins, resulting in pol CMS mitochondrial dysfunction and male infertility. The data from the joint analysis of multiple omics provided information on pol CMS’s potential molecular mechanism and will help breed B. napus hybrids.

Cytological observation of anther structure and genetic investigation of a thermo-sensitive genic male sterile line 373S in Brassica napus L

BackgroundPhotoperiod and/or thermo-sensitive male sterility is an effective pollination control system in crop two-line hybrid breeding. We previously discovered the spontaneous mutation of a partially male sterile plant and developed a thermo-sensitive genic male sterile (TGMS) line 373S in Brassica napus L. The present study characterized this TGMS line through cytological observation, photoperiod/ temperature treatments, and genetic investigation.ResultsMicroscopic observation revealed that the condensed cytoplasm and irregular exine of microspores and the abnormal degradation of tapetum are related to pollen abortion. Different temperature and photoperiod treatments in field and growth cabinet conditions indicated that the fertility alteration of 373S was mainly caused by temperature changes. The effects of photoperiod and interaction between temperature and photoperiod were insignificant. The critical temperature leading to fertility alteration ranged from 10 °C (15 °C/5 °C) to 12 °C (17 °C/7 °C), and the temperature-responding stage was coincident with anther development from pollen mother cell formation to meiosis stages. Genetic analysis indicated that the TGMS trait in 373S was controlled by one pair of genes, with male sterility as the recessive. Multiplex PCR analysis revealed that the cytoplasm of 373S is pol type.ConclusionsOur study suggested that the 373S line in B. napus has a novel thermo-sensitive gene Bnmst1 in Pol CMS cytoplasm background, and its fertility alteration is mainly caused by temperature changes. Our results will broaden the TGMS resources and lay the foundation for two-line hybrid breeding in B. napus.

Characterization of atpA and orf220 genes distinctively present in a cytoplasmic male-sterile line of tuber mustard

SummaryIn the present investigation, the atpA (Accession Number: AY211265, AY211266) gene, encoding the a subunit of F1-ATP synthase, as well as the open reading frame orf220 (Accession Number: AY208898), presumably encoding a hydrophobic membrane-associated polypeptide with deduced 220 amino acids, were found to be distinctively present in a cytoplasmic male-sterile (cms) line of tuber mustard. They were preferentially cloned by using mitochondria-specific primers and were partially characterized. The atpA gene cloned from cms and its fertile maintainer (m) lines were sequenced to be 1,454 and 1,073 bp-sized in fragments, respectively. They had a high similarity of approximately 70% in terms of nucleotide constituents. However, alignment of these two genes suggested that the atpA gene from the cms line had an insertion of 383 bp-sized fragment in 5’-flank spanning from 141th to 523th base site, compared with that of its m line. Besides, the open reading frame, orf220, was found to be exclusively present in the cms line of tuber mustard. It was deduced to possibly encode a hydrophobic N-terminus membrane-associated polypeptide with a deduced molecular weight of 26 kD. By Southern hybridization, it was further shown that orf220 existed only in the cms line and was absent from the fertile m line with no hybridization signal detected. Although the atpA gene was blotted in either cms or m line, they were of different fragment sizes when blotted with DNA digested with Hind III and Xba I, respectively. Both atpA and orf220 genes were supposed to be low coped in mitochondria genomes. In addition, by phylogenetic analysis, it was manifest that the orf220 gene had a closer genetic relationship with orf222 in Brassica nap cms than both of orf224 in Brassica pol cms and orf240 in Arabidopsis thaliana cms. The direct evidence of the involvement of these two genes in the expression of cms of tuber mustard needs further investigation.

Targeted mapping approaches to identify DNA markers linked to the Rfp1 restorer gene for the ‘Polima’ CMS of canola (Brassica napus L.)

We have used two targeting approaches [pairs of nearly isogenic lines (NILs) and bulked segregant analysis] to identify DNA markers linked to the Rfp1 restorer gene for the pol CMS of canola (Brassica napus L.). We were able to target the Rfp1 locus as efficiently by comparing NILs as by bulked segregant analysis, and it was demonstrated in this instance that double-screening strategies could significantly improve the overall targeting efficiency. The chance occurrence of shared homozygosity at specific unlinked chromosomal regions in the bulks was found to limit the efficiency of bulked segregant analysis, while the efficiency of NIL comparison was limited by residual DNA from the donor cultivar at scattered sites throughout the genome of the NILs.

Characterization of expression of a mitochondrial gene region associated with the Brassica ?Polima? CMS: developmental influences

The mitochondrial genome of the Polima (pol) male-sterile cytoplasm of Brassica napus contains a chimeric 224-codon open reading frame (orf224) that is located upstream of, and co-transcribed with, the atp6 gene. The N-terminal coding region of orf224 is derived from a conventional mitochondrial gene, orfB, while the origin of the remainder of the sequence is unknown. We show that an apparently functional copy of orfB is present in the pol mitochondrial genome, indicating that the pol CMS is not caused by the absence of an intact, expressed orfB gene. The 5' termini of orf224/atp6 transcripts present in both sterile and fertility-restored (Rf) pol cytoplasm plants are shown to map to sequences resembling mitochondrial transcription-initiation sites, whereas the 5' termini of two transcripts specific to restored lines map to sequences which resemble neither one another nor mitochondrial promoter motifs. It is suggested that the complex orf224/atp6 transcript pattern of Rf plants is generated by a combination of multiple transcription initiation and processing events and that the nuclear restorer gene acts to specifically alter orf224/atp6 transcripts by affecting RNA processing. Northern analyses demonstrate that the effect of the restorer gene on orf224/atp6 transcripts is not tissue or developmental-stage specific. However, the expression of the atp6 region is developmentally regulated in pol plants, resulting in decreased levels of monocistronic atp6 transcripts in floral tissue relative to seedlings. It is suggested that this developmental regulation may be related to the absence of overt phenotypic effects of the CMS mutation in vegetative tissues.

Hybridity and seed yield versus male sterility index (MSI) in pol CMS summer rape hybrid seed production plots

The degree of male sterility of pol A-lines, measured by a male sterility index (MSI) had significant linear relationships with both seed yield and hybridity of hybrid summer rape seed lots. Seed yield increased by 352 kg ha−1, while hybridity decreased by 6% per unit increase in MSI. A hybridity of 100% required an MSI of very nearly zero.Key words: pol CMS, Brassica napus, hybrids, MSI

RNA editing of atp6 transcripts from male-sterile and normal cytoplasms of rapeseed ( Brassica napus L.)

The complete cDNA sequence corresponding to the rapeseed atp6 gene transcript (coding for subunit 6 of F O-ATPase) has been determined by a method involving cDNA synthesis, using specific oligonucleotides as primers, followed by PCR amplification, cloning and sequencing of the amplification products. Only one modification, a C-to-U conversion, has been found when compared to the genomic mitochondrial DNA sequence. Comparison of the extent and frequency of RNA editing of the pol cytoplasmic male sterile (cms) atp6 transcript with those of normal atp6 transcript indicates that there is no variation between the editing status of the atp6 transcripts from pol cms and normal cytoplasms.

Different organization and altered transcription of the mitochondrial atp6 gene in the male-sterile cytoplasm of rapeseed (Brassica napus L.).

The Fo-ATPase subunit 6 gene (atp6) of rapeseed mitochondria has been isolated from both pol male-sterile and normal (fertile) cytoplasms in order to determine whether the rearrangements around the atp6 locus in pol male-sterile cytoplasm play a role in cytoplasmic male-sterility (cms). The pol cms and normal atp6 genes are identical and encode a 261-amino acid polypeptide. As a result of extensive rearrangement, a novel reading frame (pol-urf) was generated upstream of the atp6 gene only in pol cms mitochondria, which encoded 105 amino acids and might be co-transcribed with atp6. A 5'-portion of pol-urf shows sequence homology to the Oenothera ORFB gene associated with coxIII. A 5'-flanking region of the pol-urf also shows homology to that of ORF105 in Ogura cms radish mitochondria. These DNA rearrangements which give rise to pol-urf in the vicinity of the atp6 locus may be responsible for cms in rapeseed.

Mitochondrial genome of rapeseed (Brassica napus L.). 1. Intraspecific variation of mitochondrial DNA.

Restriction endonuclease analysis was carried out for mitochondrial (mt) DNAs of 27 accessions of rapeseed (Brassica napus L.) to detect intraspecific variation of the mitochondrial genome. MtDNA isolated from green leaves was digested with ten endonucleases and was electrophoresed using agarose slab gel. Mt genomes of the 27 accessions were classified into two major types, each of which was further divided into three subtypes based on the length differences in the restriction fragments of mtDNAs. In total, six mt genome types (type Ia, Ib, Ic, IIa, IIb and IIc) could be identified. To analyze the relationship between the mt genome types and the cytoplasmic male sterility (cms) induction, restriction fragment patterns of two male sterile cytoplasms, namely, nap cms and pol cms, were compared with those of the normal cytoplasm. The mt genome of the nap cytoplasm was found to be either of Ia, Ib and Ic types, whereas the pol cytoplasm carried the type IIb mt genome. Mitochondrial linear plasmid DNA was also observed in several accessions, but the relationship between its presence and the cms trait could not be established.