Polima Cytoplasmic Male Sterility Research Articles

Перспективные экспериментальные гибриды рапса ярового (Brassica napus L.), созданные с использованием системы ЦМС типа Polima

One of the prior directions in spring rapeseed breeding is development of heterotic hybrids. Seed growing of the most hybrids F1 used in production are often carried out by two types of cytoplasmic male sterility, they are polima and ogura. At the Lipetsk Rapeseed Research Institute, fertility restorers on sterile cytoplasm were made and estimated for the main valuable traits. The purpose of the research was to develop effective restorers, which are necessary for obtaining of highly productive hybrids on the Polima type cytoplasmic male sterility (CMS). The object of research was 56 varieties of Brassica napus L., which were selected in 2013. It is noted that hybrids F1 were more productive than the parental forms and the standards (the varieties Ratnik and Rif). The restorer line LHR-1 is of practical interest for the development of hybrids on the Polima CMS.

Поиск восстановителей фертильности для системы ЦМС типа Polima рапса ярового (Brassica napus L.) и оценка их качества

One of the prior directions in modern rapeseed breeding is making heterotic hybrids. Seed production of most hybrids F1 used in production are often carried out by two types of cytoplasmic male sterility, there are Polima and Ogura. At the All-Russian Rapeseed Research Institute, fertility restorers on sterile cytoplasm were made and estimated for the main valuable characteristics and biochemical properties. The purpose of the study was to create reducing agents necessary for obtaining highly productive hybrids on the Polima type cytoplasmic male sterility (CMS). The object of research was 56 varieties of Brassica napus L., which were chosen in 2013. The setting of experiments, observations, records and analyzes were carried out using the field plot technique, the methodology of field experiments, the method of statistical data processing. It is noted that hybrids F1 had more productivity than the parental forms and the standard (Ratnik and Rif). Promising reducing agents of cytoplasmic male sterility of the Polima type were identified, which ensured complete restoration of fertility in sterile lines with CMS of the Polima type. The reducing agent LHR-1 is of practical interest for the creation of hybrids on the Polima CMS.

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.

Comparative transcriptome analysis in Chinese cabbage (Brassica rapa ssp. pekinesis) for DEGs of Ogura-, Polima-CMS and their shared maintainer.

Cytoplasmic male sterility (CMS) is maternally inherited trait, which hinders the ability to produce viable pollen in plants. It serves as a useful tool for hybrid seed production via exploiting heterosis in crops. The molecular mechanism of CMS and fertility restoration has been investigated in different crops. However, limited number of reports is available on comparison of Ogura- and Polima-CMS with their shared maintainer in Chinese cabbage. We performed transcript profiling of sterile Ogura CMS (Tyms), Polima CMS (22m2) and their shared maintainer line (231-330) with an aim to identify genes associated with male sterility. In this work, we identified 912, 7199 and 6381 DEGs (Differentially Expressed Genes) in 22m2 Vs Tyms, 231-330 VS 22m2 and 231-330 Vs Tyms, respectively. The GO (Gene Ontology) annotation and KEGG pathway analysis suggested that most of the DEGs were involved in pollen development, carbon metabolism, lipase activity, lipid binding, penta-tricopeptide repeat (PPR), citrate cycle and oxidative phosphorylation, which were down-regulated in both CMS lines. This result will provide an important resource for further understanding of functional pollen development, the CMS mechanism and to improve molecular breeding in Chinese cabbage.

Allelism analysis of BrRfp locus in different restorer lines and map-based cloning of a fertility restorer gene, BrRfp1, for pol CMS in Chinese cabbage (Brassica rapa L.).

In Chinese cabbage, there are two Rf loci for pol CMS and one of them was mapped to a 12.6-kb region containing a potential candidate gene encoding PPR protein. In Chinese cabbage (Brassica rapa), polima cytoplasmic male sterility (pol CMS) is an important CMS type and is widely used for hybrid breeding. By extensive test crossing in Chinese cabbage, four restorer lines (92s105, 01s325, 00s109, and 88s148) for pol CMS were screened. By analyzing the allelism of the four restorer lines, it was found that 92s105, 01s325, and 00s109 had the same "restorers of fertility" (Rf) locus (designated as BrRfp1), but 88s148 had a different Rf locus (designated as BrRfp2). For fine mapping the BrRfp1 locus of 92s105, a BC1F1 population with 487 individuals and a BC1F2 population with 2485 individuals were successively constructed. Using simple sequence repeat (SSR) markers developed from Brassica rapa reference genome and InDel markers derived from whole-genome resequencing data of 94c9 and 92s105, BrRfp1 was mapped to a 12.6-kb region containing a potential candidate gene encoding pentatricopeptide repeat-containing protein. Based on the nucleotide polymorphisms of the candidate gene sequence between the restoring and nonrestoring alleles, a co-segregating marker SC718 was developed, which would be helpful for hybrid breeding by marker-assisted screening and for detecting new restorer lines.

Instability in mitochondrial membranes in Polima cytoplasmic male sterility of Brassica rapa ssp. chinensis

Cytoplasmic male sterility (CMS) is an important factor to observe heterosis in Brassica rapa. Although several studies have documented the rearrangements of mitochondrial DNA and dysfunction in the mitochondria have been observed in most types of CMS, the basis of the molecular mechanisms involved in these processes and other effects on CMS remain unclear. In this study, suppression subtractive hybridization was performed in the flowers of an alloplasmic Polima CMS system from B. rapa ssp. chinensis to identify genes that are differentially expressed between fertile and sterile plants. A total of 443 clones were isolated (156 were upregulated in fertile buds, and 287 were upregulated in sterile ones). Real-time RT-PCR further demonstrated the credibility of SSH. Among these genes, many membrane protein genes (LTP12, PIP2A, and GRP14) were inhibited in the sterile male line. Mitochondrial membrane potential (MMP) assay was then performed. Results showed that the sterile MMP was unstable and failed to create a potential difference; thus, mitochondrial dysfunction occurred. Moreover, abnormal microtubules and photosynthetic pathways were found in sterile male cells. Unstable MMP, nutritional deficiency, and abnormal microtubules were the causes of Polima CMS in Brassica campestris. H2O2, MDA, and O(2-), accumulated as byproducts of energy metabolism disorder in sterile male cells.

English

Polima cytoplasmic male sterility (pol CMS) has been widely used for exploiting heterosis in rapeseed breeding. The dominant restorer gene of pol CMS (Rfp ) is found in the nucleus and is a key component of hybrid production by achieving F1 progeny with complete fertility restoration. To identify the molecular markers associated with the Rfp gene, a near isogenic line (NIL) population of 2,000 individuals segregating for the Rfp locus was generated by crossing and backcrossing for 12 times. This NIL population was used to screen Rfp markers by AFLP technique. Based on the sequence information of AFLP markers that have been identified in previous research, we identified a homologous region of Rfp locus in chromosome 1 of Arabidopsis . Then, six sequenced Brassica rapa BAC clones corresponding to this target region were chosen to design microsatellite (SSR) primer pairs. Twenty-two SSR markers were designed and one of them, KBrDP1, was verified in the 2, 000 NILs population and proved to be strongly linked to Rfp locus. The genetic distance between KBrDP1 and Rfp was 0.2 cM. KBrDP1 marker was found located on linkage group N9 of a published DH mapping population. This SSR marker was useful in marker assisted selection breeding of the elite pol CMS restorer lines in rapeseed.

Sequence Analysis and Expression of orf224 Gene Associated with Two Types of Cytoplasmic Male Sterility in Brassica napus L.

Polima and Shaan 2A are the two most widely used forms of cytoplasmic male sterility (CMS) in the utilization of heterosis of rapeseed (Brassica napus) in China. A previous study indicated that the mitochondrial gene, orf224, was the only gene with a differential expression pattern among the normal, sterile and fertility-restored lines in rapeseed. DNA sequences of orf224, including coding sequences from Shaan 2A and Polima CMS, were then amplified and analyzed. DNA sequence alignment indicated both the coding sequences were 675 bp in length and had 99.9 and 99% homology in nucleotides and amino acids, respectively, and shared certain similarity to homologues from other Brassica spp. and Arabidopsis thaliana. The probable promoter regions of orf224 were conserved between B. napus and A. thaliana, but the upstream regions of probable promoter regions were completely divergent from each other. Additionally, analysis of the primary and secondary structure of the proteins encoded by orf224 from the two lines predicted that the proteins contain a a-helix, extended strand, and random coil. After cloning a in vitro experiment showed that these two proteins could be expressed in Escherichia coli BL21.

Cytoplasmic male sterility with self-incompatibility, a novel approach to utilizing heterosis in rapeseed (Brassica napus L.)

The Polima cytoplasmic male sterility (CMS) system has been successfully used in three/two-line hybrid production in rapeseed (Brassica napus L.). However, the sterility of the Polima (pol) CMS lines is sensitive to temperature fluctuations. Also, traces of pollen can cause self-pollination within the CMS lines, which results in reduced levels of F1 hybrid seed purity and leads to a significant yield loss. Self-incompatibility (SI) is another important approach for hybrid seed production in rapeseed. Despite having a wide range of restorers and being easily selected in a breeding program, SI system has some drawbacks. In this study, SI genes from a self-incompatible line of Brassica napus were transferred to a pol CMS line and S372A, a novel line of combined cytoplasmic male sterility with self-incompatibility was bred. Due to the SI genes, this line produced very few seeds when it was selfed at low temperature and no seeds at high temperature. This suggested that the line with CMS + SI had combined the advantages and overcome the disadvantages of both the pol CMS and SI systems. Furthermore, our results showed that most of the maintainers and all the restorers of the pol CMS system were also maintainers and restorers of the CMS + SI line, respectively. This indicates that the CMS + SI system can be easily used to establish three-line hybrids of rapeseed, and we believe this novel system could be extended to other species of Brassica.

Isolation and characterization of a male sterility gene homolog BcMS2 from Chinese cabbagge-pak-choi that expressing in an anther-specific manner

A male sterility gene homolog, designated BcMS2, was isolated from flower buds using gene-specific primer pairs and was submitted to GenBank under accession number EF093533. Comparison of BcMS2 gene with MS2 from Arabidopsis thaliana and MS2Bnap from Brassica napus revealed some differences in gene structure and evolution. The full genomic DNA sequence of BcMS2 was 2,576 bp in length containing 8 exons and 7 introns, more than those of MS2Bnap but less than MS2. RT-PCR showed that BcMS2 gene expressed only in stage III flower buds of male fertile Chinese cabbage-pak-choi 'ZUBajh97-01B' and there were no detection in all organs of Polima cytoplasmic male sterility (CMS) line 'Bpol97-05A' and Ogura CMS line 'Bogu97-06A'. Furthermore, RT-PCR revealed that BcMS2 expressed only in anthers of male fertile material and there were no expression in sepals, petals, filaments and pistils. These results suggested that BcMS2 was an anther-specific gene and might be essential for the fertility of Chinese cabbage-pak-choi.

Development and primary genetic analysis of a fertility temperature‐sensitive polima cytoplasmic male sterility restorer in Brassica napus

AbstractOver the past decade, the polima cytoplasmic male sterility (pol CMS) three‐line and two‐line systems have been developed for the production of hybrid seed in Brassica napus oilseed rape in China. The discovery of the novel pol CMS restorer line FL‐204 is described here. It restores male fertility of hybrid plants in the pol CMS system, but hybrid seed production can only be carried out under autumn sowing in Wuhan in south China under moderate temperatures at flowering. The restorer cannot be used as a male for hybrid seed production in northwestern China (Gansu) under spring sowing conditions, because there it is more or less male sterile due to high temperatures at flowering. Because of this behaviour, it is referred to as a fertility temperature‐sensitive restorer (FTSR) in this paper. F2, BC1 as well as double haploid populations were constructed to determine the inheritance of fertility restoration of FL‐204 in the autumn at Wuhan and under spring sowing conditions at Gansu, respectively. Deviations from Mendelian genetics were observed. It was hypothesized that the change of fertility was the result of the interaction between nuclear genes [restoring gene (Rf) and temperature‐sensitive genes (ts)] and the cytoplasm. The Rf gene in FL‐204 was incapable of restoring male fertility of pol CMS lines under spring sowing conditions at Gansu where it is inactivated by the recessive ts gene present in FL‐204. However, the ts gene(s) could be non‐functional under moderate temperature conditions at flowering at Wuhan which allows full expression of male fertility in FL‐204. The recessive ts gene(s) can only be expressed in plants containing the pol sterile cytoplasm. A method for the utilization of the FTSR pol CMS restorer FL‐204 for the production of hybrid seed in B. napus oilseed rape is proposed.

Inheritance of seed colour and identification of RAPD and AFLP markers linked to the seed colour gene in rapeseed (Brassica napus L.)

In China Polima cytoplasmic male sterility (cms) is currently the most important hybrid system used for the breeding of hybrids. In an effort to develop yellow-seeded Polima cms restorer lines, we used yellow-seeded, doubled haploid (DH) line No.2127-17 as the gene source in crosses with two elite black-seeded Polima cms R lines, Hui5148-2 and 99Yu42, which originated from our breeding programme. The inheritance of seed colour was investigated in the F2, BC1 and F1-derived DH progenies of the two crosses. Seed colour was found to be under the control of the maternal genotype and the yellow seed trait to be partially dominant over the black seed trait. Segregation analysis revealed a single gene locus for the partial dominance of yellow seed colour. Of 810 randomly amplified polymorphic DNA (RAPD) primers, 240 (29.6%) revealed polymorphisms between the parents. Of the 240 RAPD primers and 512 amplified fragment length polymorphism (AFLP) primer pairs, four RAPDs and 16 AFLP pairs showed polymorphisms between the bulks, with two RAPD and eight AFLP markers being identified in the vicinity of the seed-coat colour gene locus using a DH progeny population-derived from the cross Hui5148-2xNo.2127-17-of 127 individuals in combination with the bulked segregant analysis strategy. Seven of these latter ten markers were linked to the allele for yellow seed, whereas the other three were linked to the allele for black seed. The seed-coat colour gene locus was bracketed by two tightly linked markers, EA02MG08 (2.4 cM) and S1129 (3.9 cM). The partial dominance and single gene control of the yellow seed-coat colour trait together with the available molecular markers will greatly facilitate the future breeding of yellow-seeded hybrid varieties.

Sequence analysis of the gene correlated with cytoplasmic male sterility (CMS) in rape-seed (Brassica napus) Polima and Shaan 2A

orf224 is a CMS-related mitochondrial gene discovered in Polima cytoplasm. Shaan 2A CMS line is the parent of the first rapeseed hybrid cultivar Qinyou No. 2 that has been grown in many regions of China. In this work, genomic DNA of Polima CMS line and Shaan 2A CMS line were used as templates, two primers of specific oligonucleotides at 5′ and 3′ ends were used, PCR was performed, the amplification fragments were cloned into pGEM-T Easy vectors and DNA sequences were determined. The CMS-associated gene, orf224-1 present in Shaan 2A CMS line, has a sequence highly homologous to the orf224 of the Polima CMS line, except for one nucleotide at position +398. There were only one base (AAC→AGC) and one amino acid (Asn →Ser) differences between the two. The homologies of the two sequences in nucleotide and amino acid were 99.9% and 99.6%, respectively. It is concluded that orf224 in Polima CMS line and orf224-1 of Shaan 2A CMS line are the allele at the same locus in mitochondria.

Sterility and variation resulting from the transfer of polima cytoplasmic male sterility from Brassica napus into Brassica chinensis

Male sterile combinations made from interspecific crosses between the polima CMS line of Brassica napus and varieties of B. chinensis were backcrossed to BC3. Twenty-six selfing lines from B. chinensis were tested for their ability to either maintain complete sterility or to restore fertility in crosses with the polima male sterile line. Results show that four of these hybrids were completely male sterile and two were fertile. The sterility of the B. chinensis with polima cytoplasm was much more stable than male sterile lines with B. campestris and B. chinensis cytoplasm, which were sterile before full flowering but progressively became fertile as flowering proceeded. The results suggest that polima cytoplasm could be a suitable male sterile-inducing allocytoplasm for B. chinensis, as both maintainers and restorers are available, and could supply a reliable pollination control system for hybrid seed production in this species.

Genetic correlation of the orf224/atp6 gene region with Polima CMS in Brassica somatic hybrids.

To identify regions of the mitochondrial genome from the polima or pol male-sterile cytoplasm of Brassica napus that are genetically correlated with cytoplasmic male sterility (CMS) we analyzed mtDNAs of three male-sterile somatic hybrids formed by the fusion of broccoli (B. oleracea L. var. italica) and pol CMS B. napus protoplasts. Fragments characteristic of a 4.5 kb DNA segment that comprises the single organizational difference between sterile pol and fertile cam Brassica mitochondrial genomes were found in all three sterile somatic hybrids. One of these hybrids possessed a mitochondrial genome that was, apart from a limited region around this 4.5 kb CMS-associated segment, collinear with B. oleracea mtDNA. Previous studies have indicated that expression of transcripts spanning the atp6 gene and a chimeric gene, orf24, located on this 4.5 kb DNA segment, is associated with male sterility. The present results indicate that the orf224/atp6 gene region is genetically correlated with male sterility and provide significant additional support for the view that this gene region may be involved in specifying the CMS trait.

The effect of varying levels of hybridity on the performance of three pol CMS summer rape hybrid cultivars (Brassica napus L.)

The Polima (pol) cytoplasmic male sterility system (CMS) has been developed to facilitate the commercial production of hybrid summer rape cultivars. Polima A-lines usually produce small quantities of viable pollen, and further, they suffer from high-temperature-induced male-fertility reversion at temperatures of 30 °C or higher. This pollen production can result in selfing and sibing of the pol A-lines, substantially reducing the hybridity of the field-produced hybrid seed lots. The effects of 5, 15, 25 and 35% A-line contamination on seed yield, oil content and protein content for three different pol CMS summer rape hybrids grown in seven environments over 3 yr were found to be minimal in this study. It is concluded that in cases where the effect of pol A-line contamination on the oil content and (or) protein content of contaminated hybrids is significant, these effects can be predicted from a comparison of the relative oil and protein contents of the pure pol A-line and the pure hybrid. It is also concluded that the current 75% hybridity standard, set by the Canadian Seed Growers Association for the CMS hybrid summer rape seed lots is a reasonable initial minimum standard. Key words: Polima cytoplasmic male sterility, hybrid seed production

The Inheritance of Polima Cytoplasmic Male Sterility in Brassica napus L.

AbstractThe restores tested in this study have only one pair of fertility‐restoring genes (Rf) but many temperature‐sensitive genes (Ts). The Rf genes are alleles at the same locus. The cytoplasm of the restorers RC1, RC2, RC3 and ‘Huaie’ is male sterile, but that of RC4, is normal.

Studies on “Three Line” Polima Cytoplasmic Male Sterility Developed in Brassica napus L.

AbstractPolima CMS lines can be divided into three groups according to the sensitivity of male sterility to temperature: 1) high temperature CMS lines (62.9%), 2) low temperature CMS lines (28.6 %) and 3) stable CMS lines (8.5 %). The heterosis of Pol CMS hybrids is evident and common in some of the characters tested, especially in plant seed yield and siliqua number. The cytoplasm has no deleterious effects on the FI characters tested.

Inheritance of male fertility restoration and allelism of restorer genes for the Polima cytoplasmic male sterility system in oilseed rape

The inheritance of male fertility restoration for the pol cytoplasmic male sterility (CMS) system in summer rape (Brassica napus L.) was determined. Male fertility:sterility segregation ratios observed in F2 and backcross generations derived from crosses and backcrosses between two pol CMS A lines and the male fertility restorer gene(s) sources Italy and UM2353 were used in this study. Italy and UM2353 were found to possess a single Mendelian dominant gene with high male fertility restoration capabilities for the pol CMS system. Tests for allelism of the restorer genes were also conducted using male fertility:sterility segregation ratios observed in F3 families derived from crosses between F1 plants containing genes for male fertility restoration from the Italy and UM2353 restorer gene sources. The male fertility restorer gene from Italy (designated Rfp1) was found to be different (i.e., nonallelic) from the restorer gene possessed by UM2353 (designated Rfp2).Key words: Brassica napus L., oilseed rape, male fertility restoration, pol cytoplasmic male sterility.