Cytoplasmic Male Sterility Types 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.

Differentially Expressed Genes Shared by Two Distinct Cytoplasmic Male Sterility (CMS) Types of Silene vulgaris Suggest the Importance of Oxidative Stress in Pollen Abortion.

Cytoplasmic male sterility (CMS), encoded by the interacting mitochondrial and nuclear genes, causes pollen abortion or non-viability. CMS is widely used in agriculture and extensively studied in crops. Much less is known about CMS in wild species. We performed a comparative transcriptomic analysis of male sterile and fertile individuals of Silene vulgaris, a model plant for the study of gynodioecy, to reveal the genes responsible for pollen abortion in this species. We used RNA-seq datasets previously employed for the analysis of mitochondrial and plastid transcriptomes of female and hermaphrodite flower buds, making it possible to compare the transcriptomes derived from three genomes in the same RNA specimen. We assembled de novo transcriptomes for two haplotypes of S. vulgaris and identified differentially expressed genes between the females and hermaphrodites, associated with stress response or pollen development. The gene for alternative oxidase was downregulated in females. The genetic pathways controlling CMS in S. vulgaris are similar to those in crops. The high number of the differentially expressed nuclear genes contrasts with the uniformity of organellar transcriptomes across genders, which suggests these pathways are evolutionarily conserved and that selective mechanisms may shield organellar transcription against changes in the cytoplasmic transcriptome.

РЕАКЦИЯ НОВЫХ САМООПЫЛЕННЫХ ЛИНИЙ КУКУРУЗЫ НА ПАРАГВАЙСКИЙ ТИП ЦМС

It is extremely important to study cytoplasmic male sterility (CMS) in maize since it is impossible to conduct large-scale commercial seed production of hybridized maize without CMS. The purpose of the current study was to identify the response of the new self-pollinated maize lines in a sterile cytoplasm of the Paraguay type, to group the lines according to the composition of fertility restoring genes based on an optimal quantity of the analyzed crossings. The field trials were carried out in 2016–2019 in the State Scientific Establishment “Agricultural research center “Donskoy”” (SSE “ARC “Donskoy”). To obtain hybrid combinations, the method of full top-crosses, namely eight testers and ten lines, was used. The new early-ripening and mid-ripening self-pollinated maize lines (I6), developed in the ARC “Donskoy”, were grouped according to the composition of the alleles of fertility restoring genes of the Paraguay (“C”) type of CMS based on the flowering pattern of the test cross F1 hybrids. The first group included the line SP 207 (genotype rf4rf4 rf5rf5 rf6rf6); the second group included the line SP 209 (genotype rf4rf4 rf5rf5 Rf6Rf6); the third group included the line SP 194 (genotype rf4rf4 Rf5Rf5 rf6rf6); the fourth group included the line SP 198 (genotype Rf4Rf4 rf5rf5 rf6rf6); the fifth group included the lines SP 203 and SP 195 (genotype rf4rf4 Rf5Rf5 Rf6Rf6); the sixth group included the line SP 206 (genotype Rf4Rf4 rf5rf5 Rf6Rf6); the seventh group included the line SP 180 (genotype Rf4Rf4 Rf5Rf5 rf6rf6); the eighth group included the lines SP 210 and SP 197 (genotype Rf4Rf4 Rf5Rf5 Rf6Rf6). The lines SP 207, SP 210 and SP 197 were of the greatest practical value when converting maize hybrids to a sterile base: the line SP 207 was a complete sterility fixer; the lines SP 210 and SP 197 were constant natural fertility restorers. In order to optimize the quantity of the analyzed crossings for grouping the maize lines according to the composition of the alleles of fertility restoring genes of the Paraguay (“C”) type of CMS, it was necessary to use such sterile sources as KR 21C (group IV), WF9C (group V), LC (group VI), W401C (group VII). The minimum quantity of analyzers to determine the response of the lines on the Paraguay type of CMS should be at least two. However, it was necessary to use sterile sources of certain classes, such as KR 21C (group IV) and Gb834 C (group I).

Development of Fertility-Restoring Lines for 9E Type CMS of Sorghum Using Environmentally Induced Fertility Revertants

Development of reliable fertility-restoring lines is the most important prerequisite for using cytoplasmic male sterility (CMS) in plant breeding. Sorghum 9E type CMS is characterized by unstable expression of fertility-restoring genes, which require high levels of water availability for plants during microsporogenesis and flowering. In order to create reliable male-fertility-restoring lines for 9E type CMS, the progeny of test crosses of fertile revertant lines in 9E cytoplasm, which were obtained as a result of reversions in sterile F1 hybrids induced by conditions of high water availability, with CMS lines in this cytoplasm were grown in selective backgrounds: in the dry plots and in the plots with additional watering as well as in the field under natural moisture supply. In the majority of hybrid combinations, fertile plants were observed only when additionally watered during the flowering period. F1 hybrids with different CMS lines with 9E cytoplasm significantly differed in terms of the degree of restored fertility and the response to water availability conditions. Fertile plants predominated in the F2 families; in different hybrid combinations, the ratio of restored and sterile plants corresponded to 3 : 1 or 13 : 3 segregation. After selection in the drought conditions of the fertile plants from the offspring of hybrids from the cross of the CMS line 9E Pishchevoe 614 and male-fertile revertants, two lines were obtained that were able to restore fertility of F1 hybrids in field conditions.

Influence of different types of sterile cytoplasms (A3, A4, 9E) on the combining ability of CMS lines of sorghum.

Investigation of the effect of the cytoplasm on the combining ability (CA) of lines with cytoplasmic male sterility (CMS) is of considerable interest in terms of understanding the genetic functions of the cytoplasm and for practical purposes to create hybrids with improved economically valuable traits. In order to investigate the effect of different types of sterile cytoplasm (A3, A4, 9E) on CA in sorghum, we studied the manifestation of a number of biological and agronomic traits in 54 F1 hybrid combinations obtained using iso-nuclear CMS lines with the nuclear genome of the line Zheltozernoye 10, differing only in the types of sterile cytoplasm (A3, A4 and 9E). Eighteen varieties and lines of grain sorghum developed at the Russian Research and Project-technological Institute of Sorghum and Maize were used as paternal parents. The CA was determined by the topcross method. F1 hybrids and their parents were grown in 2015–2017 in conditions of insufficient (2015–2016: HTC (hydro-thermal coefficient) = 0.32–0.66), or good water availability conditions (2017: HTC = 1.00). On average, for three years of testing, a positive effect of the 9E cytoplasm on the general combining ability (GCA) (0.63) and negative effects of the A3 and A4 cytoplasms (–0.32 and –0.31) for the inflorescence length were noted. In dry seasons, significant positive effects of the 9E cytoplasm on GCA for the length of the largest leaf, and positive effects of the A3 cytoplasm on GCA for the plant height, and negative effects of the A4 cytoplasm on GCA for these traits were observed. No differences were observed during the wet season. The type of CMS did not affect the GCA for the width of the largest leaf and grain yield. The dispersion of specific combining ability (SCA) in the dry seasons was significant for the following traits: leaf length, plant height, panicle length and width, and grain yield, the 9E cytoplasm had the highest SCA dispersion, whereas the A4 cytoplasm had the smallest one. The data obtained indicate that different types of sterile cytoplasm of sorghum make a different contribution to CA under conditions of drought stress.

Identification of fertility-related genes for maize CMS-S via Bulked Segregant RNA-Seq

Cytoplasmic male sterility (CMS) is extensively used in maize hybrid production, and identification of genes related to fertility restoration for CMS is important for hybrid breeding. The fertility restoration of S type CMS is governed by several loci with major and minor effects, while the mechanism of fertility restoration for CMS-S is still unknown. In this study, BSR-Seq was conducted with two backcrossing populations with the fertility restoration genes, Rf3 and Rf10, respectively. Genetic mapping via BSR-Seq verified the positions of the two loci. A total of 353 and 176 differentially expressed genes (DEGs) between the male fertility and male sterile pools were identified in the populations with Rf3 and Rf10, respectively. In total, 265 DEGs were co-expressed in the two populations, which were up-regulated in the fertile plants, and they might be related to male fertility involving in anther or pollen development. Moreover, 35 and seven DEGs were specifically up-regulated in the fertile plants of the population with Rf3 and Rf10, respectively. Function analysis of these DEGs revealed that jasmonic acid (JA) signal pathway might be involved in the Rf3 mediated fertility restoration for CMS-S, while the small ubiquitin-related modifier system could play a role in the fertility restoration of Rf10.

Fertility restoration of Chinese wild rice-type cytoplasmic male sterility by CRISPR/Cas9-mediated genome editing of nuclear-encoded RETROGRADE-REGULATED MALE STERILITY.

Cytoplasmic male sterility (CMS) is a trait that produces nonfunctional pollen caused by the interaction between mitochondrial and nuclear genes. In Chinese-wild (CW) type CMS, CWA, in rice (Oryza sativa L.), its mitochondria enhance the expression of the nuclear gene RETROGRADE-REGULATED MALE STERILITY (RMS), which causes pollen abortion. Fertility is recovered when its expression decreases in a restorer line, CWR. The expression of RMS is controlled by the single nucleotide polymorphism (SNP) located in the promoter region 2,286 bp upstream of the start codon of RMS. However, another gene, PPR2, which encodes pentatricopeptide repeat-domain containing protein, is predicted in the reverse strand of this region and a premature stop codon is created in CWR by the SNP. To prove RMS is directly involved in restoring fertility of CW-CMS, we introduced mutations into RMS and PPR2 using CRISPR/Cas9. Fertility was recovered in the genome-edited CMS plants with reduced expression of RMS and unaltered expression of PPR2, when the mutation was introduced in the promoter regions of RMS within or outside the coding sequence (CDS) of PPR2. Fertility restoration was not obtained when the mutation was introduced within the CDS of RMS. Our results demonstrated that PPR2 is not responsible for fertility restoration, and fertility was recovered by reduced expression of RMS, providing us with a new artificial fertility restorer line for agronomical use.

QRf8-1, a Novel QTL for the Fertility Restoration of Maize CMS-C Identified by QTL-seq.

C-type cytoplasmic male sterility (CMS-C), one of the three major CMS types in maize, has a promising application prospect in hybrid seed production. However, the complex genetic mechanism underlying the fertility restoration of CMS-C remains poorly understood. The maize inbred line A619 is one of the rare strong restorer lines carrying the restorer gene Rf4, but different fertility segregation ratios are found in several F2 populations derived from crosses between isocytoplasmic allonucleus CMS-C lines and A619. In the present study, the segregation ratios of fertile to sterile plants in the (CHuangzaosi × A619) F2 and BC1F1 populations (36.77:1 and 2.36:1, respectively) did not follow a typical monogenic model of inheritance, which suggested that some F2 and BC1F1 plants displayed restored fertility even without Rf4. To determine the hidden locus affecting fertility restoration, next-generation sequencing-based QTL-seq was performed with two specific extreme bulks consisting of 30 fertile and 30 sterile rf4rf4 individuals from the F2 population. A major QTL related to fertility restoration, designated qRf8-1, was detected on the long arm of chromosome 8 in A619. Subsequently, qRf8-1 was further validated and narrowed down to a 17.93-Mb genomic interval by insertion and deletion (InDel) and simple sequence repeat (SSR) marker-based traditional QTL mapping, explaining 12.59% (LOD = 25.06) of the phenotypic variation. Thus, using genetic analyses and molecular markers, we revealed another fertility restoration system acting in parallel with Rf4 in A619 that could rescue the male sterility of CHuangzaosi. This study not only expands the original fertility restoration system but also provides valuable insights into the complex genetic mechanisms underlying the fertility restoration of CMS-C.

Utilization of Ogura CMS germplasm with the clubroot resistance gene by fertility restoration and cytoplasm replacement in Brassica oleracea L

Clubroot disease, a major plant root disease caused by Plasmodiophora brassicae, has become one of the most destructive diseases among cultivated cruciferous vegetables. However, clubroot-resistant Brassica oleracea materials are rare. A few clubroot-resistant cabbage varieties are available on the market, but all are Ogura cytoplasmic male sterile (CMS) types. Therefore, in this study, to reutilize the clubroot-resistant Ogura CMS germplasm of cabbage, a new fertility-restored Ogura CMS material, 16Q2-11, was used as a bridge to transfer the clubroot resistance (CR) gene from the Ogura CMS cytoplasm to the normal cytoplasm by a two-step method (a fertility restoration and cytoplasm replacement method). In the first cross for fertility restoration of Ogura CMS clubroot-resistant cabbage (FRCRC), 16Q2-11 was used as a restorer to cross with Ogura CMS materials containing the CR gene CRb2. Eleven Rfo-positive progenies were generated, of which four contained CRb2: F8-514, F8-620, F8-732 and F8-839. After inoculation with race 4 of P. brassicae, these four CRb2-positive individuals showed resistance. Furthermore, F8-514 and F8-839 were then used as male parents in the second cross of FRCRC to cross with cabbage inbred lines, resulting in the successful introgression of the CRb2 gene into the inbred lines. All offspring produced from this step of cross, which had a normal cytoplasm, showed a high resistance to race 4 of P. brassicae and could be utilized for the breeding of clubroot-resistant cabbage varieties in the future. This is the first time that the Ogura CMS restorer has been used to restore the fertility of Ogura CMS clubroot-resistant cabbages, which could improve germplasm diversity in cabbage and provide a reference method for using CMS germplasm in Brassica crops.

Genome-wide analysis of mRNA and lncRNA expression and mitochondrial genome sequencing provide insights into the mechanisms underlying a novel cytoplasmic male sterility system, BVRC-CMS96, in Brassicarapa.

Characterization of a novel and valuable CMS system in Brassicarapa. Cytoplasmic male sterility (CMS) is extensively used to produce F1 hybrid seeds in a variety of crops. However, it has not been successfully used in Chinese cabbage (Brassicarapa L. ssp. pekinensis) because of degeneration or temperature sensitivity. Here, we characterize a novel CMS system, BVRC-CMS96, which originated in B.napus cybrid obtained from INRAE, France and transferred by us to B.rapa. Floral morphology and agronomic characteristics indicate that BVRC-CMS96 plants are 100% male sterile and show no degeneration in the BC7 generation, confirming its suitability for commercial use. We also sequenced the BVRC-CMS96 and maintainer line 18BCM mitochondrial genomes. Genomic analyses showed the presence of syntenic blocks and distinct structures between BVRC-CMS96 and 18BCM and the other known CMS systems. We found that BVRC-CMS96 has one orf222 from 'Nap'-type CMS and two copies of orf138 from 'Ogu'-type CMS. We analyzed expression of orf222, orf138, orf261b, and the mitochondrial energy genes (atp6, atp9, and cox1) in flower bud developmental stages S1-S5 and in four floral organs. orf138 and orf222 were both highly expressed in S4, S5-stage buds, calyx, and the stamen. RNA-seq identified differentially expressed mRNAs and lncRNAs (long non-coding RNAs) that were significantly enriched in pollen wall assembly, pollen development, and pollen coat. Our findings suggest that an energy supply disorder caused by orf222/orf138/orf261b may inhibit a series of nuclear pollen development-related genes. Our study shows that BVRC-CMS96 is a valuable CMS system, and our detailed molecular analysis will facilitate its application in Chinese cabbage breeding.

Mitochondrial orf463 causing male sterility in radish is possessed by cultivars belonging to the ‘Niger’ group

In addition to Ogura cytoplasmic male sterility (CMS), which is used worldwide in radish and Brassica crops, another type of CMS was found and named DCGMS. A mitochondrial candidate gene (orf463) of DCGMS was identified, but the distribution of this gene in radish species has not been clarified to date. We found that orf463 is distributed specifically in black radish cultivars belonging to the ‘Niger’ group and a line of wild species, Raphanus raphanistrum. The progeny tests using the cultivars having orf463 as a female parent demonstrated that the cytoplasm induces male sterility in radishes. Although the DNA sequences of orf463 possessed by the ‘Niger’ group cultivars are identical to those of DCGMS, the orf463 sequence of the wild radish line contained nine nucleotide substitutions, seven of which were nonsynonymous. All radishes with orf463 had both DCGMS-type and ‘Black radish’- type mitochondrial structures according to multiplex PCR. Based on these results, the mechanism of rearrangement between the two mitochondrial types was studied.

Characterization of the mitochondrial genome of the MAX1 type of cytoplasmic male-sterile sunflower

BackgroundMore than 70 cytoplasmic male sterility (CMS) types have been identified in Helianthus, but only for less than half of them, research of mitochondrial organization has been conducted. Moreover, complete mitochondrion sequences have only been published for two CMS sources – PET1 and PET2. It has been demonstrated that other sunflower CMS sources like MAX1, significantly differ from the PET1 and PET2 types. However, possible molecular causes for the CMS induction by MAX1 have not yet been proposed. In the present study, we have investigated structural changes in the mitochondrial genome of HA89 (MAX1) CMS sunflower line in comparison to the fertile mitochondrial genome.ResultsEight significant major reorganization events have been determined in HA89 (MAX1) mtDNA: one 110 kb inverted region, four deletions of 439 bp, 978 bp, 3183 bp and 14,296 bp, respectively, and three insertions of 1999 bp, 5272 bp and 6583 bp. The rearrangements have led to functional changes in the mitochondrial genome of HA89 (MAX1) resulting in the complete elimination of orf777 and the appearance of new ORFs - orf306, orf480, orf645 and orf1287. Aligning the mtDNA of the CMS sources PET1 and PET2 with MAX1 we found some common reorganization features in their mitochondrial genome sequences.ConclusionThe new open reading frame orf1287, representing a chimeric atp6 gene, may play a key role in MAX1 CMS phenotype formation in sunflower, while the contribution of other mitochondrial reorganizations seems to appear negligible for the CMS development.

Identification of a gene responsible for cytoplasmic male-sterility in onions (Allium cepa L.) using comparative analysis of mitochondrial genome sequences of two recently diverged cytoplasms.

Almost identical mitochondrial genome sequences of two recently diverged male-fertile normal and male-sterile CMS-T-like cytoplasms were obtained in onions. A chimeric gene, orf725 , was found to be a CMS-inducing gene. In onions (Allium cepa L.), cytoplasmic male-sterility (CMS) has been widely used in hybrid seed production. Two types of CMS (CMS-S and CMS-T) have been reported in onions. A complete mitochondrial genome sequence of the CMS-S cytoplasm has been reported in our previous study. Draft mitochondrial genome sequences of male-fertile normal and CMS-T-like cytoplasms are reported in this study. Raw reads obtained from normal and CMS-T-like cytoplasms were assembled into eight and nine almost identical contigs, respectively. After connection and reorganization of contigs by PCR amplification and genome walking, four scaffold sequences with total length of 339 and 180bp were produced for the normal cytoplasm. A mitochondrial genome sequence of the CMS-T-like cytoplasm was obtained by mapping trimmed reads of CMS-T onto scaffold sequences of the normal cytoplasm. Compared with the CMS-S mitochondrial genome, the normal mitochondrial genome was highly rearranged with 31 syntenic blocks. A total of 499 single nucleotide polymorphisms (SNPs) or insertions/deletions were identified in these syntenic regions. On the other hand, normal and CMS-T-like mitochondrial genome sequences were almost identical except for orf725, a chimeric gene consisting of cox1 with other sequences. Only three SNPs were identified between normal and CMS-T-like syntenic sequences. These results indicate that orf725 is likely to be the casual gene for CMS induction in onions and that CMS-T-like cytoplasm has recently diverged from the normal cytoplasm by introduction of orf725.

Comparative transcriptome analysis reveals that tricarboxylic acid cycle-related genes are associated with maize CMS-C fertility restoration

BackgroundC-type cytoplasmic male sterility (CMS-C) is one of the three major types of cytoplasmic male sterility (CMS) in maize. Rf4 is a dominant restorer gene for CMS-C and has great value in hybrid maize breeding, but little information concerning its functional mechanism is known.ResultsTo reveal the functional mechanism of Rf4, we developed a pair of maize near-isogenic lines (NILs) for the Rf4 locus, which included a NIL_rf4 male-sterile line and a NIL_Rf4 male fertility-restored line. Genetic analysis and molecular marker detection indicated that the male fertility of NIL_Rf4 was controlled by Rf4. Whole-genome sequencing demonstrated genomic differences between the two NILs was clustered in the Rf4 mapping region. Unmapped reads of NILs were further assembled to uncover Rf4 candidates. RNA-Seq was then performed for the developing anthers of the NILs to identify critical genes and pathways associated with fertility restoration. A total of 7125 differentially expressed genes (DEGs) were identified. These DEGs were significantly enriched in 242 Gene Ontology (GO) categories, wherein 100 DEGs were involved in pollen tube development, pollen tube growth, pollen development, and gametophyte development. Homology analysis revealed 198 male fertility-related DEGs, and pathway enrichment analysis revealed that 58 DEGs were enriched in cell energy metabolism processes involved in glycolysis, the pentose phosphate pathway, and pyruvate metabolism. By querying the Plant Reactome Pathway database, we found that 14 of the DEGs were involved in the mitochondrial tricarboxylic acid (TCA) cycle and that most of them belonged to the isocitrate dehydrogenase (IDH) and oxoglutarate dehydrogenase (OGDH) enzyme complexes. Transcriptome sequencing and real-time quantitative PCR (qPCR) showed that all the above TCA cycle-related genes were up-regulated in NIL_Rf4. The results of our subsequent enzyme-linked immunosorbent assay (ELISA) experiments pointed out that the contents of both the IDH and OGDH enzymes accumulated more in the spikelets of NIL_Rf4 than in those of NIL_rf4.ConclusionThe present research provides valuable genomic resources for deep insight into the molecular mechanism underlying CMS-C male fertility restoration. Importantly, our results indicated that genes involved in energy metabolism, especially some mitochondrial TCA cycle-related genes, were associated with maize CMS-C male fertility restoration.

Mitochondrial genomes organization in alloplasmic lines of sunflower (Helianthus annuus L.) with various types of cytoplasmic male sterility.

BackgroundCytoplasmic male sterility (CMS) is a common phenotype in higher plants, that is often associated with rearrangements in mitochondrial DNA (mtDNA), and is widely used to produce hybrid seeds in a variety of valuable crop species. Investigation of the CMS phenomenon promotes understanding of fundamental issues of nuclear-cytoplasmic interactions in the ontogeny of higher plants. In the present study, we analyzed the structural changes in mitochondrial genomes of three alloplasmic lines of sunflower (Helianthus annuus L.). The investigation was focused on CMS line PET2, as there are very few reports about its mtDNA organization.MethodsThe NGS sequencing, de novo assembly, and annotation of sunflower mitochondrial genomes were performed. The comparative analysis of mtDNA of HA89 fertile line and two HA89 CMS lines (PET1, PET2) occurred.ResultsThe mtDNA of the HA89 fertile line was almost identical to the HA412 line (NC_023337). The comparative analysis of HA89 fertile and CMS (PET1) analog mitochondrial genomes revealed 11,852 bp inversion, 4,732 bp insertion, 451 bp deletion and 18 variant sites. In the mtDNA of HA89 (PET2) CMS line we determined 27.5 kb and 106.5 kb translocations, 711 bp and 3,780 bp deletions, as well as, 5,050 bp and 15,885 bp insertions. There are also 83 polymorphic sites in the PET2 mitochondrial genome, as compared with the fertile line.DiscussionThe observed mitochondrial reorganizations in PET1 resulted in only one new open reading frame formation (orfH522), and PET2 mtDNA rearrangements led to the elimination of orf777, duplication of atp6 gene and appearance of four new ORFs with transcription activity specific for the HA89 (PET2) CMS line—orf645, orf2565, orf228 and orf285. Orf228 and orf285 are the atp9 chimeric ORFs, containing transmembrane domains and possibly may impact on mitochondrial membrane potential. So orf228 and orf285 may be the cause for the appearance of the PET2 CMS phenotype, while the contribution of other mtDNA reorganizations in CMS formation is negligible.

Rf4 has minor effects on the fertility restoration of wild abortive-type cytoplasmic male sterile japonica (Oryza sativa) lines

Wild abortive (WA)-type cytoplasmic male sterility (CMS) has been exclusively used for breeding three-line hybrid indica rice, but it has not been applied for generating japonica hybrids because of the difficulties related to breeding japonica restorer lines. Determining whether the major restorer-of-fertility (Rf) gene used for indica hybrids can efficiently restore the fertility of WA-type japonica CMS lines may be useful for breeding WA-type japonica restorer lines. In this study, japonica restorer lines for Chinsurah Boro II (BT)-type CMS exhibited varying abilities to restore the fertility of ‘WA-LiuqianxinA’, which is a WA-type japonica CMS line. Additionally, Rf genes for WA-type CMS were identified in the BT-type japonica restorers. Meanwhile, ‘C9083’, which is a BT-type japonica restorer, exhibited a limited ability to restore the fertility of WA-type japonica CMS lines, and a genetic analysis revealed that the fertility restoration was controlled by one locus. The Rf gene was mapped to an approximately 370-kb physical region and was identified as Rf4. Furthermore, Rf gene dosage effects and the temperature influenced the fertility restoration of WA-type japonica CMS lines. This study is the first to confirm that Rf4 has only minor effects on the fertility restoration of WA-type japonica CMS lines. These results may be relevant for the development of WA-type japonica hybrids.

Restorer-of-Fertility Mutations Recovered in Transposon-Active Lines of S Male-Sterile Maize.

Mitochondria execute key pathways of central metabolism and serve as cellular sensing and signaling entities, functions that depend upon interactions between mitochondrial and nuclear genetic systems. This is exemplified in cytoplasmic male sterility type S (CMS-S) of Zea mays, where novel mitochondrial open reading frames are associated with a pollen collapse phenotype, but nuclear restorer-of-fertility (restorer) mutations rescue pollen function. To better understand these genetic interactions, we screened Activator-Dissociation (Ac-Ds), Enhancer/Suppressor-mutator (En/Spm), and Mutator (Mu) transposon-active CMS-S stocks to recover new restorer mutants. The frequency of restorer mutations increased in transposon-active stocks compared to transposon-inactive stocks, but most mutants recovered from Ac-Ds and En/Spm stocks were unstable, reverting upon backcrossing to CMS-S inbred lines. However, 10 independent restorer mutations recovered from CMS-S Mu transposon stocks were stable upon backcrossing. Many restorer mutations condition seed-lethal phenotypes that provide a convenient test for allelism. Eight such mutants recovered in this study included one pair of allelic mutations that were also allelic to the previously described rfl2-1 mutant. Targeted analysis of mitochondrial proteins by immunoblot identified two features that consistently distinguished restored CMS-S pollen from comparably staged, normal-cytoplasm, nonmutant pollen: increased abundance of nuclear-encoded alternative oxidase relative to mitochondria-encoded cytochrome oxidase and decreased abundance of mitochondria-encoded ATP synthase subunit 1 compared to nuclear-encoded ATP synthase subunit 2. CMS-S restorer mutants thus revealed a metabolic plasticity in maize pollen, and further study of these mutants will provide new insights into mitochondrial functions that are critical to pollen and seed development.

Choice of microsatellite markers for isolation of fertility restorers of wild abortive (WA) type cytoplasmic male sterility in rice

Simple sequence length polymorphism was examined in a set of eight effective restorers, four partial restorers, two weak maintainers and four complete maintainers of WA type CMS system in rice using a set of thirty-two microsatellite primer pairs in order to assess the nature and extent of genetic variation among them at molecular level. Altogether 248 allelic variants were detected with an average of 7.75 alleles per primer pairs. Polymorphic information content ranged from 0.508 for RM 6737 to 0.882 for RM 1 with an average value of 0.740 per primer pair. Amongst the primer pairs used, RM 1, RM 206, RM 591 RM 3873 and RM 8146 had remarkably higher number of allelic variants with greater gene diversity and genotype discrimination ability. Using similarity coefficient in numerical taxonomic approach of classification, the entries were differentiated and classified into different groups. Analysis based on molecular profiles allowed discrimination of effective fertility restorers from rest of the entries including partial fertility restorers, weak sterility maintainers and complete sterility maintainers. Principal coordinate analysis based two dimensional plotting of genetic profiles completely supported the results obtained from hierarchical classification of entries and effective fertility restorers were distinctly discriminated from the remaining entries. Four microsatellite primer pairs namely, RM 171, RM 315, RM 3873 and RM 6100, which allowed unambiguous discrimination of the eight effective fertility restorers from rest of the entries. These microsatellite primer pairs, may be further utilized in identification of effective fertility restorers of WA based CMS system in rice.

Advances in understanding the molecular mechanisms of cytoplasmic male sterility and restoration in rice.

Cytoplasmic male sterility (CMS) in plants is a male reproductive defect determined by mitochondrial genes and inherited maternally. CMS can be suppressed by nuclear restorer of fertility (Rf) genes. Therefore, CMS/Rf systems provide a classic model for the study of mitochondrial-nuclear interactions in plants. Moreover, CMS/Rf systems are economical, effective tools for the production of hybrid seeds. For example, CMS/Rf systems have been applied in over forty countries to breed hybrid rice (Oryza sativa L.) with improved yields due to hybrid vigor. The production of hybrid rice mainly depends on three types of CMS systems, namely Wild-Abortive type CMS (CMS-WA), Hong-Lian type CMS (CMS-HL) and Boro II type CMS (CMS-BT). Understanding the molecular mechanisms underlying these CMS/Rf systems will help us to understand mitochondrial-nuclear interactions, and accelerate the utilization of heterosis for improvement in yield. In the past decades, research benefitting from the availability of the high-quality, annotated mitochondrial and nuclear genome sequences of rice has isolated many CMS genes, identified the cognate nuclear Rf genes and studied the molecular mechanisms underlying CMS and restoration in rice. Here, we focus on recent advances in studies of the three major CMS/Rf systems in rice and discuss the key issues facing basic research and application of CMS/Rf systems in the future.