Summary
Type C cytoplasmic male sterility (CMS‐C) is the most commonly used form of CMS in maize hybrid seed production. Restorer of fertility 4 (Rf4), the major fertility restorer gene of CMS‐C, is ...located on chromosome 8S. To positionally clone Rf4, a large F3 population derived from a cross between a non‐restorer and restorer (n = 5104) was screened for recombinants and then phenotyped for tassel fertility, resulting in a final map‐based cloning interval of 12 kb. Within this 12‐kb interval, the only likely candidate for Rf4 was GRMZM2G021276, a basic helix−loop−helix (bHLH) transcription factor with tassel‐specific expression. The Rf4 gene product contains a nuclear localization signal and is likely to not interact directly with the mitochondria. Sequence analysis of Rf4 revealed four encoded amino acid substitutions between restoring and non‐restoring inbreds, however only one substitution, F187Y, was within the highly conserved bHLH domain. The hypothesis that Rf4 restoration is altered by a single amino acid was tested by using clustered regularly interspaced short palindromic repeat (CRISPR)‐CRISPR associated protein 9 (Cas9) homology directed repair (HDR) to create isogenic lines that varied for the F187Y substitution. In a population of these CRISPR‐Cas9 edited plants (n = 780) that was phenotyped for tassel fertility, plants containing F187 were completely fertile, indicating fertility restoration, and plants containing Y187 were sterile, indicating lack of fertility restoration. Structural modeling shows that this amino acid residue 187 is located within the four helix bundle core, a critical region for stabilizing dimer conformation and affecting interaction partner selection.
Significance Statement
Using positional cloning, we determined that Rf4, the major fertility restorer for CMS‐C, is a bHLH transcription factor also annotated as Ms23, an essential early acting factor in tapetal development, suggesting the CMS restoring ability of this gene is a secondary function. We identified and confirmed, with CRISPR‐Cas9 HDR editing, the restoration casual amino acid substitution, at the center of the bHLH dimer interface.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
The extraction of high-quality RNA from cotton (Gossypium spp.) is challenging because of the presence of high polyphenolics, polysaccharides, quinones, and other secondary metabolites. A ...high-throughput RNA extraction protocol is a prerequisite. This Triton-X-100-based RNA extraction method utilizes Polyvinyl pyrrolidone polymer (PVPP) treatment which efficiently removes phenolics, and the application of Lithium chloride (LiCl) has been found that successfully precipitated the high-quality RNA from cotton tissue. Cytoplasmic male sterility (CMS) is a maternally inherited trait associated with specific mitochondrial genome rearrangements or mutations. The suitability of RNA extracted from Cotton CMS lines was assessed. cDNA was synthesized from RNA and assayed for mitochondrial genes (cox3, nad3, nad9) associated with male sterility. This paper discuss the advantages and limitation of this protocol over existing protocol for RNA extraction for polyphenolics-rich plant tissue.
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•Efficient protocol with a minimum amount of plant material is required.•Extraction protocol is equally useful for plants having diverse lipids/fatty acids, polysaccharides, and polyphenols.•High throughput protocol is simple, economic, less time-consuming, efficient and applicable to cytoplasmic genome analysis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
•This study determined that the cellulose synthase-like gene CaCslD1 was specifically expressed in fertile anthers of pepper.•Down-regulation of CaCslD1 expression caused anther crumpling and pollen ...grain shrinking and decreased pollen viability of pepper.•CaCslD1 was involved in the regulation of male fertility in pepper.
Cytoplasmic male sterility (CMS) plays a pivotal role in harnessing heterosis, which is of great significance for improving the yield and quality of hybrids. In our previous study, a cellulose synthase-like genes, CaCslD1, was characterized to be related to the fertility of CMS by bulked segregant RNA sequencing (BSR-seq) in pepper. In this study, CaCslD1 exhibited strongly specific expression in anther compared to the other tissues of flower buds in pepper. The expressing profiles of CaCslD1 was highest in the hybrid F1 (H1, H2 and H3) of CMS lines and restorer lines, followed by restorer lines (R1, R2, and R3) and maintainer line (M1, M2 and M3), while it showed negligible expression in CMS lines. The CaCslD1-silenced plants of maintainer line 5B, R1 and F1 showed abnormal phenotypic characterized by crumpled anthers, reduced and defective pollen grains and decreased pollen activities, which suggested that CaCslD1 played an important role in anther and pollen development and was involved in the fertility regulation in pepper. These results provide new insights into the molecular mechanism of fertility in pepper.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Plant cytoplasmic male sterility is caused by mutations and rearrangements of mitochondrial genes. It serves as a significant way to utilize hybrid vigor to enhance crop yield. Ogu CMS is a natural ...cytoplasmic male sterility type discovered in radishes, being successfully transferred to rapeseed and cruciferous vegetables. However, current studies lack depth in exploring the molecular mechanisms of its male sterility. In this study, we confirmed orf138 is the causal gene for Ogu CMS through genetic transformation in Arabidopsis. Transcriptome analysis of aborted anthers in different stages suggested differentially expressed genes (DEGs) are mainly enriched in pathways such as glycerophospholipid metabolism and arginine and proline metabolism. It reveals that key genes involved in lipid metabolism pathways are significantly down-regulated in the sterile line (OguA), including BnaGPAT1, localized within the tapetum mitochondrial and endoplasmic reticulum. This could lead to changes in the metabolism of substances like acylglycerols within the tapetum, causing disruptions in lipid metabolism. This is consistent with morphological and subcellular structural changes in the tapetum and microspore cells as observed in the transmission electron microscopy. This abnormal lipid metabolism may trigger specific ROS accumulation in an oxidative stress response, ultimately leading to aborted microspore. Our study based on transcriptome has deepened our understanding of the molecular mechanisms in Ogu CMS.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The discovery of a wild abortive-type (WA) cytoplasmic male sterile (CMS) line and breeding its restorer line have led to the commercialization of three-line hybrid rice, contributing considerably to ...global food security. However, the molecular mechanisms underlying fertility abortion and the restoration of CMS-WA lines remain largely elusive. In this study, we cloned a restorer gene, Rf20, following a genome-wide association study analysis of the core parent lines of three-line hybrid rice. We found that Rf20 was present in all core parental lines, but different haplotypes and structural variants of its gene resulted in differences in Rf20 expression levels between sterile and restored lines. Rf20 could restore pollen fertility in the CMS-WA line and was found to be responsible for fertility restoration in some CMS lines under high temperatures. In addition, we found that Rf20 encodes a pentatricopeptide repeat protein that competes with WA352 for binding with COX11. This interaction enhances COX11’s function as a scavenger of reactive oxygen species, which in turn restores pollen fertility. Collectively, our study suggests a new action mode for pentatricopeptide repeat proteins in the fertility restoration of CMS lines, providing an essential theoretical basis for breeding robust restorer lines and for overcoming high temperature-induced fertility recovery of some CMS lines.
Cytoplasmic male sterility (CMS) is an important genetic tool for breeding and exploiting hybrid vigor in crops. Rf genes can make CMS lines fertile. Song et al. show that structural variations of a new fertility restorer gene, Rf20, underlie the restoration of wild abortive-type CMS in rice. Mechanistically, RF20 can compete with WA352 for binding with COX11 to enhance the scavenging of reactive oxygen species for the fertility restoration.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Photoperiod-sensitive cytoplasmic male sterility (PCMS) caused by Aegilops crassa cytoplasm under long-day (LD) photoperiods (≧15 h) has been proposed as a two-line system for producing hybrid ...varieties of common wheat (Triticum aestivum). PCMS lines are maintained by self-pollination under short-day (SD) conditions (<15 h), while outcrossing PCMS plants to a T. aestivum pollinator line under LD conditions produces hybrid seeds. In this system, it is not necessary to have maintainer lines in order to sustain the PCMS lines. Several promising elite PCMS lines with a Japanese wheat cultivar ‘Fukuotome’ genetic background have been developed; these lines show high cross-pollination fertility and high male sterility under LD conditions, and high seed fertility under SD conditions. In addition, the European wheat cultivar ‘Fortunato’ was found to be suitable for use in the PCMS system. Through the use of the PCMS elite lines and the selected pollinator line, F1 seeds have been produced under LD conditions in Hokkaido, Japan, and the agronomic characters of F1 hybrids have been examined under SD conditions in Fukui, Japan. The results indicate the practicality of using this PCMS system for hybrid wheat production in Japan.
In sorghum Sorghum bicolor (L.) Moench, combining ability and heterosis analysis are commonly used to evaluate superior parental lines and to screen for strongly heterotic hybrids, which helps in ...sorghum variety selection and breeding. In this context, combining ability and heterosis analysis were assessed using 14 restorer lines and seven cytoplasmic male sterile (CMS) lines in 2019 and 2020. The analysis of variance of all cross combinations had highly significant differences for all characters studied, which indicated a wide variation across the parents, lines, testers, and crosses. Combining ability analysis showed that the general combining ability (GCA) and specific combining ability (SCA) of the different parents were differed significantly among different traits. Most combinations with high SCA also showed high GCA in their parent lines. The heritability in the narrow sense of grain weight per panicle and grain yield was relatively low, indicating that the ability of these traits to be directly inherited by offspring was weak, that they were greatly affected by the environment. The better-parent heterosis for plant height, grain weight per panicle, panicle length, and 1000-grain weight was consistent with the order of mid-parent heterosis from strong to weak. The GCA effects of two lines 10480A, 3765A and three testers 0-30R, R111, and JY15R were significant for the majority of the agronomic traits including grain yield and might be used for improving the yield of grains in sorghum as parents of excellent specific combining ability. Seven strongly heterotic F.sub.1 hybrids were screened; of these, hybrids 3765A x R111, 1102A x L2R, and 3765A x JY15R showed significant increases in seed iristectorigenin A content and will feature into the creation of new sorghum varieties rich in iristectorigenin A.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Restorer-of-fertility (Rf) genes encode pentatricopeptide repeat (PPR) proteins that are targeted to mitochondria where they specifically bind to transcripts that induce cytoplasmic male sterility ...and repress their expression. In searching for a molecular signature unique to this class of proteins, we found that a majority of known Rf proteins have a distinct domain, which we called RfCTD (Restorer-of-fertility C-terminal domain), and its presence correlates with the ability to induce cleavage of the mitochondrial RNA target. A screen of 219 angiosperm genomes from 123 genera using a sequence profile that can quickly and accurately identify RfCTD sequences revealed considerable variation in RFL/RfCTD gene numbers across flowering plants. We observed that plant genera with bisexual flowers have significantly higher numbers of RFL genes compared to those with unisexual flowers, consistent with a role of these proteins in restoration of male fertility. We show that removing the RfCTD from the RFL protein RNA PROCESSING FACTOR 2-nad6 prevented cleavage of its RNA target, the nad6 transcript, in Arabidopsis thaliana mitochondria. We provide a simple way of identifying putative Rf candidates in genome sequences, new insights into the molecular mode of action of Rf proteins and the evolution of fertility restoration in flowering plants.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
In many plant species, male-sterile female lines with cytoplasmic male sterility (CMS) or nuclear-controlled environment-sensitive genic male sterility (EGMS) have long been used to efficiently ...produce hybrids that harness hybrid vigor or heterosis. However, the underlying molecular mechanisms for these applications have only recently been uncovered in a few species. We provide here an update on the understanding of cytoplasmic–nuclear communication based on the discovery of mitochondrial CMS genes and their corresponding nuclear fertility determinants. Recent findings that uncover diverse mechanisms such as epigenetic, transcriptional, and post-transcriptional controls of EGMS by temperature and photoperiod signals are also reviewed. Furthermore, translational research that applies basic knowledge of plant male fertility control to hybrid seed production practice is highlighted.
CMS lines used for three-line hybrid system can only be recovered by a specific restorer of male fertility (Rf) gene.
CMS genes may be formed during evolution through gene duplication and multi-recombination, and the corresponding nuclear Rf genes appear to have undergone coevolution.
EGMS lines, mainly caused by nuclear gene mutations, have been developed for the two-line breeding system, particularly in rice.
Complex and diverse interactions between genetic control and environmental temperature and photoperiods may determine plant male fertility.
The identification and utilization of nuclear male-sterile genes in combination with new technologies have achieved successful male fertility control of crops.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
In plants, male sterility can be caused either by mitochondrial genes with coupled nuclear genes or by nuclear genes alone; the resulting conditions are known as cytoplasmic male sterility (CMS) and ...genic male sterility (GMS), respectively. CMS and GMS facilitate hybrid seed production for many crops and thus allow breeders to harness yield gains associated with hybrid vigor (heterosis). In CMS, layers of interaction between mitochondrial and nuclear genes control its male specificity, occurrence, and restoration of fertility. Environment-sensitive GMS (EGMS) mutants may involve epigenetic control by noncoding RNAs and can revert to fertility under different growth conditions, making them useful breeding materials in the hybrid seed industry. Here, we review recent research on CMS and EGMS systems in crops, summarize general models of male sterility and fertility restoration, and discuss the evolutionary significance of these reproductive systems.