Identification and cytological observation of CMS in cotton

Identification and cytological observation of CMS in cotton | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Identification and cytological observation of CMS in cotton Qingyu PEI, Jinshan LIU, Chunping GUO, Xiaomei MA, Xiaoyan LIU, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3690132/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Journal of Cotton Research → Version 1 posted 5 You are reading this latest preprint version Abstract Background The utilization of male sterility is essential for producing hybrid seeds, and comprehending the mechanism of male sterility is the foundation for developing sterile germplasm resources. However, only a few cytoplasmic male sterility (CMS) lines of cotton have been produced due to various hindrances. Inadequate agronomic traits and incomplete sterility, weak resilience of restorer lines and the difficulty in combining strong dominance all serve as primary impediments to the advancement of CMS in cotton. Therefore, identifying and cytologically observing of CMS in cotton will provide new insights for heterosis utilization. Results Two F 2 segregating populations of cotton were constructed from cytoplasmic sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal). Genetic analysis of F 2 segregation populations showed that the separation ratio of fertile to sterile plants was 3:1. Phenotypic analysis indicated that there was no significant difference in the development process of flower buds between sterile and fertile plants. Compared to fertile plants, sterile plants exhibit smaller floral organs, shortened filament length and anther atrophy on the day of flowering. Scanning electron microscopy (SEM) of two F2 populations revealed morphological variations in the folds of the anther epidermis. Analysis of cell level differences showed that there was no significant difference in pollen development before the pollen maturation stage. However, between pollen maturation stage and flowering stage, the tapetum layer of sterile plants degenerated prematurely, the pollen grains were abnormal, pollen cytoplasmic degradation and gradually hollowed out. Conclusion The sterile trait is controlled by a single recessive gene. The floral morphology of the sterile plant was characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs from the time of pollen maturity until flowering. Degradation of pollen grains and the formation of empty pollen grains due to the premature degradation of the tapetum are presumed to be the main cause for pollen abortion. Cotton Cytoplasmic male sterility Genetic analysis Morphology Cytology Figures Figure 1 Figure 2 Figure 3 Background Cotton, a plant belonging to the Gossypium genus within the Malvaceae family, approximately 46 species are diploid (2n = 2x = 26) including Gossypium herbaceum and Gossypium arboretum , and about 7 species are allotetraploid (2n = 4x = 52) including Gossypium hirsutum and Gossypium barbadense (Wendel and Grover 2015 ; Zhang et al. 2020 ; Kushanov et al. 2021 ; Lima et al. 2021 ; Jan et al. 2022 ). Cotton exhibits clear heterosis, heterosis refers to the offspring of hybrid varieties of a species or crosses between different species that possess greater biomass, faster developmental rate, and higher fertility compared to their parent plants (Birchler et al. 2010 ; Groszmann et al. 2013 ; Fu et al. 2015 ). Hybrid seed production requires minimizing self-pollination and promoting cross-breeding between parent plants. To achieve this, techniques such as artificial and chemical emasculation, as well as male sterility, can be utilized (Kempe and Gils 2011 ; Colombo and Galmarini 2017 ; Yahaya et al. 2020 ; Zhang et al. 2023 ). Male sterility is an essential tool for heterosis utilization in breeding (Zhang et al. 2021 ). In 1973, the German botanist Joseph Gottlieb Kölreuter first observed the phenomenon of male sterility (Mayr 1986 ). Plant male sterility refers to the inability to produce dehiscent anthers, functional pollen, and viable male gametes, while female fertility remains unaffected (Schnable and Wise 1998 ; Chen and Liu 2014 ; Farinati et al. 2023 ). According to its causes, male sterility can be classified into three categories: cytoplasmic male sterility (CMS), which results from mitochondrial genes interact with nuclear genes; genic male sterility (GMS) is caused by nuclear genes alone; and environment-sensitive GMS (EGMS) is attributed to the collaboration between nuclear genes and environmental factors, including photoperiod-sensitive GMS (PGMS), temperature-sensitive GMS (TGMS), and photoperiod and temperature-sensitive GMS (PTGMS) (Vedel et al. 1994 ; Chen and Liu 2014 ; Sun et al. 2022 ; Ranaware et al. 2023 ). Cotton flower is a hermaphrodite flower, which is composed of six parts: peduncle, receptacle, sepal, petal, stamen and pistil. Previous study showed that Zhong41A and Zhong41B had no obvious differences in flower appearance, except for the smaller corolla of Zhong41A. While after removing the petals, the shorter stamen filaments and stigma of the CMS line Zhong41A were apparent, and the anthers of Zhong41A were indehiscent and light yellow (Yang et al. 2018 ). Cheng et al. ( 2020 ) studied shida98-6A and shida98-6 and found no morphological difference between vegetative growth and mature plants. During the period of reproductive growth, shida98-6A and shida98-6 plants showed an obvious difference in anther appearance and development. Sanders et al. ( 1999 ) argued that abnormal anther structure and abnormal pollen development could affect the fertility of plants. Laser and Lersten ( 1972 ) believed that pollen abortion may occur at any stage of pollen development. Pollen development comprises three major stages: (i) microsporogenesis (differentiation of the sporogenous cells and meiosis); (ii) postmeiotic development of microspores; (iii) microspore mitosis and (iv) microgametogenesis (Chaudhury 1993 ; Gómez et al. 2015 ; Halbritter et al. 2018 ). Li et al. ( 2021 ) found that differences between J4A and J4B began to appear at the start of meiosis, the middle cell layers of J4A did not degenerate and tapetum cells did not undergo mitosis, unable to provided nutrients for microspore development, led to microspore abortion. Kong et al. ( 2017 ) through the H276A microspore observations indicated that neither the degradation of nuclei in microspores during the tetrad stage nor the degradation of tapetal cells transpired during the microspore development. In this study, the cytoplasmic sterile line was used as the maternal parent and the restorer line was used as the paternal parent. Two different F 2 segregating populations were constructed as experimental materials. Genetic analysis showed that the fertile and sterile plants followed a 3:1 segregation ratio, indicating that the sterile trait is controlled by a single recessive gene. There were variations in morphological size, with sterile plants of Y66 and Y73 having significantly smaller floral organs, smaller petals and smaller sepals in contrast to fertile plants. SEM showed that the anthers of sterile plants were smaller than those of fertile plants, the anthers crumpled were different, and the morphology of the folds in the anther epidermis were different. Further observation of anther sections showed that the degradation degree of tapetum was different between sterile and fertile plants. Meanwhile, the pollen grains of the sterile plants degraded over time, resulting in empty pollen grains. In conclusion, this experiment aims to provide the theoretical basis for in-depth investigations of the dominance configuration of cotton hybrid combinations and the functions of sterile genes and restorer genes. Materials and methods Plant materials Two F 2 segregating populations of cotton were constructed from cytoplasmic sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal), designated Y66 (HaA × HaR) and Y73 (01A × 26R). The cultivation of all experimental materials was conducted in the teaching test field of Shihezi University (Shihezi, Xinjiang Uygur Autonomous Region, China). The experimental materials managed based on the production mode, regular fertilization, and irrigation. The populations were constructed in 2019, crossed to get F 1 generation, F 1 generation self-crossed to get F 2 generation; the F 2 segregating populations were evaluated for fertility identification, morphological and cytological observation in 2021. Fertility investigation and flower morphological characteristics observation The fertility of each F 2 segregating populations were assessed during the flowering stage. Depending on whether pollen was produced, fertility was assessed and labeled, and the number of sterile and fertile plants was recorded. To ensure accuracy, the time, weather, and temperature of phenotype identifications were as consistent as possible each time, the survey was repeated five times, with at least three flowers per plant examined each time (Liu et al. 2018 ). The fertility data were analyzed using IBM SPSS Statistics 26 for the χ 2 test and P -value. The flower buds and blooming flowers of sterile and fertile plants were sampled the same growth state to avoid the incomplete development and deformity of flower organs caused by growth, nutrient and other reasons of individual plants, and the morphology of the floral organs were observed and photographed three times at various stages of development. Sampling and fixation According to the flower bud development process, we randomly selected the sterile and fertile buds at several stages, including the pollen maturation stage (PMS), pre-flowering stage and flowering stage. The samples for paraffin sections were immersed in FAA fixative solution (anhydrous ethanol: glacial acetic acid: 37% formaldehyde solution: distilled water = 10:1:2:7 v/v), vacuumed, slowly deflated, repeated thrice, each time for 5–10 min. After vacuuming, replaced with FAA fixative solution and temporarily stored at 4℃. Following 24 h, the samples were transferred to 70% ethanol and stored at 4℃ (Yang et al. 2012 ). The SEM samples were soaked in 2.5% glutaraldehyde fixed solution, vacuumed, slowly deflated, repeated thrice, each time for 5–10 min, and then replaced with 2.5% glutaraldehyde fixed solution once after vacuuming and stored at 4℃ (Wu et al. 2015 ). Scanning electron microscopy observation of cotton anthers Fixed anthers were immersed in a 2.5% glutaraldehyde fixed solution, followed by soaking in 0.1 M phosphate buffer (pH = 7.2) for 20 min, repeated three times at room temperature. Anthers were dehydrated in a stepwise manner using 50%, 70%, 80%, 90%, and 100% ethanol, and each gradient was maintained at 4℃ for 20 min. A fully automated critical point dryer (Leica EM CPD300) was used for CO 2 drying. Treated anthers were mounted on the sample platform with double-sided adhesive tape. The surface of each anther was coated with a layer of gold using sputter deposition (Chang et al. 2016 ; Tian and Wan 2018; Liu et al. 2019 ). Observations and micrograph acquisition were performed using a scanning electron microscope (Hitachi SU8010) at the Analysis and Testing Center (Shihezi University). Paraffin section observation of cotton anthers Take anther had been immersed in FAA Fixative Solution, stepwise dehydrated and waxed leaching. 75% alcohol 5 h, 85% alcohol 2.5 h, 90% alcohol 2.5 h, 95% alcohol 1.5 h, anhydrous ethanol repeated twice for 30 min each time, xylenes soaked twice for 10 min each time, and paraffin immersed thrice for 1 h each time. The wax-soaked samples were embedded in the embedding machine. The melted wax was placed into the embedding frame. Cool it at -20℃ on a freezing table. After the wax solidified, the samples were sliced to 4–6µm with a microtome. Tissue slides were placed on the slides and dried for 1 h. The samples were dewaxed in xylene two times for 8–10 min each time and rehydrated in ethanol in steps of 10 min each stage. Subsequently, the samples were placed into 0.5% Toluidine Blue O staining solution for 5 min and dehydrated in a gradient alcohol series (Min et al. 2014 ). Put the samples rinsed in xylene three times for 5 min each time, and the tissue sections were mounted. Finally, a Panoramic MIDI automatic digital slide scanner (3DHISTECH Ltd., Budapest, Hungary) was used for observation images (Shao et al. 2022 ). Results Genetic analysis of the CMS In the F 2 segregating population, we observed two phenotypes of Y66 and Y73 plants, one with pollen and the other without pollen. The fertility phenotype was investigated based on pollen dispersal during the flowering stage (Table 1 ). We found that the F 2 population Y66 derived from the hybridization and self-crossing of HaA and HaR had 224 fertile plants and 71 sterile plants, consistent with a 3:1 segregation ratio ( χ 2 = 0.137 0.05). And the F 2 population Y73 was derived from the hybridization and self-crossing of 01A and 26R had 239 fertile plants and 88 sterile plants, consistent with a 3:1 segregation ratio ( χ 2 = 0.637 0.05). In conclusion, these results indicate that the sterile trait is controlled by a single recessive gene. Table 1 Separation ratio of fertile plants and sterile plants in F2 populations. Materials Total Fertility Sterility Expect separation ratio χ 2 -value P -value Y66 295 224 71 3:1 0.137 0.712 Y73 327 239 88 3:1 0.637 0.425 Phenotype observation of the F 2 populations Cotton flowers are hermaphroditic flowers composed of six parts: peduncle, receptacle, calyx, corolla, stamen and pistil. No difference in the morphological composition of floral organs were observed between fertile plants and sterile plants during the growth and development process. However, the flower organs of sterile plants exhibited significant male sterility traits. At flowering day, there was no obvious difference in the color of the floral organs between sterile plants and fertile plants, and all petals were light yellow (Fig. 1 A-D). There were variations in morphological size, with sterile plants of Y66 and Y73 having significantly smaller floral organs, smaller petals and smaller sepals in contrast to fertile plants (Fig. 1 ). After the removal petals, noticeable differences in anther histology were observed between sterile plants and fertile plants. Sterile plants possessed small and shriveled anthers with short and tiny filaments. No pollen was observed, and the surface of the anthers appeared concave and uneven in a lackluster light yellow or light brown (Fig. 1 H, P). Nevertheless, compared to the corresponding fertile plants, we found that the developing anthers were plump and spherical, and dispersed pollen, eventually achieving a glossy yellow color (Fig. 1 L, T). Anther epidermis SEM observation of the F 2 populations According to the phenotypic observations, there were significant differences between the sterile and the fertile plant anthers of Y66 and Y73 at flowering day, and the anthers of the sterile plants were deformed. Further observation by SEM revealed that the anthers of the sterile plants were smaller than those of the fertile plants. Moreover, there were obvious differences in the morphological characteristics of anther epidermis folds, including the peaks and valleys of fold formation (Fig. 2 ). The anther epidermis of Y66 sterile plants exhibited tight wrinkled and indented, the chamber epidermis displayed wavy, irregular folds with a loose fold structure, and the smooth peaks and valleys (Fig. 2 A-C). The anther epidermis of Y66 fertile plants was fully expanded, the chamber epidermis exhibited prominent scale-like folds arranged in a close structure, with high peaks and deep valleys, resulting in a relatively rough surface (Fig. 2 D-F). The area cell where the vascular bundle and anther chamber converge in Y73 sterile plants contracted tightly. This caused an epidermal bump in the anther chamber, irregular wavy folds in the chamber's epidermis, a loose fold structure, and relatively mild peaks and valleys (Fig. 2 G-I). The anther epidermis of fully matured Y73 plants was expanded, revealing clearly recognizable wavy folds of the chamber epidermis. The fold structure was closely packed, with steep peaks and deep valleys (Fig. 2 J-L). Anthers paraffin section observation of the F 2 populations To further analyze the sterile characteristics of the F 2 segregating population CMS, the anthers of sterile plants and fertile plants were selected to make paraffin sections at the pollen maturation stage (PMS), pre-flowering stage and flowering stage (Fig. 3 ). Observed the anther paraffin sections of PMS, we found that the degradation degree of the tapetum of the sterile plants of Y66 and Y73 was similar to that of the fertile plants of Y66 and Y73. We observed clear pollen grains, spinules on the surface of pollen grains, and the pollen grains were not significantly different in morphology (Fig. 3 A, E, I, M). It can be inferred that there was no significant difference between the sterile plants of Y66 and Y73 and the fertile plants of Y66 and Y73 in pollen grain development before PMS. Upon observing the sterile (Y66 and Y73) and fertile (Y66 and Y73) plant at pre-flowering stage, noticeable discrepancies were found in the degree of degradation of the anther tapetum. It was noted that the anther chamber of the sterile plants almost lacked tapetum degradation residue. In sterile plants, the pollen grains gradually exhibited abnormalities and inclusions gradually disintegrated leading to the formation of voids. Furthermore, the nucleolus and cytoplasm disappeared, eventually culminating in the formation of empty pollen grains and the pollen grain spinules had lost their sharpness. In addition, it was observed that the cells in the anther chamber and anther vascular bundle area had atrophied (Fig. 3 B, J, C, K). In fertile plants, exhibited full pollen grains, developed normally with good morphology and structure, and evenly distributed tapetum degradation products (Fig. 3 F, N, G, O). Observed the anthers of Y66 and Y73 at flowering day, it was observed that the anther chamber of the sterile plants had shrunken inward and the anther chamber of the fertile plants had fully expanded (Fig. 3 D, H, L, P). Discussion Heterosis utilization of CMS in cotton Meyer ( 1975 ) was the first to report the CMS of cotton and realized the "three-line" matching of the CMS of cotton. "Three-line" is composed of a sterile line, restorer line and maintainer line (Zheng et al. 2020 ). In the application of heterosis utilization, employing the "three-line" method of CMS can significantly economize on labor, materials, and finances resources compared to manual or chemical emasculation, as well as the "two-line" method. Additionally, this method affords straightforward, effective, and productive high-yielding seed production process. A high-quality cytoplasmic sterile line is essential for hybridization using the "three-line" method (Wang 2019 ). The more complete the sterility, the higher the purity of the hybrid line. Thus, many researchers are focusing on the in-depth exploration of excellent germplasm resources and restoring genes of cytoplasmic sterile lines or the genetic improvement of existing sterile lines and restorer lines through genetic engineering technology to promote the heterosis utilization of sterile lines. This study analyzed the cytoplasmic male sterility F 2 segregating population using fertility identification and genetic analysis. The results showed that Y66 and Y73, the experimental materials, followed a 3:1 separation ratio (Table 1 ). The study revealed that inheritance of the gene cause sterile trait is controlled by a single recessive gene. Which offers valuable material for thorough exploration into gene localization and functional analysis. Abortion characteristics of the F 2 populations The morphological changes of flower organs in male sterile plants are complex, exhibiting various phenotypes and variations in different fertility levels. The stamen atrophy, abnormal anther development, degeneration of sporangia and microspores, and functional infertility of the pollen are morphological and functional features of abortive flower organs (Zhang et al. 2014 ; Jiang et al. 2020 ). Many studies have shown that there are no significant differences in flower organ composition and developmental processes between various sterile lines of cotton and the materials used in this experiment. However, significant differences in the pollen development process exist between the corresponding fertile plants and maintainer lines of different sterile lines (Wu et al. 2015 ; Kong et al. 2017 ; Yang et al. 2018 ; Cheng et al. 2020 ; Li et al. 2021 ). In this experiment, there was no difference in flower organ composition and petal color between sterile and fertile plants on the day of flowering. The flower organs of sterile plants were smaller than those of fertile plants. Following petal removal, significant differences in filament length, anther morphology and anther color were also observed in the Y66 and Y73 sterile plants compared to the fertile ones (Fig. 1 ). SEM showed that the anthers of sterile plants were smaller than those of fertile plants, the anthers crumpled were different, and the morphology of the folds in the anther epidermis were different (Fig. 2 ). In summary, the abortive floral organs of Y66 and Y73 exhibit characteristics of stamen atrophy and anther abnormality. Pollen abortion period, features and reasons of the F 2 population CMS Laser and Lersten ( 1972 ) believed that pollen abortion could occur at all stages of pollen development, and the period, mode and characteristics of abortion varied in different plants, species and sources of male sterility. Any mutation in a gene that affects stamen development, sporogonium differentiation, meiosis, mitosis, microspore development, or flower differentiation has the potential to cause male sterility in plants (Glover et al. 1998 ; Niu et al. 2013 ; Ko et al. 2014 ; Han et al. 2018 ). Xie et al. ( 2006 ) reviewed that there are various factors causing plant male sterility, and the process of anther abortion is intricate, including tapetal development, ATPase, Ca 2+ concentration, cytoskeleton and programmed cell death. The tapetum is the innermost anther wall directly connected with pollen mother cells, and it plays a crucial role in pollen development (Falasca et al. 2013 ; Singh et al. 2015 ; Sun et al. 2019 ). Precise regulation is necessary for tapetum degradation, as it provides essential enzymes and nutrients for microspore development. Premature or delayed degradation may lead to microspore abortion (Balk and Leaver 2001 ; Ariizumi and Toriyama 2011 ; Wan et al. 2011 ). According to the observation of anther tissue sections (Fig. 3 ), there was no significant difference in pollen development between the sterile and fertile plants at the pollen maturity stage, but the pollen of the sterile plants showed abnormal development, and the pollen abortion process was similar between Y66 sterile plants and Y73 sterile plants. Further observation showed that the degradation degree of tapetum was different between sterile and fertile plants. Before flowering, the sterile plants had almost no tapetum degradation residue in the anther chamber, but the fertile plants had more tapetum degradation residue and uniform distribution. Meanwhile, the pollen grains of the sterile plants degraded over time, resulting in empty pollen grains, and the cells in the connecting area between the vascular bundles and the anther chamber underwent atrophied. In summary, it can be concluded that pollen abortion occurred in Y66 and Y73 between the time of pollen maturity until flowering. The cause of this phenomenon may be due to premature degradation of the tapetum and insufficient nutrient supply to maintain pollen activity in the anther chamber, resulting in pollen grain degradation and eventually male sterility. Conclusion The F 2 segregating populations derived from the cytoplasmic sterile lines HaA and 01A of cotton exhibited similar genetic, morphological and cytological characteristics. Genetic analysis showed that the fertile and sterile plants followed a 3:1 segregation ratio, indicating that the gene cause sterile trait was controlled by a single recessive gene. The floral morphology of the sterile plant was characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs between the time of pollen maturity until flowering. The degradation and formation of empty pollen grains, caused by the premature degradation of the tapetum, might be the main cause for pollen abortion. Declarations Acknowledgements We thank the Key Laboratory of Oasis Ecology Agricultural of Xinjiang Production and Construction Corps, Agricultural College, Shihezi University, Shihezi, Xinjiang, China, the Analysis and Testing Center, Shihezi University, Shihezi, Xinjiang, China ,and the Cotton Research Institute of the Shihezi Academy of Agriculture Science, Shihezi, Xinjiang, China, which provided us the experimental fields and experimental platform. Authors’ contributions Nie XH, Pan ZY, Guo CP and Liu JS designed and supervised the experiments. Nie XH provided the funding. You CY provided the cytoplasmic male sterility lines. Guo CP, Liu JS, Ma SM and Liu XY constructed the segregating population, investigated the fertility and phenotype observation. Lin HR, Li ZB, Zhao RH, Pan ZY and Pei QY performed the experiments. Pei QY sorted out the results, wrote the main manuscript and prepared all figures. Wu YL, Nie XH，Pan ZY and Guo CP revised and polished the manuscript. All authors contributed to the article and approved the final manuscript. Funding This work was supported by the Fund for National Natural Science Foundation of China (31960439), the Key Core Technology Projects of the Xinjiang production and Construction Crops, China, the Technology Innovation Team Project of the Xinjiang production and Construction Crops, China, the Science and Technology Plan of Shuanghe city, Xinjiang production and Construction Crops, China (2021NY02), the Science and Technology Plan of Shuanghe city, Xinjiang production and Construction Crops, China (2021NY04) and the Key Programs for Science and Technology Development of Shihezi city, Xinjiang production and Construction Crops, China (2022NY01). Supplementary Information Not applicable. Availability of data and materials The datasets used during the current study are available from the corresponding author on reasonable request. Ethics approval and consent to participate Not applicable. Consent for publication All authors have provided ethical approval and consent to participate as well as consent for publication. Competing interests The authors declare that they have no competing interests. Author details 1 Key Laboratory of Oasis Ecology Agricultural of Xinjiang Production and Construction Corps, Agricultural College, Shihezi University, Shihezi 832003, Xinjiang, China. 2 Seed Management Station of Xinjiang Production and Construction Corps, Urumqi 830000, Xinjiang, China. 3 Cotton Institute, Xinjiang Academy of Agricultural and Reclamation Science, Shihezi, Xinjiang 832000, China. 4 Xinjiang Jinbo Seed Industry Co., Ltd, Bole 833400, Xinjiang, China. 5 National Key Laboratory of Crop Genetic Improvement, College of Plant Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China. 6 Cotton Research Institute, Shihezi Academy of Agriculture Science, Shihezi 832011, Xinjiang, China. 7 Agricultural Science Research Institute of the 5th Division of Xinjiang Production and Construction Corps, Shuanghe 833408, Xinjiang, China. References Ariizumi T, Toriyama K. Genetic Regulation of Sporopollenin Synthesis and Pollen Exine Development. Annu Rev Plant Biol. 2011;62. https://doi.org/10.1146/annurev-arplant-042809-112312 . :437 – 60. Balk J, Leaver CJ. The PET1-CMS Mitochondrial Mutation in Sunflower Is Associated with Premature Programmed Cell Death and Cytochrome c Release. Plant Cell. 2001;13:1803–18. https://doi.org/10.1105/TPC.010116 . Birchler JA, Yao H, Chudalayandi S, et al. Heterosis Plant Cell. 2010;22:2105–12. https://doi.org/10.1105/tpc.110.076133 . Chang Z, Chen Z, Wang N, et al. Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene. Proc Natl Acad Sci USA. 2016;113:14145–50. https://doi.org/10.1073/pnas.1613792113 . Chaudhury AM. Nuclear Genes Controlling Male Fertility. Plant Cell. 1993;5:1277–83. https://doi.org/10.1105/tpc.5.10.1277 . Chen L, Liu Y. Male Sterility and Fertility Restoration in Crops. Annu Rev Plant Biol. 2014;65:579–606. https://doi.org/10.1146/annurev-arplant-050213-040119 . Cheng X, Zhang X, Xue F, et al. Characterization and transcriptome analysis of a dominant genic male sterile cotton mutant. BMC Plant Biol. 2020;20:312. https://doi.org/10.1186/s12870-020-02522-0 . Colombo N, Galmarini CR. The use of genetic, manual and chemical methods to control pollination in vegetable hybrid seed production: a review. Plant Breed. 2017;136:287–99. https://doi.org/https://doi.org/10.1111/pbr.12473 . Falasca G, D'Angeli S, Biasi R, et al. Tapetum and middle layer control male fertility in Actinidia deliciosa. Ann Bot. 2013;112:1045–55. https://doi.org/10.1093/aob/mct173 . Farinati S, Draga S, Betto A, et al. Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications. Front Plant Sci. 2023;14. https://doi.org/10.3389/fpls.2023.1223861 . Fu W, Shen Y, Hao J, et al. Acyl-CoA N-acyltransferase influences fertility by regulating lipid metabolism and jasmonic acid biogenesis in cotton. Sci Rep. 2015;5:11790. https://doi.org/10.1038/srep11790 . Glover J, Grelon M, Craig S, et al. Cloning and characterization of MS5 from Arabidopsis : a gene critical in male meiosis. Plant J. 1998;15:345–56. https://doi.org/10.1046/j.1365-313x.1998.00216.x . Gómez JF, Talle B, Wilson ZA. Anther and pollen development: A conserved developmental pathway. J Integr Plant Biol. 2015;57. https://doi.org/https://doi.org/10.1111/jipb.12425 . :876 – 91. Groszmann M, Greaves IK, Fujimoto R, et al. The role of epigenetics in hybrid vigour. Trends Genet. 2013;29. https://doi.org/10.1016/j.tig.2013.07.004 . :684 – 90. Halbritter H, Ulrich S, Grímsson F, et al. Pollen Dev. 2018;23–35. https://doi.org/10.1007/978-3-319-71365-6_2 . Han Y, Zhao F, Gao S, et al. Fine mapping of a male sterility gene ms-3 in a novel cucumber ( Cucumis sativus L.) mutant. Theor Appl Genet. 2018;131:449–60. https://doi.org/10.1007/s00122-017-3013-2 . Jan M, Liu Z, Guo C, et al. An Overview of Cotton Gland Development and Its Transcriptional Regulation. Int J Mol Sci. 2022;23. https://doi.org/10.3390/ijms23094892 . Jiang H, Yang S, Liu Y, et al. Biological characteristics and cytological studies on anther abortion of male sterile Camellia crassocolumn . Acta Agron Sinica. 2020;46:1076–86. https://doi.org/10.3724/SP.J.1006.2020.94159 . (in Chinese). Kempe K, Gils M. Pollination control technologies for hybrid breeding. Mol Breed. 2011;27:417–37. https://doi.org/10.1007/s11032-011-9555-0 . Ko S, Li M, Sun-Ben Ku M, et al. The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice. Plant Cell. 2014;26:2486–504. https://doi.org/10.1105/tpc.114.126292 . Kong X, Liu D, Liao X, et al. Comparative Analysis of the Cytology and Transcriptomes of the Cytoplasmic Male Sterility Line H276A and Its Maintainer Line H276B of Cotton ( Gossypium barbadense L). Int J Mol Sci. 2017;18:2240. https://doi.org/10.3390/ijms18112240 . Kushanov FN, Turaev OS, Ernazarova DK, et al. Genetic Diversity, QTL Mapping, and Marker-Assisted Selection Technology in Cotton ( Gossypium spp). Front Plant Sci. 2021;12. https://doi.org/10.3389/fpls.2021.779386 . Laser KD, Lersten NR. Anatomy and cytology of microsporogenesis in cytoplasmic male sterile angiosperms. Bot Rev. 1972;38:425–54. https://doi.org/10.1007/BF02860010 . Li M, Chen L, Khan A, et al. Transcriptome and MiRNAomics Analyses Identify Genes Associated with Cytoplasmic Male Sterility in Cotton ( Gossypium hirsutum L). Int J Mol Sci. 2021;22. https://doi.org/10.3390/ijms22094684 . Lima LFD, Oliveira JOD, Carneiro JNP, et al. Ethnobotanical and antimicrobial activities of the Gossypium (Cotton) genus: A review. J Ethnopharmacol. 2021;279:114363. https://doi.org/10.1016/j.jep.2021.114363 . Liu F, Ma L, Wang Y, et al. GhFAD2 – 3 is required for anther development in Gossypium hirsutum . BMC Plant Biol. 2019;19:393. https://doi.org/10.1186/s12870-019-2010-9 . Liu L, Guo L, Qi T. SSCP Marker Development and Analysis of Candidate Restorer Gene Rf 1 for Cotton Cytoplasmic Male Sterility. Cotton Sci. 2018;30:12–20. https://doi.org/10.11963/1002-7807.llcxcz.20180104 . (in Chinese). Mayr E. Joseph Gottlieb Kolreuter's Contributions to Biology. Osiris. 1986;2:135–76. https://doi.org/10.1086/368655 . Meyer VG. Male Sterility From Gossypium harknessii . J Hered. 1975;66:23–7. https://doi.org/10.1093/oxfordjournals.jhered.a108566 . Min L, Li Y, Hu Q, et al. Sugar and Auxin Signaling Pathways Respond to High-Temperature Stress during Anther Development as Revealed by Transcript Profiling Analysis in Cotton. Plant Physiol. 2014;164:1293–308. https://doi.org/10.1104/pp.113.232314 . Niu N, Liang W, Yang X, et al. EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice. Nat Commun. 2013;4:1445. https://doi.org/10.1038/ncomms2396 . Ranaware AS, Kunchge NS, Lele SS, Ochatt SJ. Protoplast Technology and Somatic Hybridisation in the Family Apiaceae. Plants. 2023;12:1060. https://doi.org/10.3390/plants12051060 . Sanders PM, Bui AQ, Weterings K, et al. Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod. 1999;11:297–322. https://doi.org/10.1007/s004970050158 . Schnable PS, Wise RP. The molecular basis of cytoplasmic male sterility and fertility restoration. Trends Plant Sci. 1998;3:175–80. https://doi.org/10.1016/S1360-1385(98)01235-7 . Shao P, Peng Y, Wu Y, et al. Genome-wide association study and transcriptome analysis reveal key genes controlling fruit branch angle in cotton. Front Plant Sci. 2022;13. https://doi.org/10.3389/fpls.2022.988647 . Singh SP, Singh SP, Pandey T, et al. A novel male sterility-fertility restoration system in plants for hybrid seed production. Sci Rep. 2015;5:11274. https://doi.org/10.1038/srep11274 . Sun L, Sui X, Lucas WJ, et al. Down-regulation of the Sucrose Transporter CsSUT1 Causes Male Sterility by Altering Carbohydrate Supply. Plant Physiol. 2019;180:986–97. https://doi.org/10.1104/pp.19.00317 . Sun Y, Fu M, Wang L, et al. OsSPLs Regulate Male Fertility in Response to Different Temperatures by Flavonoid Biosynthesis and Tapetum PCD in PTGMS Rice. Int J Mol Sci. 2022;23. https://doi.org/10.3390/ijms23073744 . Tian Y, Wan X, Cytobiology, Molecular Genetics Research Methods on Maize Anther Development. China Biotechnol. 2018;38:88–99. https://doi.org/10.13523/j.cb.20180111 . (in Chinese). Vedel F, Pla M, Vitart V, et al. Molecular basis of nuclear and cytoplasmic male sterility in higher plants. Plant Physiol Biochem. 1994;32:601–8. Wan L, Zha W, Cheng X, et al. A rice β-1,3-glucanase gene Osg1 is required for callose degradation in pollen development. Planta. 2011;233:309–23. https://doi.org/10.1007/s00425-010-1301-z . Wang X. Overview of the Study and Application of Cytoplasmic Male Sterility in Cotton. Scientia Agricultura Sinica. 2019;52:1341–54. https://doi.org/10.3864/j.issn.0578-1752.2019.08.005 . (in Chinese). Wendel JF, Grover CE. Taxonomy and Evolution of the Cotton Genus. Gossypium. 2015;25–44. https://doi.org/10.2134/agronmonogr57.2013.0020 . Wu Y, Min L, Wu Z, et al. Defective pollen wall contributes to male sterility in the male sterile line 1355A of cotton. Sci Rep. 2015;5:9608. https://doi.org/10.1038/srep09608 . Xie C, Wei D, Tian H. Advances in Cell Biological Researches on Male Sterility of Higher Plants. J Plant Physiol Mol Biology. 2006;32:17–23. https://doi.org/10.3321/j.issn:1671-3877.2006.01.003 . (in Chinese). Yahaya MA, Shimelis H, Laing M, et al. Methodologies for hybridization in predominantly self-pollinating crops: a review. J Crop Improv. 2020;34:268–89. https://doi.org/10.1080/15427528.2019.1698483 . Yang L, Wu Y, Zhang M, et al. Transcriptome, cytological and biochemical analysis of cytoplasmic male sterility and maintainer line in CMS-D8 cotton. Plant Mol Biol. 2018;97:537–51. https://doi.org/10.1007/s11103-018-0757-2 . Yang X, Zhang X, Yuan D, et al. Transcript profiling reveals complex auxin signalling pathway and transcription regulation involved in dedifferentiation and redifferentiation during somatic embryogenesis in cotton. BMC Plant Biol. 2012;12:110. https://doi.org/10.1186/1471-2229-12-110 . Zhang C, Zhang M, Zhu Q, et al. Cytological observation of pollen development in 'Ougan' ( Citrus suavissima Hort. ex Tanaka) and its seedless mutant. J Fruit Sci. 2014;31:265–9. https://doi.org/10.13925/j.cnki.gsxb.2014.02.022 . (in Chinese). Zhang R, Chang J, Li J, et al. Disruption of the bHLH transcription factor Abnormal Tapetum 1 causes male sterility in watermelon. Hortic Res. 2021;8:258. https://doi.org/10.1038/s41438-021-00695-9 . Zhang T, Xuan L, Mao Y, Hu Y. Cotton heterosis and hybrid cultivar development. Theor Appl Genet. 2023;136:89. https://doi.org/10.1007/s00122-023-04334-w . Zhang X, Zhang Z, Zhou R, et al. Ratooning Annual Cotton ( Gossypium spp.) for Perennial Utilization of Heterosis. Front Plant Sci. 2020;11:554970. https://doi.org/10.3389/fpls.2020.554970 . Zheng W, Ma Z, Zhao M, et al. Research and Development Strategies for Hybrid japonica Rice. Rice. 2020;13:36. https://doi.org/10.1186/s12284-020-00398-0 . Cite Share Download PDF Status: Published Journal Publication published 05 Aug, 2024 Read the published version in Journal of Cotton Research → Version 1 posted Editorial decision: Major revision 18 Mar, 2024 Reviewers agreed at journal 11 Dec, 2023 Reviewers invited by journal 08 Dec, 2023 Editor assigned by journal 01 Dec, 2023 First submitted to journal 29 Nov, 2023 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3690132","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":257746415,"identity":"e4cc6d67-d07c-49e0-bea9-e3f6ad910a66","order_by":0,"name":"Qingyu PEI","email":"","orcid":"","institution":"Shihezi University College of Agriculture","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Qingyu","middleName":"","lastName":"PEI","suffix":""},{"id":257746416,"identity":"30ad00ce-663e-4d51-b33b-52653f67533a","order_by":1,"name":"Jinshan LIU","email":"","orcid":"","institution":"Seed Management Station of Xinjiang Production and Construction Corps","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jinshan","middleName":"","lastName":"LIU","suffix":""},{"id":257746417,"identity":"78cfd221-f5f5-4c98-bdf2-a4fbecdb1a98","order_by":2,"name":"Chunping GUO","email":"","orcid":"","institution":"Shihezi University College of Agriculture","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunping","middleName":"","lastName":"GUO","suffix":""},{"id":257746418,"identity":"6b7134e3-f04f-43b7-8ee2-4ce88441b870","order_by":3,"name":"Xiaomei MA","email":"","orcid":"","institution":"Xinjiang Academy of Agricultural and Reclamation Science","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaomei","middleName":"","lastName":"MA","suffix":""},{"id":257746419,"identity":"d562d1ad-c4fb-4f48-a53e-004ddb6617ff","order_by":4,"name":"Xiaoyan LIU","email":"","orcid":"","institution":"Xinjiang Jinbo Seed Industry","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaoyan","middleName":"","lastName":"LIU","suffix":""},{"id":257746420,"identity":"58b7d04a-400a-4b39-8c17-6a7ae0b8deaf","order_by":5,"name":"Chunyuan YOU","email":"","orcid":"","institution":"Huazhong University of Science and Technology，National Key Laboratory of crop Genetic Improvement;Shihezi Academy of Agriculture Science, Cotton Research Institute","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Chunyuan","middleName":"","lastName":"YOU","suffix":""},{"id":257746421,"identity":"41d77914-d1fe-483e-9553-edbc4774879d","order_by":6,"name":"Hairong LIN","email":"","orcid":"","institution":"Shihezi University College of Agriculture","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Hairong","middleName":"","lastName":"LIN","suffix":""},{"id":257746422,"identity":"762d7859-8a5d-4665-ba34-77ccd7a308d3","order_by":7,"name":"Zhibo LI","email":"","orcid":"","institution":"Shihezi University College of Agriculture","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhibo","middleName":"","lastName":"LI","suffix":""},{"id":257746423,"identity":"f7f56356-8293-4aff-b140-3bbd2c55ab31","order_by":8,"name":"Ruihai ZHAO","email":"","orcid":"","institution":"Shihezi University College of Agriculture","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Ruihai","middleName":"","lastName":"ZHAO","suffix":""},{"id":257746424,"identity":"5e3444bc-334e-421a-8ca6-50efb3784749","order_by":9,"name":"Bo ZHU","email":"","orcid":"","institution":"Agricultural Science Research Institute of the 5th Division of Xinjiang Production and Construction Corps","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Bo","middleName":"","lastName":"ZHU","suffix":""},{"id":257746425,"identity":"60a54d12-931b-46a3-9f15-ee4e1a19bdd8","order_by":10,"name":"Yuanlong WU","email":"","orcid":"","institution":"Shihezi University College of Agriculture","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuanlong","middleName":"","lastName":"WU","suffix":""},{"id":257746426,"identity":"c5d8ef32-3b62-4847-83f4-a88b10a3f1e1","order_by":11,"name":"Zhenyuan PAN","email":"","orcid":"","institution":"Shihezi University College of Agriculture","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhenyuan","middleName":"","lastName":"PAN","suffix":""},{"id":257746427,"identity":"c33f06ba-9459-4d95-88ff-f683d8016bee","order_by":12,"name":"Xinhui Nie","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIie2RsUoDQRCGZzm4a0au3bBwzzBh4bC4h7lF2CpIwCblwYI2Str4FvoErgyezWqdVnyBlBaS5EJK4TZlwP3q/+PnnwFIJM4SDxksJApXeL+hpjpRCU2V3aH5Ws2tPqknE7dWF0vQGjdsonF6/ejVPGfz4MCqhrIWCn57GlMm3eeVXiGbRyd6NaP8GtDa9ZhSQph+o2TzfGiZEd6AxHpUySEQI7F5YajVJUnTxZRjS2u1cMN8IIorky4Mh/JNJdxw5HtqdR7bQutQq4vt8Mrlu/c/v7uqLLgfVUD6P+tilF00kkgkEv+dPbhBSd7XK0SVAAAAAElFTkSuQmCC","orcid":"","institution":"Shihezi University College of Agriculture","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xinhui","middleName":"","lastName":"Nie","suffix":""}],"badges":[],"createdAt":"2023-12-01 04:25:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3690132/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3690132/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s42397-024-00189-8","type":"published","date":"2024-08-05T15:58:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":48068842,"identity":"e484b976-1e22-43d5-9132-e10f6d3b2385","added_by":"auto","created_at":"2023-12-12 15:20:05","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":192182,"visible":true,"origin":"","legend":"\u003cp\u003ePhenotypic observation of Y66 and Y73. (A). Flower field phenotype of Y66 sterile plant; (B). Flower field phenotypes of Y66 fertile plants; (C). Flower field phenotype of Y73 sterile plants; (D). Flower field phenotype of Y73 fertile plants; (E). Flower morphology of Y66 sterile plant; (F). Petals of Y66 sterile plant; (G). Calyx of Y66 sterile plant; (H). Y66 sterile plants at the developmental phase and flowering day flowers without petals; (I). Flower morphology of Y66 fertile plant; (J). Petals of Y66 fertile plant; (K). Calyx of Y66 fertile plant; (L). Y66 fertile strains at the developmental phase and flowering day flowers without petals; (M). Flower morphology of Y73 sterile plant; (N). Petals of Y73 sterile plant; (O). Calyx of Y73 sterile plant; (P). Y73 sterile plants at the developmental phase and flowering day flowers without petals; (Q). Flower morphology of Y73 fertile plant; (R). Petals of Y73 fertile plant; (S). Calyx of Y73 fertile plant; (T). Y73 fertile plants at the developmental phase and flowering day flowers without petals. Scale bars: 1 cm.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3690132/v1/457baabeb8c1e9ea15e08f2b.jpeg"},{"id":48068844,"identity":"17927775-2b4d-48bc-8ad3-18139b6c4958","added_by":"auto","created_at":"2023-12-12 15:20:05","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":293647,"visible":true,"origin":"","legend":"\u003cp\u003eAnther epidermis SEM observation of Y66 and Y73 at flowering day. (A). Anthers of Y66 sterile plant at flowering day, magnification factor ×60; (B). Anther epidermis of Y66 sterile plant at flowering day, magnification factor ×1000; (C). Local amplification of the anther epidermis of Y66 sterile plant at flowering day, magnification factor ×3000; (D). Anthers of a Y66 fertile plant at flowering day, magnification factor ×60; (E). Anther epidermis of Y66 fertile plant at flowering day, magnification factor ×1000; (F). Local amplification of the anther epidermis of Y66 fertile plant at flowering day, magnification factor ×3000; (G). Anthers of Y73 sterile plant at flowering day, magnification factor ×60; (H). Anther epidermis of Y73 sterile plant at flowering day, magnification factor ×1000; (I). Local amplification of the anther epidermis of Y73 sterile plant at flowering day, magnification factor ×3000; (J). Anthers of Y73 fertile plant at flowering day, magnification factor ×60; (K). Anther epidermis of Y73 fertile plant at flowering day, magnification factor ×1000; (L). Local amplification of the anther epidermis of Y73 fertile plant at flowering day, magnification factor ×3000. Scale bars: A, D, G and J, 500 μm; B, E, H and K, 50 μm; C, F, I and L, 10 μm.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3690132/v1/174234d92bb4b55ece725b39.jpeg"},{"id":48068841,"identity":"003b0fb8-bac2-4aa4-aacb-1c13897097d5","added_by":"auto","created_at":"2023-12-12 15:20:04","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":459560,"visible":true,"origin":"","legend":"\u003cp\u003eObservation of anther paraffin section of Y66 and Y73 at different developmental stages. (A). Y66 sterile plant anther section at PMS; (B and C). Y66 sterile plant anther section at pre-flowering stage; (D). Y66 sterile plant anther section at flowering stage; (E). Y66 fertile plant anther section at PMS; (F and G). Y66 fertile plant anther section at pre-flowering stage; (H). Y66 fertile plant anther section at flowering stage; (I). Y73 sterile plant anther section at PMS; (J and K). Y73 sterile plant anther section at pre-flowering stage; (L). Y73 sterile plant anther section at flowering stage; (M). Y73 fertile plant anther section at PMS; (N and O). Y73 fertile plant anther section at pre-flowering stage; (P). Y73 fertile plant anther section at flowering stage. Ep: epidermis; En: endothecium; Ta: tapetum; PG: pollen grain; EPG: empty pollen grain; St: stomium; V: vascular bundle; C: connective. Scale bars: A, B, E, F, I, J, M and N, 100 μm; C, D, G, H, K, L, O and P, 200 μm.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-3690132/v1/a7df07cb7b1e4f054d0ea4b6.jpeg"},{"id":62299082,"identity":"3ac6c915-f6ba-42ae-9c04-a5fcc71f17ed","added_by":"auto","created_at":"2024-08-12 16:18:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1555048,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3690132/v1/0fb7c48d-7288-4ca8-81dd-5a01ec5084a0.pdf"}],"financialInterests":"","formattedTitle":"Identification and cytological observation of CMS in cotton","fulltext":[{"header":"Background","content":"\u003cp\u003eCotton, a plant belonging to the \u003cem\u003eGossypium\u003c/em\u003e genus within the \u003cem\u003eMalvaceae\u003c/em\u003e family, approximately 46 species are diploid (2n\u0026thinsp;=\u0026thinsp;2x\u0026thinsp;=\u0026thinsp;26) including \u003cem\u003eGossypium herbaceum\u003c/em\u003e and \u003cem\u003eGossypium arboretum\u003c/em\u003e, and about 7 species are allotetraploid (2n\u0026thinsp;=\u0026thinsp;4x\u0026thinsp;=\u0026thinsp;52) including \u003cem\u003eGossypium hirsutum\u003c/em\u003e and \u003cem\u003eGossypium barbadense\u003c/em\u003e (Wendel and Grover \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Kushanov et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Lima et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e; Jan et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). Cotton exhibits clear heterosis, heterosis refers to the offspring of hybrid varieties of a species or crosses between different species that possess greater biomass, faster developmental rate, and higher fertility compared to their parent plants (Birchler et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Groszmann et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Fu et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Hybrid seed production requires minimizing self-pollination and promoting cross-breeding between parent plants. To achieve this, techniques such as artificial and chemical emasculation, as well as male sterility, can be utilized (Kempe and Gils \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Colombo and Galmarini \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yahaya et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Zhang et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMale sterility is an essential tool for heterosis utilization in breeding (Zhang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In 1973, the German botanist Joseph Gottlieb K\u0026ouml;lreuter first observed the phenomenon of male sterility (Mayr \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e1986\u003c/span\u003e). Plant male sterility refers to the inability to produce dehiscent anthers, functional pollen, and viable male gametes, while female fertility remains unaffected (Schnable and Wise \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Chen and Liu \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Farinati et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). According to its causes, male sterility can be classified into three categories: cytoplasmic male sterility (CMS), which results from mitochondrial genes interact with nuclear genes; genic male sterility (GMS) is caused by nuclear genes alone; and environment-sensitive GMS (EGMS) is attributed to the collaboration between nuclear genes and environmental factors, including photoperiod-sensitive GMS (PGMS), temperature-sensitive GMS (TGMS), and photoperiod and temperature-sensitive GMS (PTGMS) (Vedel et al. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e1994\u003c/span\u003e; Chen and Liu \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Ranaware et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eCotton flower is a hermaphrodite flower, which is composed of six parts: peduncle, receptacle, sepal, petal, stamen and pistil. Previous study showed that Zhong41A and Zhong41B had no obvious differences in flower appearance, except for the smaller corolla of Zhong41A. While after removing the petals, the shorter stamen filaments and stigma of the CMS line Zhong41A were apparent, and the anthers of Zhong41A were indehiscent and light yellow (Yang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Cheng et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) studied shida98-6A and shida98-6 and found no morphological difference between vegetative growth and mature plants. During the period of reproductive growth, shida98-6A and shida98-6 plants showed an obvious difference in anther appearance and development.\u003c/p\u003e \u003cp\u003eSanders et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1999\u003c/span\u003e) argued that abnormal anther structure and abnormal pollen development could affect the fertility of plants. Laser and Lersten (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1972\u003c/span\u003e) believed that pollen abortion may occur at any stage of pollen development. Pollen development comprises three major stages: (i) microsporogenesis (differentiation of the sporogenous cells and meiosis); (ii) postmeiotic development of microspores; (iii) microspore mitosis and (iv) microgametogenesis (Chaudhury \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e1993\u003c/span\u003e; G\u0026oacute;mez et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Halbritter et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Li et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that differences between J4A and J4B began to appear at the start of meiosis, the middle cell layers of J4A did not degenerate and tapetum cells did not undergo mitosis, unable to provided nutrients for microspore development, led to microspore abortion. Kong et al. (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) through the H276A microspore observations indicated that neither the degradation of nuclei in microspores during the tetrad stage nor the degradation of tapetal cells transpired during the microspore development.\u003c/p\u003e \u003cp\u003eIn this study, the cytoplasmic sterile line was used as the maternal parent and the restorer line was used as the paternal parent. Two different F\u003csub\u003e2\u003c/sub\u003e segregating populations were constructed as experimental materials. Genetic analysis showed that the fertile and sterile plants followed a 3:1 segregation ratio, indicating that the sterile trait is controlled by a single recessive gene. There were variations in morphological size, with sterile plants of Y66 and Y73 having significantly smaller floral organs, smaller petals and smaller sepals in contrast to fertile plants. SEM showed that the anthers of sterile plants were smaller than those of fertile plants, the anthers crumpled were different, and the morphology of the folds in the anther epidermis were different. Further observation of anther sections showed that the degradation degree of tapetum was different between sterile and fertile plants. Meanwhile, the pollen grains of the sterile plants degraded over time, resulting in empty pollen grains. In conclusion, this experiment aims to provide the theoretical basis for in-depth investigations of the dominance configuration of cotton hybrid combinations and the functions of sterile genes and restorer genes.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant materials\u003c/h2\u003e \u003cp\u003eTwo F\u003csub\u003e2\u003c/sub\u003e segregating populations of cotton were constructed from cytoplasmic sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal), designated Y66 (HaA \u0026times; HaR) and Y73 (01A \u0026times; 26R). The cultivation of all experimental materials was conducted in the teaching test field of Shihezi University (Shihezi, Xinjiang Uygur Autonomous Region, China). The experimental materials managed based on the production mode, regular fertilization, and irrigation. The populations were constructed in 2019, crossed to get F\u003csub\u003e1\u003c/sub\u003e generation, F\u003csub\u003e1\u003c/sub\u003e generation self-crossed to get F\u003csub\u003e2\u003c/sub\u003e generation; the F\u003csub\u003e2\u003c/sub\u003e segregating populations were evaluated for fertility identification, morphological and cytological observation in 2021.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eFertility investigation and flower morphological characteristics observation\u003c/h2\u003e \u003cp\u003eThe fertility of each F\u003csub\u003e2\u003c/sub\u003e segregating populations were assessed during the flowering stage. Depending on whether pollen was produced, fertility was assessed and labeled, and the number of sterile and fertile plants was recorded. To ensure accuracy, the time, weather, and temperature of phenotype identifications were as consistent as possible each time, the survey was repeated five times, with at least three flowers per plant examined each time (Liu et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The fertility data were analyzed using IBM SPSS Statistics 26 for the \u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e test and \u003cem\u003eP\u003c/em\u003e-value. The flower buds and blooming flowers of sterile and fertile plants were sampled the same growth state to avoid the incomplete development and deformity of flower organs caused by growth, nutrient and other reasons of individual plants, and the morphology of the floral organs were observed and photographed three times at various stages of development.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSampling and fixation\u003c/h2\u003e \u003cp\u003e According to the flower bud development process, we randomly selected the sterile and fertile buds at several stages, including the pollen maturation stage (PMS), pre-flowering stage and flowering stage. The samples for paraffin sections were immersed in FAA fixative solution (anhydrous ethanol: glacial acetic acid: 37% formaldehyde solution: distilled water\u0026thinsp;=\u0026thinsp;10:1:2:7 v/v), vacuumed, slowly deflated, repeated thrice, each time for 5\u0026ndash;10 min. After vacuuming, replaced with FAA fixative solution and temporarily stored at 4℃. Following 24 h, the samples were transferred to 70% ethanol and stored at 4℃ (Yang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The SEM samples were soaked in 2.5% glutaraldehyde fixed solution, vacuumed, slowly deflated, repeated thrice, each time for 5\u0026ndash;10 min, and then replaced with 2.5% glutaraldehyde fixed solution once after vacuuming and stored at 4℃ (Wu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eScanning electron microscopy observation of cotton anthers\u003c/h2\u003e \u003cp\u003eFixed anthers were immersed in a 2.5% glutaraldehyde fixed solution, followed by soaking in 0.1 M phosphate buffer (pH\u0026thinsp;=\u0026thinsp;7.2) for 20 min, repeated three times at room temperature. Anthers were dehydrated in a stepwise manner using 50%, 70%, 80%, 90%, and 100% ethanol, and each gradient was maintained at 4℃ for 20 min. A fully automated critical point dryer (Leica EM CPD300) was used for CO\u003csub\u003e2\u003c/sub\u003e drying. Treated anthers were mounted on the sample platform with double-sided adhesive tape. The surface of each anther was coated with a layer of gold using sputter deposition (Chang et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2016\u003c/span\u003e; Tian and Wan 2018; Liu et al. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Observations and micrograph acquisition were performed using a scanning electron microscope (Hitachi SU8010) at the Analysis and Testing Center (Shihezi University).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eParaffin section observation of cotton anthers\u003c/h2\u003e \u003cp\u003eTake anther had been immersed in FAA Fixative Solution, stepwise dehydrated and waxed leaching. 75% alcohol 5 h, 85% alcohol 2.5 h, 90% alcohol 2.5 h, 95% alcohol 1.5 h, anhydrous ethanol repeated twice for 30 min each time, xylenes soaked twice for 10 min each time, and paraffin immersed thrice for 1 h each time. The wax-soaked samples were embedded in the embedding machine. The melted wax was placed into the embedding frame. Cool it at -20℃ on a freezing table. After the wax solidified, the samples were sliced to 4\u0026ndash;6\u0026micro;m with a microtome. Tissue slides were placed on the slides and dried for 1 h. The samples were dewaxed in xylene two times for 8\u0026ndash;10 min each time and rehydrated in ethanol in steps of 10 min each stage. Subsequently, the samples were placed into 0.5% Toluidine Blue O staining solution for 5 min and dehydrated in a gradient alcohol series (Min et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Put the samples rinsed in xylene three times for 5 min each time, and the tissue sections were mounted. Finally, a Panoramic MIDI automatic digital slide scanner (3DHISTECH Ltd., Budapest, Hungary) was used for observation images (Shao et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eGenetic analysis of the CMS\u003c/h2\u003e \u003cp\u003eIn the F\u003csub\u003e2\u003c/sub\u003e segregating population, we observed two phenotypes of Y66 and Y73 plants, one with pollen and the other without pollen. The fertility phenotype was investigated based on pollen dispersal during the flowering stage (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). We found that the F\u003csub\u003e2\u003c/sub\u003e population Y66 derived from the hybridization and self-crossing of HaA and HaR had 224 fertile plants and 71 sterile plants, consistent with a 3:1 segregation ratio (\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.137\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003csub\u003e0.05\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.841, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.712\u0026thinsp;\u0026gt;\u0026thinsp;0.05). And the F\u003csub\u003e2\u003c/sub\u003e population Y73 was derived from the hybridization and self-crossing of 01A and 26R had 239 fertile plants and 88 sterile plants, consistent with a 3:1 segregation ratio (\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.637\u0026thinsp;\u0026lt;\u0026thinsp;\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003csub\u003e0.05\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.841, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.425\u0026thinsp;\u0026gt;\u0026thinsp;0.05). In conclusion, these results indicate that the sterile trait is controlled by a single recessive gene.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSeparation ratio of fertile plants and sterile plants in F2 populations.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMaterials\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFertility\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eSterility\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eExpect separation ratio\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cem\u003eχ\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e-value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cem\u003eP\u003c/em\u003e-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY66\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e295\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e224\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.137\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.712\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e327\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e239\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e88\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3:1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.637\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.425\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePhenotype observation of the F\u003csub\u003e2\u003c/sub\u003e populations\u003c/h2\u003e \u003cp\u003eCotton flowers are hermaphroditic flowers composed of six parts: peduncle, receptacle, calyx, corolla, stamen and pistil. No difference in the morphological composition of floral organs were observed between fertile plants and sterile plants during the growth and development process. However, the flower organs of sterile plants exhibited significant male sterility traits. At flowering day, there was no obvious difference in the color of the floral organs between sterile plants and fertile plants, and all petals were light yellow (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-D). There were variations in morphological size, with sterile plants of Y66 and Y73 having significantly smaller floral organs, smaller petals and smaller sepals in contrast to fertile plants (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). After the removal petals, noticeable differences in anther histology were observed between sterile plants and fertile plants. Sterile plants possessed small and shriveled anthers with short and tiny filaments. No pollen was observed, and the surface of the anthers appeared concave and uneven in a lackluster light yellow or light brown (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eH, P). Nevertheless, compared to the corresponding fertile plants, we found that the developing anthers were plump and spherical, and dispersed pollen, eventually achieving a glossy yellow color (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eL, T).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eAnther epidermis SEM observation of the F\u003csub\u003e2\u003c/sub\u003e populations\u003c/h2\u003e \u003cp\u003e According to the phenotypic observations, there were significant differences between the sterile and the fertile plant anthers of Y66 and Y73 at flowering day, and the anthers of the sterile plants were deformed. Further observation by SEM revealed that the anthers of the sterile plants were smaller than those of the fertile plants. Moreover, there were obvious differences in the morphological characteristics of anther epidermis folds, including the peaks and valleys of fold formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The anther epidermis of Y66 sterile plants exhibited tight wrinkled and indented, the chamber epidermis displayed wavy, irregular folds with a loose fold structure, and the smooth peaks and valleys (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-C). The anther epidermis of Y66 fertile plants was fully expanded, the chamber epidermis exhibited prominent scale-like folds arranged in a close structure, with high peaks and deep valleys, resulting in a relatively rough surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD-F). The area cell where the vascular bundle and anther chamber converge in Y73 sterile plants contracted tightly. This caused an epidermal bump in the anther chamber, irregular wavy folds in the chamber's epidermis, a loose fold structure, and relatively mild peaks and valleys (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-I). The anther epidermis of fully matured Y73 plants was expanded, revealing clearly recognizable wavy folds of the chamber epidermis. The fold structure was closely packed, with steep peaks and deep valleys (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eJ-L).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAnthers paraffin section observation of the F\u003csub\u003e2\u003c/sub\u003e populations\u003c/h2\u003e \u003cp\u003eTo further analyze the sterile characteristics of the F\u003csub\u003e2\u003c/sub\u003e segregating population CMS, the anthers of sterile plants and fertile plants were selected to make paraffin sections at the pollen maturation stage (PMS), pre-flowering stage and flowering stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Observed the anther paraffin sections of PMS, we found that the degradation degree of the tapetum of the sterile plants of Y66 and Y73 was similar to that of the fertile plants of Y66 and Y73. We observed clear pollen grains, spinules on the surface of pollen grains, and the pollen grains were not significantly different in morphology (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, E, I, M). It can be inferred that there was no significant difference between the sterile plants of Y66 and Y73 and the fertile plants of Y66 and Y73 in pollen grain development before PMS. Upon observing the sterile (Y66 and Y73) and fertile (Y66 and Y73) plant at pre-flowering stage, noticeable discrepancies were found in the degree of degradation of the anther tapetum. It was noted that the anther chamber of the sterile plants almost lacked tapetum degradation residue. In sterile plants, the pollen grains gradually exhibited abnormalities and inclusions gradually disintegrated leading to the formation of voids. Furthermore, the nucleolus and cytoplasm disappeared, eventually culminating in the formation of empty pollen grains and the pollen grain spinules had lost their sharpness. In addition, it was observed that the cells in the anther chamber and anther vascular bundle area had atrophied (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, J, C, K). In fertile plants, exhibited full pollen grains, developed normally with good morphology and structure, and evenly distributed tapetum degradation products (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF, N, G, O). Observed the anthers of Y66 and Y73 at flowering day, it was observed that the anther chamber of the sterile plants had shrunken inward and the anther chamber of the fertile plants had fully expanded (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD, H, L, P).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eHeterosis utilization of CMS in cotton\u003c/h2\u003e \u003cp\u003eMeyer (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e1975\u003c/span\u003e) was the first to report the CMS of cotton and realized the \"three-line\" matching of the CMS of cotton. \"Three-line\" is composed of a sterile line, restorer line and maintainer line (Zheng et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). In the application of heterosis utilization, employing the \"three-line\" method of CMS can significantly economize on labor, materials, and finances resources compared to manual or chemical emasculation, as well as the \"two-line\" method. Additionally, this method affords straightforward, effective, and productive high-yielding seed production process. A high-quality cytoplasmic sterile line is essential for hybridization using the \"three-line\" method (Wang \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The more complete the sterility, the higher the purity of the hybrid line. Thus, many researchers are focusing on the in-depth exploration of excellent germplasm resources and restoring genes of cytoplasmic sterile lines or the genetic improvement of existing sterile lines and restorer lines through genetic engineering technology to promote the heterosis utilization of sterile lines. This study analyzed the cytoplasmic male sterility F\u003csub\u003e2\u003c/sub\u003e segregating population using fertility identification and genetic analysis. The results showed that Y66 and Y73, the experimental materials, followed a 3:1 separation ratio (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The study revealed that inheritance of the gene cause sterile trait is controlled by a single recessive gene. Which offers valuable material for thorough exploration into gene localization and functional analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eAbortion characteristics of the F\u003csub\u003e2\u003c/sub\u003e populations\u003c/h2\u003e \u003cp\u003eThe morphological changes of flower organs in male sterile plants are complex, exhibiting various phenotypes and variations in different fertility levels. The stamen atrophy, abnormal anther development, degeneration of sporangia and microspores, and functional infertility of the pollen are morphological and functional features of abortive flower organs (Zhang et al. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Jiang et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Many studies have shown that there are no significant differences in flower organ composition and developmental processes between various sterile lines of cotton and the materials used in this experiment. However, significant differences in the pollen development process exist between the corresponding fertile plants and maintainer lines of different sterile lines (Wu et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Kong et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Cheng et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Li et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In this experiment, there was no difference in flower organ composition and petal color between sterile and fertile plants on the day of flowering. The flower organs of sterile plants were smaller than those of fertile plants. Following petal removal, significant differences in filament length, anther morphology and anther color were also observed in the Y66 and Y73 sterile plants compared to the fertile ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). SEM showed that the anthers of sterile plants were smaller than those of fertile plants, the anthers crumpled were different, and the morphology of the folds in the anther epidermis were different (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). In summary, the abortive floral organs of Y66 and Y73 exhibit characteristics of stamen atrophy and anther abnormality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003ePollen abortion period, features and reasons of the F\u003csub\u003e2\u003c/sub\u003e population CMS\u003c/h2\u003e \u003cp\u003eLaser and Lersten (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e1972\u003c/span\u003e) believed that pollen abortion could occur at all stages of pollen development, and the period, mode and characteristics of abortion varied in different plants, species and sources of male sterility. Any mutation in a gene that affects stamen development, sporogonium differentiation, meiosis, mitosis, microspore development, or flower differentiation has the potential to cause male sterility in plants (Glover et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Niu et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Ko et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Han et al. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Xie et al. (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) reviewed that there are various factors causing plant male sterility, and the process of anther abortion is intricate, including tapetal development, ATPase, Ca\u003csup\u003e2+\u003c/sup\u003e concentration, cytoskeleton and programmed cell death. The tapetum is the innermost anther wall directly connected with pollen mother cells, and it plays a crucial role in pollen development (Falasca et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Singh et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2015\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Precise regulation is necessary for tapetum degradation, as it provides essential enzymes and nutrients for microspore development. Premature or delayed degradation may lead to microspore abortion (Balk and Leaver \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Ariizumi and Toriyama \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Wan et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). According to the observation of anther tissue sections (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), there was no significant difference in pollen development between the sterile and fertile plants at the pollen maturity stage, but the pollen of the sterile plants showed abnormal development, and the pollen abortion process was similar between Y66 sterile plants and Y73 sterile plants. Further observation showed that the degradation degree of tapetum was different between sterile and fertile plants. Before flowering, the sterile plants had almost no tapetum degradation residue in the anther chamber, but the fertile plants had more tapetum degradation residue and uniform distribution. Meanwhile, the pollen grains of the sterile plants degraded over time, resulting in empty pollen grains, and the cells in the connecting area between the vascular bundles and the anther chamber underwent atrophied. In summary, it can be concluded that pollen abortion occurred in Y66 and Y73 between the time of pollen maturity until flowering. The cause of this phenomenon may be due to premature degradation of the tapetum and insufficient nutrient supply to maintain pollen activity in the anther chamber, resulting in pollen grain degradation and eventually male sterility.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThe F\u003csub\u003e2\u003c/sub\u003e segregating populations derived from the cytoplasmic sterile lines HaA and 01A of cotton exhibited similar genetic, morphological and cytological characteristics. Genetic analysis showed that the fertile and sterile plants followed a 3:1 segregation ratio, indicating that the gene cause sterile trait was controlled by a single recessive gene. The floral morphology of the sterile plant was characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs between the time of pollen maturity until flowering. The degradation and formation of empty pollen grains, caused by the premature degradation of the tapetum, might be the main cause for pollen abortion.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe thank the Key Laboratory of Oasis Ecology Agricultural of Xinjiang Production and Construction Corps, Agricultural College, Shihezi University, Shihezi, Xinjiang, China, the Analysis and Testing Center, Shihezi University, Shihezi, Xinjiang, China ,and the Cotton Research Institute of the Shihezi Academy of Agriculture Science, Shihezi, Xinjiang, China, which provided us the experimental fields and experimental platform.\u0026nbsp;\u003c/p\u003e\n\u003ch2\u003eAuthors\u0026rsquo; contributions\u003c/h2\u003e\n\u003cp\u003eNie XH, Pan ZY, Guo CP and Liu JS designed and supervised the experiments. Nie XH provided the funding. You CY provided the cytoplasmic male sterility lines. Guo CP, Liu JS, Ma SM and Liu XY constructed the segregating population, investigated the fertility and phenotype observation. Lin HR, Li ZB, Zhao RH, Pan ZY and Pei QY performed the experiments. Pei QY sorted out the results, wrote the main manuscript and prepared all figures. Wu YL, Nie XH，Pan ZY and Guo CP revised and polished the manuscript. All authors contributed to the article and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis work was supported by the Fund for National Natural Science Foundation of China (31960439), the Key Core Technology Projects of the Xinjiang production and Construction Crops, China, the Technology Innovation Team Project of the Xinjiang production and Construction Crops, China, the Science and Technology Plan of Shuanghe city, Xinjiang production and Construction Crops, China (2021NY02), the Science and Technology Plan of Shuanghe city, Xinjiang production and Construction Crops, China (2021NY04) and the Key Programs for Science and Technology Development of Shihezi city, Xinjiang production and Construction Crops, China (2022NY01).\u003c/p\u003e\n\u003ch2\u003eSupplementary Information\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eAll authors have provided ethical approval and consent to participate as well as consent for publication.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003ch2\u003eAuthor details\u003c/h2\u003e\n\u003cp\u003e\u003csup\u003e1\u0026nbsp;\u003c/sup\u003eKey Laboratory of Oasis Ecology Agricultural of Xinjiang Production and Construction Corps, Agricultural College, Shihezi University, Shihezi 832003, Xinjiang, China. \u003csup\u003e2\u0026nbsp;\u003c/sup\u003eSeed Management Station of Xinjiang Production and Construction Corps, Urumqi 830000, Xinjiang, China. \u003csup\u003e3\u0026nbsp;\u003c/sup\u003eCotton Institute, Xinjiang Academy of Agricultural and Reclamation Science, Shihezi, Xinjiang 832000, China. \u003csup\u003e4\u003c/sup\u003e Xinjiang Jinbo Seed Industry Co., Ltd, Bole 833400, Xinjiang, China. \u003csup\u003e5\u003c/sup\u003e National Key Laboratory of Crop Genetic Improvement, College of Plant Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei, China. \u003csup\u003e6\u003c/sup\u003e Cotton Research Institute, Shihezi Academy of Agriculture Science, Shihezi 832011, Xinjiang, China. \u003csup\u003e7\u003c/sup\u003e Agricultural Science Research Institute of the 5th Division of Xinjiang Production and Construction Corps, Shuanghe 833408, Xinjiang, China.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAriizumi T, Toriyama K. Genetic Regulation of Sporopollenin Synthesis and Pollen Exine Development. Annu Rev Plant Biol. 2011;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-arplant-042809-112312\u003c/span\u003e\u003cspan address=\"10.1146/annurev-arplant-042809-112312\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. :437\u0026thinsp;\u0026ndash;\u0026thinsp;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBalk J, Leaver CJ. The PET1-CMS Mitochondrial Mutation in Sunflower Is Associated with Premature Programmed Cell Death and Cytochrome c Release. Plant Cell. 2001;13:1803\u0026ndash;18. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/TPC.010116\u003c/span\u003e\u003cspan address=\"10.1105/TPC.010116\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBirchler JA, Yao H, Chudalayandi S, et al. Heterosis Plant Cell. 2010;22:2105\u0026ndash;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.110.076133\u003c/span\u003e\u003cspan address=\"10.1105/tpc.110.076133\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChang Z, Chen Z, Wang N, et al. Construction of a male sterility system for hybrid rice breeding and seed production using a nuclear male sterility gene. Proc Natl Acad Sci USA. 2016;113:14145\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1073/pnas.1613792113\u003c/span\u003e\u003cspan address=\"10.1073/pnas.1613792113\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChaudhury AM. Nuclear Genes Controlling Male Fertility. Plant Cell. 1993;5:1277\u0026ndash;83. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.5.10.1277\u003c/span\u003e\u003cspan address=\"10.1105/tpc.5.10.1277\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen L, Liu Y. Male Sterility and Fertility Restoration in Crops. Annu Rev Plant Biol. 2014;65:579\u0026ndash;606. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1146/annurev-arplant-050213-040119\u003c/span\u003e\u003cspan address=\"10.1146/annurev-arplant-050213-040119\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCheng X, Zhang X, Xue F, et al. Characterization and transcriptome analysis of a dominant genic male sterile cotton mutant. BMC Plant Biol. 2020;20:312. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12870-020-02522-0\u003c/span\u003e\u003cspan address=\"10.1186/s12870-020-02522-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eColombo N, Galmarini CR. The use of genetic, manual and chemical methods to control pollination in vegetable hybrid seed production: a review. Plant Breed. 2017;136:287\u0026ndash;99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1111/pbr.12473\u003c/span\u003e\u003cspan address=\"10.1111/pbr.12473\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFalasca G, D'Angeli S, Biasi R, et al. Tapetum and middle layer control male fertility in Actinidia deliciosa. Ann Bot. 2013;112:1045\u0026ndash;55. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/aob/mct173\u003c/span\u003e\u003cspan address=\"10.1093/aob/mct173\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFarinati S, Draga S, Betto A, et al. Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications. Front Plant Sci. 2023;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2023.1223861\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2023.1223861\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFu W, Shen Y, Hao J, et al. Acyl-CoA N-acyltransferase influences fertility by regulating lipid metabolism and jasmonic acid biogenesis in cotton. Sci Rep. 2015;5:11790. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep11790\u003c/span\u003e\u003cspan address=\"10.1038/srep11790\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlover J, Grelon M, Craig S, et al. Cloning and characterization of \u003cem\u003eMS5\u003c/em\u003e from \u003cem\u003eArabidopsis\u003c/em\u003e: a gene critical in male meiosis. Plant J. 1998;15:345\u0026ndash;56. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1046/j.1365-313x.1998.00216.x\u003c/span\u003e\u003cspan address=\"10.1046/j.1365-313x.1998.00216.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eG\u0026oacute;mez JF, Talle B, Wilson ZA. Anther and pollen development: A conserved developmental pathway. J Integr Plant Biol. 2015;57. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/https://doi.org/10.1111/jipb.12425\u003c/span\u003e\u003cspan address=\"10.1111/jipb.12425\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. :876\u0026thinsp;\u0026ndash;\u0026thinsp;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGroszmann M, Greaves IK, Fujimoto R, et al. The role of epigenetics in hybrid vigour. Trends Genet. 2013;29. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.tig.2013.07.004\u003c/span\u003e\u003cspan address=\"10.1016/j.tig.2013.07.004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. :684\u0026thinsp;\u0026ndash;\u0026thinsp;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalbritter H, Ulrich S, Gr\u0026iacute;msson F, et al. Pollen Dev. 2018;23\u0026ndash;35. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/978-3-319-71365-6_2\u003c/span\u003e\u003cspan address=\"10.1007/978-3-319-71365-6_2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHan Y, Zhao F, Gao S, et al. Fine mapping of a male sterility gene ms-3 in a novel cucumber (\u003cem\u003eCucumis sativus\u003c/em\u003e L.) mutant. Theor Appl Genet. 2018;131:449\u0026ndash;60. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00122-017-3013-2\u003c/span\u003e\u003cspan address=\"10.1007/s00122-017-3013-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJan M, Liu Z, Guo C, et al. An Overview of Cotton Gland Development and Its Transcriptional Regulation. Int J Mol Sci. 2022;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms23094892\u003c/span\u003e\u003cspan address=\"10.3390/ijms23094892\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJiang H, Yang S, Liu Y, et al. Biological characteristics and cytological studies on anther abortion of male sterile \u003cem\u003eCamellia crassocolumn\u003c/em\u003e. Acta Agron Sinica. 2020;46:1076\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3724/SP.J.1006.2020.94159\u003c/span\u003e\u003cspan address=\"10.3724/SP.J.1006.2020.94159\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (in Chinese).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKempe K, Gils M. Pollination control technologies for hybrid breeding. Mol Breed. 2011;27:417\u0026ndash;37. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11032-011-9555-0\u003c/span\u003e\u003cspan address=\"10.1007/s11032-011-9555-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKo S, Li M, Sun-Ben Ku M, et al. The bHLH142 Transcription Factor Coordinates with TDR1 to Modulate the Expression of EAT1 and Regulate Pollen Development in Rice. Plant Cell. 2014;26:2486\u0026ndash;504. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1105/tpc.114.126292\u003c/span\u003e\u003cspan address=\"10.1105/tpc.114.126292\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKong X, Liu D, Liao X, et al. Comparative Analysis of the Cytology and Transcriptomes of the Cytoplasmic Male Sterility Line H276A and Its Maintainer Line H276B of Cotton (\u003cem\u003eGossypium barbadense\u003c/em\u003e L). Int J Mol Sci. 2017;18:2240. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms18112240\u003c/span\u003e\u003cspan address=\"10.3390/ijms18112240\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKushanov FN, Turaev OS, Ernazarova DK, et al. Genetic Diversity, QTL Mapping, and Marker-Assisted Selection Technology in Cotton (\u003cem\u003eGossypium\u003c/em\u003e spp). Front Plant Sci. 2021;12. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2021.779386\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2021.779386\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLaser KD, Lersten NR. Anatomy and cytology of microsporogenesis in cytoplasmic male sterile angiosperms. Bot Rev. 1972;38:425\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/BF02860010\u003c/span\u003e\u003cspan address=\"10.1007/BF02860010\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Chen L, Khan A, et al. Transcriptome and MiRNAomics Analyses Identify Genes Associated with Cytoplasmic Male Sterility in Cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e L). Int J Mol Sci. 2021;22. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms22094684\u003c/span\u003e\u003cspan address=\"10.3390/ijms22094684\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLima LFD, Oliveira JOD, Carneiro JNP, et al. Ethnobotanical and antimicrobial activities of the \u003cem\u003eGossypium\u003c/em\u003e (Cotton) genus: A review. J Ethnopharmacol. 2021;279:114363. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jep.2021.114363\u003c/span\u003e\u003cspan address=\"10.1016/j.jep.2021.114363\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu F, Ma L, Wang Y, et al. \u003cem\u003eGhFAD2\u003c/em\u003e\u0026ndash;\u003cem\u003e3\u003c/em\u003e is required for anther development in \u003cem\u003eGossypium hirsutum\u003c/em\u003e. BMC Plant Biol. 2019;19:393. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12870-019-2010-9\u003c/span\u003e\u003cspan address=\"10.1186/s12870-019-2010-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLiu L, Guo L, Qi T. SSCP Marker Development and Analysis of Candidate Restorer Gene \u003cem\u003eRf\u003c/em\u003e\u003csub\u003e1\u003c/sub\u003e for Cotton Cytoplasmic Male Sterility. Cotton Sci. 2018;30:12\u0026ndash;20. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.11963/1002-7807.llcxcz.20180104\u003c/span\u003e\u003cspan address=\"10.11963/1002-7807.llcxcz.20180104\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (in Chinese).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMayr E. Joseph Gottlieb Kolreuter's Contributions to Biology. Osiris. 1986;2:135\u0026ndash;76. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1086/368655\u003c/span\u003e\u003cspan address=\"10.1086/368655\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMeyer VG. Male Sterility From \u003cem\u003eGossypium harknessii\u003c/em\u003e. J Hered. 1975;66:23\u0026ndash;7. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/oxfordjournals.jhered.a108566\u003c/span\u003e\u003cspan address=\"10.1093/oxfordjournals.jhered.a108566\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMin L, Li Y, Hu Q, et al. Sugar and Auxin Signaling Pathways Respond to High-Temperature Stress during Anther Development as Revealed by Transcript Profiling Analysis in Cotton. Plant Physiol. 2014;164:1293\u0026ndash;308. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.113.232314\u003c/span\u003e\u003cspan address=\"10.1104/pp.113.232314\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNiu N, Liang W, Yang X, et al. EAT1 promotes tapetal cell death by regulating aspartic proteases during male reproductive development in rice. Nat Commun. 2013;4:1445. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/ncomms2396\u003c/span\u003e\u003cspan address=\"10.1038/ncomms2396\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRanaware AS, Kunchge NS, Lele SS, Ochatt SJ. Protoplast Technology and Somatic Hybridisation in the Family Apiaceae. Plants. 2023;12:1060. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/plants12051060\u003c/span\u003e\u003cspan address=\"10.3390/plants12051060\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSanders PM, Bui AQ, Weterings K, et al. Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Reprod. 1999;11:297\u0026ndash;322. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s004970050158\u003c/span\u003e\u003cspan address=\"10.1007/s004970050158\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchnable PS, Wise RP. The molecular basis of cytoplasmic male sterility and fertility restoration. Trends Plant Sci. 1998;3:175\u0026ndash;80. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/S1360-1385(98)01235-7\u003c/span\u003e\u003cspan address=\"10.1016/S1360-1385(98)01235-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShao P, Peng Y, Wu Y, et al. Genome-wide association study and transcriptome analysis reveal key genes controlling fruit branch angle in cotton. Front Plant Sci. 2022;13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2022.988647\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2022.988647\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSingh SP, Singh SP, Pandey T, et al. A novel male sterility-fertility restoration system in plants for hybrid seed production. Sci Rep. 2015;5:11274. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep11274\u003c/span\u003e\u003cspan address=\"10.1038/srep11274\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun L, Sui X, Lucas WJ, et al. Down-regulation of the Sucrose Transporter CsSUT1 Causes Male Sterility by Altering Carbohydrate Supply. Plant Physiol. 2019;180:986\u0026ndash;97. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1104/pp.19.00317\u003c/span\u003e\u003cspan address=\"10.1104/pp.19.00317\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSun Y, Fu M, Wang L, et al. OsSPLs Regulate Male Fertility in Response to Different Temperatures by Flavonoid Biosynthesis and Tapetum PCD in PTGMS Rice. Int J Mol Sci. 2022;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/ijms23073744\u003c/span\u003e\u003cspan address=\"10.3390/ijms23073744\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTian Y, Wan X, Cytobiology, Molecular Genetics Research Methods on Maize Anther Development. China Biotechnol. 2018;38:88\u0026ndash;99. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13523/j.cb.20180111\u003c/span\u003e\u003cspan address=\"10.13523/j.cb.20180111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (in Chinese).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVedel F, Pla M, Vitart V, et al. Molecular basis of nuclear and cytoplasmic male sterility in higher plants. Plant Physiol Biochem. 1994;32:601\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWan L, Zha W, Cheng X, et al. A rice β-1,3-glucanase gene Osg1 is required for callose degradation in pollen development. Planta. 2011;233:309\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00425-010-1301-z\u003c/span\u003e\u003cspan address=\"10.1007/s00425-010-1301-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWang X. Overview of the Study and Application of Cytoplasmic Male Sterility in Cotton. Scientia Agricultura Sinica. 2019;52:1341\u0026ndash;54. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3864/j.issn.0578-1752.2019.08.005\u003c/span\u003e\u003cspan address=\"10.3864/j.issn.0578-1752.2019.08.005\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (in Chinese).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWendel JF, Grover CE. Taxonomy and Evolution of the Cotton Genus. Gossypium. 2015;25\u0026ndash;44. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.2134/agronmonogr57.2013.0020\u003c/span\u003e\u003cspan address=\"10.2134/agronmonogr57.2013.0020\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWu Y, Min L, Wu Z, et al. Defective pollen wall contributes to male sterility in the male sterile line 1355A of cotton. Sci Rep. 2015;5:9608. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/srep09608\u003c/span\u003e\u003cspan address=\"10.1038/srep09608\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie C, Wei D, Tian H. Advances in Cell Biological Researches on Male Sterility of Higher Plants. J Plant Physiol Mol Biology. 2006;32:17\u0026ndash;23. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3321/j.issn:1671-3877.2006.01.003\u003c/span\u003e\u003cspan address=\"10.3321/j.issn:1671-3877.2006.01.003\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (in Chinese).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYahaya MA, Shimelis H, Laing M, et al. Methodologies for hybridization in predominantly self-pollinating crops: a review. J Crop Improv. 2020;34:268\u0026ndash;89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/15427528.2019.1698483\u003c/span\u003e\u003cspan address=\"10.1080/15427528.2019.1698483\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang L, Wu Y, Zhang M, et al. Transcriptome, cytological and biochemical analysis of cytoplasmic male sterility and maintainer line in CMS-D8 cotton. Plant Mol Biol. 2018;97:537\u0026ndash;51. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11103-018-0757-2\u003c/span\u003e\u003cspan address=\"10.1007/s11103-018-0757-2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang X, Zhang X, Yuan D, et al. Transcript profiling reveals complex auxin signalling pathway and transcription regulation involved in dedifferentiation and redifferentiation during somatic embryogenesis in cotton. BMC Plant Biol. 2012;12:110. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/1471-2229-12-110\u003c/span\u003e\u003cspan address=\"10.1186/1471-2229-12-110\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang C, Zhang M, Zhu Q, et al. Cytological observation of pollen development in 'Ougan' (\u003cem\u003eCitrus suavissima\u003c/em\u003e Hort. ex Tanaka) and its seedless mutant. J Fruit Sci. 2014;31:265\u0026ndash;9. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.13925/j.cnki.gsxb.2014.02.022\u003c/span\u003e\u003cspan address=\"10.13925/j.cnki.gsxb.2014.02.022\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. (in Chinese).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang R, Chang J, Li J, et al. Disruption of the bHLH transcription factor Abnormal Tapetum 1 causes male sterility in watermelon. Hortic Res. 2021;8:258. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41438-021-00695-9\u003c/span\u003e\u003cspan address=\"10.1038/s41438-021-00695-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang T, Xuan L, Mao Y, Hu Y. Cotton heterosis and hybrid cultivar development. Theor Appl Genet. 2023;136:89. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00122-023-04334-w\u003c/span\u003e\u003cspan address=\"10.1007/s00122-023-04334-w\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhang X, Zhang Z, Zhou R, et al. Ratooning Annual Cotton (\u003cem\u003eGossypium\u003c/em\u003e spp.) for Perennial Utilization of Heterosis. Front Plant Sci. 2020;11:554970. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3389/fpls.2020.554970\u003c/span\u003e\u003cspan address=\"10.3389/fpls.2020.554970\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZheng W, Ma Z, Zhao M, et al. Research and Development Strategies for Hybrid \u003cem\u003ejaponica\u003c/em\u003e Rice. Rice. 2020;13:36. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s12284-020-00398-0\u003c/span\u003e\u003cspan address=\"10.1186/s12284-020-00398-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"journal-of-cotton-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cotn","sideBox":"Learn more about [Journal of Cotton Research](https://jcottonres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cotn/default.aspx","title":"Journal of Cotton Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cotton, Cytoplasmic male sterility, Genetic analysis, Morphology, Cytology","lastPublishedDoi":"10.21203/rs.3.rs-3690132/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3690132/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e The utilization of male sterility is essential for producing hybrid seeds, and comprehending the mechanism of male sterility is the foundation for developing sterile germplasm resources. However, only a few cytoplasmic male sterility (CMS) lines of cotton have been produced due to various hindrances. Inadequate agronomic traits and incomplete sterility, weak resilience of restorer lines and the difficulty in combining strong dominance all serve as primary impediments to the advancement of CMS in cotton. Therefore, identifying and cytologically observing of CMS in cotton will provide new insights for heterosis utilization.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eTwo F\u003csub\u003e2\u003c/sub\u003e segregating populations of cotton were constructed from cytoplasmic sterile lines (HaA and 01A, maternal) and restorer lines (HaR and 26R, paternal).\u0026nbsp; Genetic analysis of F\u003csub\u003e2\u003c/sub\u003e segregation populations showed that the separation ratio of fertile to sterile plants was 3:1. Phenotypic analysis indicated that there was no significant difference in the development process of flower buds between sterile and fertile plants. Compared to fertile plants, sterile plants exhibit smaller floral organs, shortened filament length and anther atrophy on the day of flowering. Scanning electron microscopy (SEM) of two F2 populations revealed morphological variations in the folds of the anther epidermis. Analysis of cell level differences showed that there was no significant difference in pollen development before the pollen maturation stage. However, between pollen maturation stage and flowering stage, the tapetum layer of sterile plants degenerated prematurely, the pollen grains were abnormal, pollen cytoplasmic degradation and gradually hollowed out.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e The sterile trait is controlled by a single recessive gene. The floral morphology of the sterile plant was characterized by stamen atrophy and abnormal anthers. Pollen abortion occurs from the time of pollen maturity until flowering. Degradation of pollen grains and the formation of empty pollen grains due to the premature degradation of the tapetum are presumed to be the main cause for pollen abortion.\u003c/p\u003e","manuscriptTitle":"Identification and cytological observation of CMS in cotton","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-12 15:19:59","doi":"10.21203/rs.3.rs-3690132/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-03-18T22:28:46+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2023-12-11T09:30:26+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2023-12-08T06:36:52+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2023-12-01T05:47:58+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Cotton Research","date":"2023-11-29T21:09:00+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-cotton-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cotn","sideBox":"Learn more about [Journal of Cotton Research](https://jcottonres.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cotn/default.aspx","title":"Journal of Cotton Research","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d2b7137f-e076-4a5b-bb33-068d1eceb0bc","owner":[],"postedDate":"December 12th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-12T16:14:46+00:00","versionOfRecord":{"articleIdentity":"rs-3690132","link":"https://doi.org/10.1186/s42397-024-00189-8","journal":{"identity":"journal-of-cotton-research","isVorOnly":false,"title":"Journal of Cotton Research"},"publishedOn":"2024-08-05 15:58:00","publishedOnDateReadable":"August 5th, 2024"},"versionCreatedAt":"2023-12-12 15:19:59","video":"","vorDoi":"10.1186/s42397-024-00189-8","vorDoiUrl":"https://doi.org/10.1186/s42397-024-00189-8","workflowStages":[]},"version":"v1","identity":"rs-3690132","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3690132","identity":"rs-3690132","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}