Sterile Lines Research Articles

Male sterility in plants: an overview of advancements from natural CMS to genetically manipulated systems for hybrid seed production.

The male sterility system in plants has traditionally been utilized for hybrid seed production. In last three decades, genetic manipulation for male sterility has revolutionized this area of research related to hybrid seed production technology. Here, we have surveyed some of the natural cytoplasmic male sterility (CMS) systems that existed/ were developed in different crop plants for developing male sterility-fertility restoration systems used in hybrid seed production and highlighted some of the recent biotechnological advancements in the development of genetically engineered systems that occurred in this area. We have indicated the possible future directions toward the development of engineered male sterility systems. Cytoplasmic male sterility (CMS) is an important trait that is naturally prevalent in many plant species, which has been used in the development of hybrid varieties. This is associated with the use of appropriate genes for fertility restoration provided by the restorer line that restores fertility on the corresponding CMS line. The development of hybrids based on a CMS system has been demonstrated in several different crops. However, there are examples of species, which do not have usable cytoplasmic male sterility and fertility restoration systems (Cytoplasmic Genetic Male Sterility Systems-CGMS) for hybrid variety development. In such plants, it is necessary to develop usable male sterile lines through genetic engineering with the use of heterologous expression of suitable genes that control the development of male gametophyte and fertile male gamete formation. They can also be developed through gene editing using the recently developed CRISPR-Cas technology to knock out suitable genes that are responsible for the development of male gametes. The present review aims at providing an insight into the development of various technologies for successful production of hybrid varieties and is intended to provide only essential information on male sterility systems starting from naturally occurring ones to the genetically engineered systems obtained through different means.

Evaluation of Sweet Sorghum Hybrids for Juce Yield, Ethanol Yield and its Attributing Traits

An investigation was carried out to assess the extent of heterosis for juice yield and its attributing traits in 45 new hybrids of sweet sorghum developed by crossing 3 male sterile lines with 15 restorer in L x T mating design and were sown in randomized block design (RBD) with three replications during Kharif 2018 at Botany Farm, Post Graduate Institute, M.P.K.V., Rahuri. The significant average heterosis, heterobeltiosis and standard heterosis recorded for total biomass, juice yield and its contributing characters in fourty five crosses. The cross CMS-1409 x RSSV-512 showed the highest significant average heterosis (224.84%) and heterobeltiosis (286.36%) for juice yield. The extent of standard heterosis observed for juice yield over the check, CSH-22SS was upto 59.15 per cent. The highest significant standard heterosis was recorded by the cross CMS-1409 x RSSV-512 (59.15%), followed by cross ICMS-479 x RSSV-355 (43.58%) and CMS-1409 x RSSV-355 (24.30%). Based on per se performance and heterotic performance for yield and its principal components in the F1 hybrids the cross combinations viz; CMS-1409 x RSSV-512, ICMS-479 x RSSV-355 and CMS-1409 x RSSV-499 CMS-1409 x RSSV-498 and CMS-1409 x RSSV-355 were appeared to be promising.

Genetic analysis of the 1000 grain weight trait in interlinear hybrids of winter rye (secale cereale l.)

The aim of the study is to quantify the contribution of additive, dominant and epistatic dispersions to the overall genetic variation of the 1000 grain weight trait to increase the efficiency of breeding heterotic hybrids of winter rye. The experiments were carried out at the experimental base of the Federal Research Center «Nemchinovka» (Moscow region). The starting material was 10 male sterile homozygous inbred lines carrying the Pump-type cytoplasm. These lines were crossed with two male fertile test lines: mf H-842 and mf H-1247. Test crosses were carried out in 2019 on two isolated plots according to the topcross scheme. The resulting hybrids and their parent forms were tested in 2020 and 2021 on plots of 8 m2 in two-fold repetition. The test for epistasis and decomposition of the genetic varianсе into additive and dominant components was carried out by the method of Jinks J., Perkins L.M., Brees E.L. (1969). The data show that the additive interaction of genes, as well as incomplete dominance, plays the main role in determining the trait of granularity. A stable ratio of these interactions was established over the years: the proportion of additive effects varied at the level of 83.9-84.7%, and the proportion of dominant effects at the level of 15.3-16.1%. The indicator of the degree of dominance √(H1/D) was less than one and varied slightly over the years. It follows from this that dominant alleles affecting gross grain do not completely suppress the effect of their recessive alleles. For this reason, simple interlinear hybrids inherit an intermediate phenotype compared to the parent forms. The most coarse-grained hybrids were obtained by crossing lines ms H-1054, ms H-649 and ms H-1090 with the coarsegrained tester mf H-1247. It is concluded that, in the synthesis of commercial rye hybrids, it is necessary to strive to ensure that all parent forms are gross-grained. To obtain such lines it is advisable to use methods of recurrent selection as well as convergent and cumulative breeding.

Superoxide dismutase promotes early flowering in Triticum aestivum L.

Superoxide dismutase (SOD) is a first-line-defense antioxidant enzyme that plays a crucial role in scavenging reactive oxygen species (ROS) to maintain homeostasis in plants. SOD catalyzes the conversion of superoxide (O2-) into oxygen (O2) and hydrogen peroxide (H2O2), and besides its role in stress resistance, SOD also impacts plant growth and development. Here, we cloned and characterized a TaCSOD gene from the wheat photo-thermosensitive genic male sterile line BS366. Phylogenetic and motif analyses identified TaCSOD as a Cu/Zn-dependent SOD due to the presence of conserved Cu2+ and Zn2+ binding sites. Overexpression of TaCSOD enhanced drought and salt tolerance in both Arabidopsis thaliana and yeast. In addition, seed germination rate, primary root length, and fresh weight of the transgenic plants were higher than those of the wild-type under drought- and salt-stressed conditions. The Arabidopsis TaCSOD overexpression lines also exhibited an early flowering phenotype, with fewer leaves and shorter flowering period. Nitroblue tetrazolium (NBT) and 3, 3-diaminobenzidine (DAB) staining, along with transcriptome analysis, demonstrated that TaCSOD regulates ROS homeostasis and flowering time through carbohydrate signaling, aging, vernalization, and gibberellic acid pathways. Our study provides valuable insights into the functions of SOD genes in regulating flowering through the regulation of ROS homeostasis in plants.

Fine mapping and cloning of the sterility gene Bra2Ms in non-heading Chinese cabbage (Brassica rapa ssp. chinensis)

The application of a male-sterile line is an ideal approach for hybrid seed production in non-heading Chinese cabbage (Brassica rapa ssp. chinensis). However, the molecular mechanisms underlying male sterility in B. rapa are still largely unclear. We previously obtained the natural male sterile line WS24-3 of non-heading Chinese cabbage and located the male sterile locus, Bra2Ms, on the A2 chromosome. Cytological observations revealed that the male sterility of WS24-3 resulted from disruption of the meiosis process during pollen formation. Fine mapping of Bra2Ms delimited the locus within a physical distance of about 129 kb on the A2 chromosome of B. rapa. The Bra039753 gene encodes a plant homeodomain (PHD)-finger protein and is considered a potential candidate gene for Bra2Ms. Bra039753 was significantly downregulated in sterile line WS24-3 compared to the fertile line at the meiotic anther stage. Sequence analysis of Bra039753 identified a 369 bp fragment insertion in the first exon in male sterile plants, which led to an amino acid insertion in the Bra039753 protein. In addition, the 369 bp fragment insertion was found to cosegregate with the male sterility trait. This study identified a novel locus related to male sterility in non-heading Chinese cabbage, and the molecular marker obtained in this study will be beneficial for the marker-assisted selection of excellent sterile lines in non-heading Chinese cabbage and other Brassica crops.

Deciphering the inheritance of fertility restoration and influence of environmental factors on maldandi source of male sterility in rabi sorghum [Sorghum bicolor (L.) Moench

AbstractThis study aimed to investigate the inheritance pattern of fertility restoration in hybrids resulting from crosses between a male sterile line (M31‐2A), a maldandi cytoplasm, and two promising restorers (DSMR‐4 and DSMR‐8). The evaluation of hybrids was conducted in two sets of experiments, including the F2, BC1F1 and F2, F3 generations. The segregation pattern was analysed in the F2 and BC1F1 generations, resulting in a 54:10 ratio of fertile to sterile plants and a 1:1 ratio of fertile to sterile plants, respectively. This indicated the involvement of three genes, at least two of which must be in a dominant condition for restoration to occur, acting in a duplicate complementary manner. In the F3 generation, both crosses were grown to confirm the stability of restorers, and they segregated in the ratio of 10 true‐breeding families and 44 segregating families, which was consistent with the F2 ratio. Additionally, this study observed the influence of rainfall on the partial restoration pattern over seasons, as restorer genes are up or down‐regulated depending on the availability of water.

Utilizing differences in bTH tolerance between the parents of two-line hybrid rice to improve the purity of hybrid rice seed.

Two-line hybrid rice based on Photoperiod/thermo-sensitive genic male sterile (P/TGMS) lines has been developed and applied widely in agriculture due to the freedom in making hybrid combinations, less difficulty in breeding sterile lines, and simpler procedures for breeding and producing hybrid seed. However, there are certain risks associated with hybrid seed production; if the temperature during the P/TGMS fertility-sensitive period is lower than the critical temperature, seed production will fail due to self-pollination. In a previous study, we found that the issue of insufficient purity of two-line hybrid rice seed could be initially addressed by using the difference in tolerance to β-triketone herbicides (bTHs) between the female parent and the hybrid seeds. In this study, we further investigated the types of applicable herbicides, application methods, application time, and the effects on physiological and biochemical indexes and yield in rice. The results showed that this method could be used for hybrid purification by soaking seeds and spraying plants with the bTH benzobicylon (BBC) at safe concentrations in the range of 37.5-112.5 mg/L, and the seeds could be soaked in BBC at a treatment rate of 75.0 mg/L for 36-55 h without significant negative effects. The safe concentration for spraying in the field is 50.0-400.0 mg/L BBC at the three-leaf stage. Unlike BBC, Mesotrione (MST) can only be sprayed to achieve hybrid purification at concentrations between 10.0 and 70.0 mg/L without affecting yield. The three methods of hybrid seed purification can reach 100% efficiency without compromising the nutritional growth and yield of hybrid rice. Moreover, transcriptome sequencing revealed that 299 up-regulated significant differentially expressed genes (DEGs) in the resistant material (Huazhan) poisoned by BBC, were mainly enriched in phenylalanine metabolism and phenylpropanoid biosynthesis pathway, it may eliminate the toxic effects of herbicides through this way. Our study establishes a foundation for the application of the bTH seed purification strategy and the three methods provide an effective mechanism for improving the purity of two-line hybrid rice seeds.

Variability of the main breeding traits of sorgo CMS lines depending on the weather conditions of the Saratov region

Determining the nature of the interaction between the genotype and the environment is one of the critical stages of breeding practice for selecting effective parental forms of hybrids of the first generation of sorghum and their stable seed reproduction in the arid conditions of the Saratov region. The contribution of regulated and unregulated factors to the overall variability of a trait can be determined using a two-factor analysis of variance used in this work. The research objects were 11 sterile lines of grain sorghum grown on the experimental field of the Federal State Budgetary Research Institution RosRISM (Russian Research Institute of Sorghum and Maize) “Rossorgo” during 2009–2018. The meteorological conditions of plant cultivation varied significantly: in 2013 and 2017, they were characterised as sufficiently humid (Hydrothermal coefficient - HTC = 0.98–1.01), 2011 – acutely dry (HTC = 0.26), other years – dry (HTC = 0.45–0.68). As a result of the tests, it was found that the CMS lines differed in economically valuable traits. The sterile line A1 Efremovskoe 2 should be classified as tall (131.2 cm) and more productive (4.41 t/ha of seeds) with a well-developed leaf apparatus (the area of the giant leaf is 238.0 cm2). Low-growing CMS lines M35 and 9E Pishchevoe 614 (94.9–98.5 cm) were distinguished by the protrusion of the inflorescence stem - 21.6 cm, and A3, A4 and 9E Zheltozernoe 10 (109.8–110.2 cm) - with a long leaf (50.7–51.5 cm). Medium-sized lines A2 Vostorga and A4 KP 70 had a long inflorescence (23.0–24.5 cm), while A3 Feterita 14 had a short inflorescence (13.7 cm). It was found that the genotype (41.4–61.6%) had a more substantial effect on the overall variability of plant height, length and protrusion of the stem of the inflorescence, and leaf length, while the environmental factor had a more substantial effect on seed yield (55.3%).

Genetic analysis and fine mapping of a grain size QTL in the small-grain sterile rice line Zhuo201S

The development and application of the small-grain rice sterile line Zhuo201S (Z201S) has demonstrated its potential for mechanized hybrid rice seed production, leading to significant cost reductions. However, the molecular mechanism responsible for the small-grain size characteristic of Z201S remains unclear. In this study, we conducted a genetic analysis using near-isogenic lines constructed from Z210S, a small-grain rice sterile line, and R2115, a normal-grain variety. The results revealed that the small-grain trait in Z201S is governed by a single partially dominant gene which also enhances grain number. Through mapping, we localized the causal gene to the short arm of chromosome 2, within a 113 kb physical region delimited by the molecular markers S2-4-1 and LB63. Transgenic analysis and gene expression assays indicated LOC_Os02g14760 as the most likely candidate gene, suggesting that the small-grain size trait of Z201S is controlled by a novel locus that has not been previously identified.

Use of olive pomace extract as a pollinator attractant to increase onion (Allium cepa L.) seed crop production

Onion is an allogamous species with protandry, as anthers release pollen before the stigma is receptive. Thus, insect pollination is needed to produce seeds. When male sterile lines are used, an efficient pollinator is important to ensure pollen transfer to the stigma. Therefore, increasing seed yield depends on increasing bee activity. Seed yield is variable in hybrid lines and lower than open-pollinated varieties, with a decrease of up to 60%. Bee attractants sometimes are used to influence worker bees towards flowers for pollination. The objective of this work is to use a food industry waste, such as olive pomace, as bee attractant upon application of the water extract on onion umbels. The effectiveness of this by-product in enhancing pollination efficiency was evaluated and compared to untreated controls. Male-sterile lines, as well as a fertile line, were used in commercial fields and environmental variables were registered. Pollination effectiveness was measured by seed yield. Olive pomace water extracts (OPWE), rich in bioactive compounds, from local industries were evaluated. Some compounds present in the OPWE were analyzed, identified and quantified. The OPWEs were applied during flowering, at different times and correlated with seed yield and the frequency of bee visits. After 3 years of study, we can assert that the application of OPWE enhances honeybee pollination efficiency, and as consequence improves onion seed yield.

Natural variation of MS2 confers male fertility and drives hybrid breeding in soybean.

A complete and genetically stable male sterile line with high outcrossing rate is a prerequisite for the development of commercial hybrid soybean. It was reported in the last century that the soybean male sterile ms2 mutant has the highest record with seed set. Here we report the cloning and characterization of the MS2 gene in soybean, which encodes a protein that is specifically expressed in the anther. MS2 functions in the tapetum and microspore by directly regulating genes involved in the biosynthesis of secondary metabolites and the lipid metabolism, which is essential for the formation of microspore cell wall. Through comparison of the field performance with the widely used male sterile mutants in the same genetic background, we demonstrated that the ms2 mutant conducts the best in outcrossing rate and makes it an ideal tool in building a cost-effective hybrid system for soybean.

Estimating reliability for evaluating nitrate accumulation in sorghum (Sorghum bicolor L. Moench) inbred A‐lines

AbstractSorghum is an important forage crop in many cropping systems. Nitrate can accumulate in the vegetative plant tissue to levels high enough that result in fatality when fed to ruminant livestock when grown under high nitrate conditions. If variation among inbred lines can be documented for nitrate concentration, it may be possible to reduce nitrate accumulation within forage sorghum hybrids by selecting inbred lines having lower nitrate accumulation. The goal of this study was to evaluate a diverse set of male‐sterile sorghum inbred lines grown under high nitrate conditions, to observe if genotypic variation exists among lines used to produce forage sorghum hybrids. Forty replicates of 20 sorghum A‐lines were grown in a greenhouse and received either 272 kg ha−1 or 408 kg ha−1 of actual nitrogen per plant through applications of ammonium nitrate. Variance components were extracted from linear regression models and reliability was estimated on an entry‐mean basis. Leaf and stem reliability estimates were 0.95 and 0.93, respectively. These reliability estimates indicate the genetic variance comprised most of the phenotypic variance, and that our measurements were consistent. Significant differences were also observed between genotypes for both leaf and stem tissue. Therefore, sufficient genetic variance exists for developing male sterile sorghum inbred lines having leaf nitrate concentrations below the potentially lethal toxicity threshold of 10,000 μg g−1.

Combined transcriptome and proteome analyses between the cytoplasmic male sterile line and its maintainer line in Welsh onion (Allium fistulosum)

AbstractCytoplasmic male sterility (CMS) is dispensable for research of heterosis and investigation of nuclear–cytoplasmic interaction in Welsh onion. The molecular mechanism of the cytoplasmic male sterile line and its maintainer line was investigated by combined transcriptome and proteome analyses in Welsh onion. The 410,585 full‐length non‐chimeric (FLNC, full‐length readsnon‐Chimeric) sequences were obtained, and AS events and lncRNAs were identified by full‐length transcriptome. Towards a comprehensive understanding and identification of related genes for male sterility, the transcriptome and proteome sequencing were utilized to explore the differences between the cytoplasmic male sterile line and its maintainer line. The 287 differentially expressed genes (DEGs) and 49 differentially expressed proteins (DEPs) were identified, and we furthermore explored the function of genes and proteins. The heat map preliminarily demonstrated the expression change of the key genes and proteins in two cultivars. CMS‐related genes were screened form differentially expressed genes, and verified by quantitative real‐time quantitative polymerase chain reaction (qRT‐PCR). Our research found the CMS regulatory genes, proteins and related pathways. The transcriptome and proteome datasets contribute to accelerate the development of CMS gene clones and functional genomics research on Welsh onions.

Development of alloplasmic male sterile lines and their assessment for heterotic potential in eggplant (Solanum melongena L.)

ABSTRACT The use of Solanum aethiopicum as the female parent with S. melongena led to the development of a cytoplasmic-genic male sterility system (CGMS) in eggplant. The male sterility was complete and expressed as rudimentary anthers with no pollen grains. Ten backcross progenies of male sterile lines in diverse genetic backgrounds of S. melongena were characterised after BC7 generation for 14 horticultural traits. It was observed that male sterile lines and their respective maintainer parents were isogenic for all the traits, except stamen size and days to 50% flowering. The fertility of male sterile lines was restored after hybridising with plants derived from an S. melongena × S. aethiopicum cross. The fertility restoration was governed by a single dominant gene. The maternal cytoplasm of S. aethiopicum in male sterile lines and of S. melongena in maintainers and restorers was confirmed with mitochondria and chloroplast DNA-specific molecular markers. Further, to assess the heterotic potential, 10 cytoplasmic male sterility lines were crossed with three restorers to develop 30 F1 hybrids, out of which D−67A × R−6 and D−104A × R−6 were found consistently better than the standard check for yield plant−1 over two years. In this manuscript a comprehensive study on development and utilisation of an alloplasmic CGMS system in eggplant has been discussed.

A review of rice male sterility types and their sterility mechanisms

Male sterility plays an important role in the utilization of heterosis in rice. The establishment of male sterile lines in rice is one of the key technologies in hybrid rice production systems. The currently widely used male sterile line breeding systems mainly include: three-line hybrid rice based on cytoplasmic male sterility, two-line hybrid rice based on environmental sensitive gene male sterility, and third-generation hybrid rice based on nuclear gene male sterility Seed production system. This study reviewed the types and mechanisms of male sterility in rice, and looked forward to the development direction of hybrid rice.

Comparative Transcriptome Profiling of CMS-D2 and CMS-D8 Systems Characterizes Fertility Restoration Genes Network in Upland Cotton

Resolving the genetic basis of fertility restoration for cytoplasmic male sterility (CMS) can improve the efficiency of three-line hybrid breeding. However, the genetic determinants of male fertility restoration in cotton are still largely unknown. This study comprehensively compared the full-length transcripts of CMS-D2 and CMS-D8 systems to identify potential genes linked with fertility restorer genes Rf1 or Rf2. Target comparative analysis revealed a higher percentage of differential genes in each restorer line as compared to their corresponding sterile and maintainer lines. An array of genes with specific expression in the restorer line of CMS-D2 had functional annotations related to floral development and pathway enrichments in various secondary metabolites, while specifically expressed genes in the CMS-D8 restorer line showed functional annotations related to anther development and pathway enrichment in the biosynthesis of secondary metabolites. Further analysis identified potentially key genes located in the target region of fertility restorer genes Rf1 or Rf2. In particular, Ghir_D05G032450 can be the candidate gene related to restorer gene Rf1, and Ghir_D05G035690 can be the candidate gene associated with restorer gene Rf2. Further gene expression validation with qRT-PCR confirmed the accuracy of our results. Our findings provide useful insights into decoding the potential regulatory network that retrieves pollen fertility in cotton and will help to further reveal the differences in the genetic basis of fertility restoration for two CMS systems.

Irradiated Pollen-Induced Parthenogenesis for Doubled Haploid Production in Sunflowers (Helianthus spp.).

Doubled haploid (DH) technology is a tool used to develop large numbers of inbred lines and increase the rate of genetic gain by shortening the breeding cycles. However, previous attempts to produce DH sunflower plants (Helianthus spp.) have resulted in limited success. In this research, we applied gamma-induced parthenogenesis to assist the production of DH sunflowers. The objectives of the study included (1) identifying optimal gamma ray doses for inducing DH sunflowers using two cytoplasmic male sterility (CMS) lines as female plants and two male pollinators with recognizable morphological markers, (2) selecting new male pollinators from wild sunflower varieties, and (3) testing the efficacy of the selected male pollinators using emasculated non-male sterile sunflower lines as female plants. In these experiments, pollen grains were irradiated with gamma ray doses ranging from 50 to 200 Gy. The optimal gamma ray dose for pollen grain irradiation and DH plant production was identified to be 100 Gy. In addition, a cultivated (G11/1440) and a wild-type (ANN1811) sunflower line can be used as common male pollinators for their distinctive morphological markers and wide capacity for DH induction by gamma-irradiated pollen grains.

Genetic analysis of sunflower fatty acids under optimum and water stressed conditions

Abstract In order to study the genetic control of sunflower fatty acids under optimum and water limited conditions a set of 12 sunflower hybrids were evaluated as randomized complete block design with three replications in two separate experiments during two years (2019 and 2020) in Karaj, Iran. The hybrids were obtained by crossing of four male sterile lines and three restorer lines as testers. Water limitation was imposed by water withholding during reproductive stage. Palmitic acid content was governed by both additive and non-additive effects and linoleic acid content by non-additive gene action under optimum and water limited conditions. Stearic acid content was controlled by both additive and non-additive effects in optimum but by additive effects under water limited condition. Oleic acid content was governed by non-additive factors in optimum, while by both additive and non-additive effects in water limited condition. Oil content and leaf temperature were inherited by additive and seed yield by additive and non-additive effects in both conditions. Relative water content and chlorophyll content were controlled by both additive and non-additive effects under optimum but by non-additive effects in water limited condition. According to these results hybrid breeding is suggested for improvement of main sunflower fatty acids under optimum and water limited condition except that selection-based methods for improvement of stearic acid content.

Gibberellin confers to the expression of TaGA-6D and negatively regulates the fertility of wheat with Aegilops juvenalis cytoplasm

As the trace signal molecules widely existing in plants, plant hormones can regulate physiological responses of plants at low concentrations. At present, the effect of plant endogenous hormones on wheat male fertility has attracted attention, but the molecular mechanism underlying fertility regulation is unclear. Given this, the anthers of five isonuclear alloplasmic male sterile lines and their maintainer line were RNA-sequenced. A gene TaGA-6D encoding gibberellin (GA) regulated protein was isolated, which was located to the nucleus, cell wall and/or cell membrane, and predominantly highly expressed in the anther of Ju706A, a male sterile line with Aegilops juvenalis cytoplasm. By spraying assay of GA with different concentrations on fertility line Ju706R, it was found that with the increase of exogenous GA concentration, the content of endogenous GA and expression level of TaGA-6D in anther gradually increased, and the fertility decreased. However, silencing of TaGA-6D partially restore the fertility of Ju706R sprayed with 1000 ng/μl GA, and indicating that gibberellin can promote the expression of TaGA-6D and negatively regulates the fertility of wheat with Aegilops juvenalis cytoplasm, which providing new insights for understanding hormone regulation of male fertility in wheat.

Industrial chicory genome gives insights into the molecular timetable of anther development and male sterility.

Industrial chicory (Cichorium intybus var. sativum) is a biannual crop mostly cultivated for extraction of inulin, a fructose polymer used as a dietary fiber. F1 hybrid breeding is a promising breeding strategy in chicory but relies on stable male sterile lines to prevent self-pollination. Here, we report the assembly and annotation of a new industrial chicory reference genome. Additionally, we performed RNA-Seq on subsequent stages of flower bud development of a fertile line and two cytoplasmic male sterile (CMS) clones. Comparison of fertile and CMS flower bud transcriptomes combined with morphological microscopic analysis of anthers, provided a molecular understanding of anther development and identified key genes in a range of underlying processes, including tapetum development, sink establishment, pollen wall development and anther dehiscence. We also described the role of phytohormones in the regulation of these processes under normal fertile flower bud development. In parallel, we evaluated which processes are disturbed in CMS clones and could contribute to the male sterile phenotype. Taken together, this study provides a state-of-the-art industrial chicory reference genome, an annotated and curated candidate gene set related to anther development and male sterility as well as a detailed molecular timetable of flower bud development in fertile and CMS lines.