Main conclusion Characterization of anther and ovule developmental programs and expression analyses of stage-specific floral marker genes in Gossypium hirsutum allowed to build a comprehensive portrait of cotton flower development before fiber initiation. Abstract Gossypium hirsutum is the most important cotton species that is cultivated worldwide. Although cotton reproductive development is important for fiber production, since fiber is formed on the epidermis of mature ovules, cotton floral development remains poorly understood. Therefore, this work aims to characterize the cotton floral morphoanatomy by performing a detailed description of anther and ovule developmental programs and identifying stage-specific floral marker genes in G. hirsutum . Using light microscopy and scanning electron microscopy, we analyzed anther and ovule development during 11 stages of flower development. To better characterize the ovule development in cotton, we performed histochemical analyses to evaluate the accumulation of phenolic compounds, pectin, and sugar in ovule tissues. After identification of major hallmarks of floral development, three key stages were established in G. hirsutum floral development: in stage 1 (early—EF), sepal, petal, and stamen primordia were observed; in stage 2 (intermediate—IF), primordial ovules and anthers are present, and the differentiating archesporial cells were observed, marking the beginning of microsporogenesis; and in stage 6 (late—LF), flower buds presented initial anther tapetum degeneration and microspore were released from the tetrad, and nucellus and both inner and outer integuments are developing. We used transcriptome data of cotton EF, IF and LF stages to identify floral marker genes and evaluated their expression by real-time quantitative PCR (qPCR). Twelve marker genes were preferentially expressed in a stage-specific manner, including the putative homologs for AtLEAFY , AtAPETALA 3 , AtAGAMOUS-LIKE 19 and AtMALE STERILITY 1 , which are crucial for several aspects of reproductive development, such as flower organogenesis and anther and petal development. We also evaluated the expression profile of B-class MADS-box genes in G. hirsutum floral transcriptome (EF, IF, and LF). In addition, we performed a comparative analysis of developmental programs between Arabidopsis thaliana and G. hirsutum that considered major morphoanatomical and molecular processes of flower, anther, and ovule development. Our findings provide the first detailed analysis of cotton flower development.

中文翻译：

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花形态解剖学特征和棉花花发育特定阶段优先表达的标记基因的鉴定

主要结论 陆地棉中花药和胚珠发育程序的表征和阶段特异性花标记基因的表达分析允许建立棉花纤维起始前发育的全面画像。摘要 陆地棉是世界范围内种植的最重要的棉花品种。尽管棉花的生殖发育对纤维的生产很重要，但由于纤维是在成熟胚珠的表皮上形成的，因此人们对棉花的花发育仍然知之甚少。因此，这项工作旨在通过详细描述陆地棉中的花药和胚珠发育程序并鉴定阶段特异性花标记基因来表征棉花花的形态解剖学。使用光学显微镜和扫描电子显微镜，我们分析了花发育的 11 个阶段的花药和胚珠发育。为了更好地表征棉花胚珠的发育，我们进行了组织化学分析以评估酚类化合物、果胶和糖在胚珠组织中的积累。在确定了花发育的主要标志后，陆地棉花发育的三个关键阶段被确定：在第 1 阶段（早期-EF），观察到萼片、花瓣和雄蕊原基；在第2阶段（中间-IF），存在原始胚珠和花药，并观察到分化的始孢子细胞，标志着小孢子发生的开始；在第6阶段（晚-LF），花芽出现初始花药绒毡层退化，小孢子从四分体中释放出来，珠心和内外珠被都发育。我们使用棉花 EF、IF 和 LF 阶段的转录组数据来鉴定花标记基因，并通过实时定量 PCR (qPCR) 评估它们的表达。十二个标记基因以特定阶段的方式优先表达，包括 AtLEAFY 、 AtAPETALA 3 、 AtAGAMOUS-LIKE 19 和 AtMALE STERILITY 1 的推定同源物，它们对生殖发育的几个方面至关重要，例如花器官发生和花药和花瓣发展。我们还评估了 G. hirsutum 花转录组（EF、IF 和 LF）中 B 类 MADS-box 基因的表达谱。此外，我们对拟南芥和陆地棉之间的发育程序进行了比较分析，考虑了花、花药和胚珠发育的主要形态解剖学和分子过程。