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Acta Agronomica Sinica ›› 2025, Vol. 51 ›› Issue (10): 2570-2580.doi: 10.3724/SP.J.1006.2025.54038

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

Construction of a meiotic progression identification system in cotton and analysis of its response to high-temperature stress

LI Ya-Wei(), XU Ying-Ying, ZUO Chun-Yang, LIU Ruo-Nan, LIANG Ya-Jun, KONG Jie, ZHANG Xian-Long, MIN Ling()   

  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University / Hubei Hongshan Laboratory, Wuhan 430070, Hubei, China
  • Received:2025-03-20 Accepted:2025-07-09 Online:2025-10-12 Published:2025-07-15
  • Contact: *E-mail: lingmin@mail.hzau.edu.cn E-mail:liyawei@webmail.hzau.edu.cn;lingmin@mail.hzau.edu.cn
  • About author:First author contact:**Contributed equally to this work
  • Supported by:
    “Unveiling and Commanding” Major Agronomic Traits and Molecular Design Breeding in Cotton Varieties of Xinjiang Uyghur Autonomous Region(2023A01-3);Projects Sponsored by the Development Fund for Xinjiang Talents “Exploration and Breeding Application of Key Genes for High-Quality, Heat-Resistant Cotton”(202403-03);Hubei Provincial Outstanding Youth Fund-Genetic Mechanism Analysis and Breeding Application of Cotton in Response to High Temperature Stress(2024AFA059)

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Abstract:

High-temperature stress has become a critical factor disrupting cotton meiosis and reducing yield. In this study, using the cotton transformation receptor material ‘Jin 668', we systematically established a reliable method for identifying meiotic progression in cotton by optimizing protocols for hydrochloric acid hydrolysis, enzymatic digestion, and acetic acid maceration of cotton flower buds. This optimized system effectively minimized interference from impurities during slide preparation. By examining the morphology of microspore mother cells and chromosomes in buds of varying lengths, we clarified the relationship between bud length and meiotic stage, and characterized cellular and chromosomal features across key stages, including prophase I, metaphase I, anaphase I, telophase I, metaphase II, anaphase II, and telophase II. Our results indicate that high-temperature stress significantly affects the early stages of prophase I, leading to abnormal chromosome condensation, failure of homologous pairing, disrupted crossover recombination, and ultimately defective microspore development. The identification system we developed provides a robust technical foundation for further investigation into cotton meiosis and its response to high-temperature stress, offering valuable strategies for breeding heat-tolerant cotton varieties and facilitating genetic improvement efforts.

Key words: cotton, high-temperature stress, meiosis, chromosome behavior, slide preparation technique

Fig. 1

Relationship between anther developmental stages and bud lengths in upland cotton ‘Jin 668' A-J represent semi-thin sections corresponding to different lengths of flower buds as follows, respectively. Annotations in the semi-thin sections are as follows, E: epidermis; Ar: archesporial cell; 1°P: primary parietal cell; 2°P: secondary parietal cell; 1°Sp: primary sporogenous cell; Sp: sporogenous cell; En: endothecium; ML: middle layer; T: tapetum; MMC: microspore mother cells; Msp: microspore. Vertical scale bar = 0.5 cm; horizontal scale bar=50 μm."

Fig. 2

Exploration of the optimal HCl acidolysis treatment time for meiosis in cotton anthers A-D show microscopic images of microspore mother cells from anthers after being treated with 1 mol L-1 hydrochloric acid for five minutes during different meiotic stages, pre-meiotic interphase (A), meiotic prophase I (B), meiotic telophase I (C), and the tetrad stage (D). E-K represent the exploration of acid hydrolysis duration for samples at the tetrad stage, with treatment times of 0 min (E), 1 min (F), 3 min (G), 5 min (H), 7 min (I), 10 min (J), and 20 min (K) using 1 mol L-1 hydrochloric acid. Scale bar=50 μm."

Fig. 3

Relationship between flower bud length and the morphology of sexual mother cells in cotton A: mitosis; B: interphase of meiosis; C: leptotene stage of prophase I of meiosis; D: zygotene stage of prophase I of meiosis; E: pachytene stage of prophase I of meiosis; F: diplotene stage of prophase I of meiosis; G: metaphase I of meiosis; H: anaphase I of meiosis; I: telophase I of meiosis; J: metaphase II of meiosis; K: anaphase II of meiosis; L: tetrad stage. Scale bar=50 μm."

Fig. 4

Optimization of enzymatic digestion time in cotton A-E depict the sample conditions in the enzymatic solution after different durations of enzymatic digestion, scale bar=1.5 cm; F-J show the sample conditions after washing with 100 μL of 0.01 mol L-1 sodium citrate buffer (pH 4.5) following enzymatic digestion, scale bar=1.5 cm; K-O present the individual anthers observed under a microscope after enzymatic digestion and rinsing with 0.01 mol L-1 sodium citrate buffer, scale bar=1000 μm; P-T illustrate the corresponding chromosome conditions at different enzymatic digestion durations, scale bar=20 μm."

Fig. 5

Optimization of the acetic acid hydrolysis system for chromosome visualization during meiosis in cotton anthers A-F: exhibit the conditions of cotton anthers where the chromosomes are covered by impurities after treatment with different concentrations of acetic acid for three minutes. G-L: demonstrate the conditions of cotton anthers where the chromosomes are covered by impurities after treatment with 70% acetic acid for varying durations. The durations of treatment are 3 min (G), 5 min (H), 7 min (I), 10 min (J), 12 min (K), and 15 min (L), respectively. Scale bar=20 μm."

Fig. 6

Chromosome dynamics throughout the meiotic process in cotton anthers A: prophase I interval of meiosis; B: leptotene stage of prophase I of meiosis; C: zygotene stage of prophase I of meiosis; D: pachytene stage of prophase I of meiosis; E: diplotene stage of prophase I of meiosis; F: diakinesis stage of prophase I of meiosis; G: metaphase I of meiosis; H: anaphase I of meiosis; I: telophase I of meiosis; J: metaphase II of meiosis; K: anaphase II of meiosis; L: tetrad stage. Scale bar as indicated in the lower right corner of the image; the upper right corner shows a magnified view. Scale bar=20 μm."

Fig. 7

Morphology of microspore mother cells during meiosis in cotton under normal and high temperature conditions A: displays the morphological characteristics of microspore mother cells during meiosis I under normal temperature conditions. B-D show the morphological characteristics of microspore mother cells during meiosis I under high temperature conditions. E illustrates the morphological characteristics of microspore mother cells at the tetrad stage under normal temperature conditions. F-H depict the morphological characteristics of microspore mother cells at the tetrad stage under high temperature conditions. NT: normal temperature; HT: high temperature. Scale bar=50 μm."

Fig. 8

Chromosome morphology at different meiotic stages in cotton under normal and high temperature conditions A and G depict the behavioral characteristics of the premeiotic interphase under normal and high temperature conditions, respectively. B and H illustrate the behavioral characteristics of the leptotene stage of prophase I of meiosis under normal and high temperature conditions, respectively. C and I show the behavioral characteristics of the zygotene stage of prophase I of meiosis under normal and high temperature conditions, respectively. D and J present the behavioral characteristics of the pachytene stage of prophase I of meiosis under normal and high temperature conditions, respectively. E and K exhibit the behavioral characteristics of the diplotene stage of prophase I of meiosis under normal and high temperature conditions, respectively. F and L depict the behavioral characteristics of the tetrad stage under normal and high temperature conditions, respectively. NT: normal temperature; HT: high temperature. Scale bar=20 μm."

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