Preparing high-quality chromosome spreads from Crocus species for karyotyping and FISH.

IF 1.3 4区生物学 Q4 GENETICS & HEREDITY Molecular Cytogenetics Pub Date : 2025-02-20 DOI:10.1186/s13039-025-00706-7

Abdullah El-Nagish, Susan Liedtke, Sarah Breitenbach, Tony Heitkam

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Abstract

Background: The saffron-producing Crocus sativus (L.) and its wild relative C. cartwrightianus (Herb.) are key species for understanding genetic evolution in this genus. Molecular-cytogenetic methods, especially fluorescent in situ hybridization (FISH), are essential for exploring the genetic relationships in this genus. Yet, preparing high-quality chromosomes for FISH analysis across Crocus species remains difficult. A standardized protocol for achieving clear and well-separated mitotic chromosomes is still lacking. This study aimed to assess the effectiveness of pretreatments with four chromosome synchronization methods for optimal chromosome spread preparation in Crocus. Root tips of different Crocus species were treated with four chromosome preparation methods namely hydroxyurea-colchicine (HC), nitrous oxide (NO), hydroxyquinoline (HQ), and ice water (IW) pretreatments to investigate their effectiveness in producing high-quality mitotic chromosome spreads. Metaphases obtained by the four methods were analyzed to assess their quality and metaphase index.

Results: Evaluation of 22,507 cells allowed us to confidently recommend a protocol for Crocus chromosome preparation. Among the methods, ice water pretreatment yielded the highest metaphase index (2.05%), more than doubling the results of HC (1.08%), NO (1.15%), and HQ (1.16%). Ice water-treated chromosomes exhibited better chromosome morphology, with relatively proper size, and non-overlapping chromosomes that were optimal for FISH analysis. Ice water pretreatment was also applied to C. cartwrightianus, the diploid progenitor of C. sativus, where it demonstrated similar efficacy. DAPI staining of chromosomes in both species allowed for clear visualization of intercalary and terminal heterochromatin. FISH analysis using 18S-5.8S-25S and 5S rDNA probes confirmed the utility of IW-prepared chromosome spreads for cytogenetic studies.

Conclusions: We strongly recommend ice water pretreatment as a suitable and effective method for obtaining many metaphase spreads of high-quality in C. sativus and related species, particularly for applications involving a detailed cytogenetic analysis.

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背景：产藏红花的 Crocus sativus (L.) 及其野生近缘种 C. cartwrightianus (Herb.) 是了解该属遗传进化的关键物种。分子-细胞遗传学方法，尤其是荧光原位杂交（FISH），对于探索该属的遗传关系至关重要。然而，要制备高质量的染色体用于对鳄鱼属各物种进行 FISH 分析仍然很困难。目前仍缺乏一个标准化的方案来获得清晰、分离良好的有丝分裂染色体。本研究旨在评估四种染色体同步方法的预处理效果，以优化有丝分裂染色体的扩散制备。研究人员用四种染色体制备方法，即羟基脲-秋水仙碱（HC）、氧化亚氮（NO）、羟基喹啉（HQ）和冰水（IW）预处理不同番红花品种的根尖，以考察它们在产生高质量有丝分裂染色体扩散方面的效果。对四种方法获得的有丝分裂片进行分析，以评估其质量和有丝分裂期指数：对 22,507 个细胞进行的评估使我们能够有把握地推荐一种用于制备番红花染色体的方案。在这几种方法中，冰水预处理产生的分裂相指数最高（2.05%），是HC（1.08%）、NO（1.15%）和HQ（1.16%）的两倍多。冰水处理后的染色体形态更好，大小相对合适，染色体无重叠，最适于进行 FISH 分析。冰水预处理也适用于 C. cartwrightianus（C. sativus 的二倍体祖先），其效果类似。对这两个物种的染色体进行 DAPI 染色，可以清楚地看到闰层和末端异染色质。使用 18S-5.8S-25S 和 5S rDNA 探针进行的 FISH 分析证实，冰水预处理染色体涂片可用于细胞遗传学研究：我们强烈建议将冰水预处理作为一种合适而有效的方法，用于获得许多高质量的荠菜及相关物种的移相平展片，尤其适用于涉及详细细胞遗传学分析的应用。

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来源期刊

Molecular Cytogenetics GENETICS & HEREDITY-

CiteScore

2.60

自引率

7.70%

发文量

审稿时长

>12 weeks

期刊介绍： Molecular Cytogenetics encompasses all aspects of chromosome biology and the application of molecular cytogenetic techniques in all areas of biology and medicine, including structural and functional organization of the chromosome and nucleus, genome variation, expression and evolution, chromosome abnormalities and genomic variations in medical genetics and tumor genetics. Molecular Cytogenetics primarily defines a large set of the techniques that operate either with the entire genome or with specific targeted DNA sequences. Topical areas include, but are not limited to: -Structural and functional organization of chromosome and nucleus- Genome variation, expression and evolution- Animal and plant molecular cytogenetics and genomics- Chromosome abnormalities and genomic variations in clinical genetics- Applications in preimplantation, pre- and post-natal diagnosis- Applications in the central nervous system, cancer and haematology research- Previously unreported applications of molecular cytogenetic techniques- Development of new techniques or significant enhancements to established techniques. This journal is a source for numerous scientists all over the world, who wish to improve or introduce molecular cytogenetic techniques into their practice.