Abstract
BackgroundDetailed karyotyping using metaphase chromosomes in melon (Cucumis melo L.) remains a challenge because of their small chromosome sizes and poor stainability. Prometaphase chromosomes, which are two times longer and loosely condensed, provide a significantly better resolution for fluorescence in situ hybridization (FISH) than metaphase chromosomes. However, suitable method for acquiring prometaphase chromosomes in melon have been poorly investigated.ResultsIn this study, a modified Carnoy’s solution II (MC II) [6:3:1 (v/v) ethanol: acetic acid: chloroform] was used as a pretreatment solution to obtain prometaphase chromosomes. We demonstrated that the prometaphase chromosomes obtained using the MC II method are excellent for karyotyping and FISH analysis. We also observed that a combination of MC II and the modified air dry (ADI) method provides a satisfactory meiotic pachytene chromosome preparation with reduced cytoplasmic background and clear chromatin spreads. Moreover, we demonstrated that pachytene and prometaphase chromosomes of melon and Abelia × grandiflora generate significantly better FISH images when prepared using the method described. We confirmed, for the first time, that Abelia × grandiflora has pairs of both strong and weak 45S ribosomal DNA signals on the short arms of their metaphase chromosomes.ConclusionThe MC II and ADI method are simple and effective for acquiring prometaphase and pachytene chromosomes with reduced cytoplasm background in plants. Our methods provide high-resolution FISH images that can help accelerate molecular cytogenetic research in plants.
Highlights
Detailed karyotyping using metaphase chromosomes in melon (Cucumis melo L.) remains a challenge because of their small chromosome sizes and poor stainability
The root tips were pretreated with freshly prepared 6:3:1 (v/v) ethanol: acetic acid: chloroform for 3–4 h at room temperature (RT), and fixed in 3:1 (v/v) ethanol: acetic acid solution (C3:1) for 5 days at 4 ° C. (2) Root tips were pre-treated with 0.002 M 8-Hq for 4 h at RT and fixed in C3:1 (v/v) for 5 days at 4 °C. (3) Root tips were pretreated with Ice water (Ice) for 24 h and fixed in C3:1 for 5 days at 4 °C. (4) Root tips were directly fixed in C3:1 for 5 days at 4 °C
Modified Carnoy’s solution II increases the prometaphase index Four methods, namely, modified Carnoy’s solution II (MC II), 0.002 M 8-Hq for 4 h, ice treatment for 24 h, and C3:1 were compared for their effectiveness in obtaining properly dispersed prometaphase and metaphase chromosomes in melon
Summary
Detailed karyotyping using metaphase chromosomes in melon (Cucumis melo L.) remains a challenge because of their small chromosome sizes and poor stainability. Suitable method for acquiring prometaphase chromosomes in melon have been poorly investigated. Fluorescence in situ hybridization (FISH), a molecular cytogenetic technique, requires properly dispersed metaphase or prometaphase chromosomes for its application. Melon (Cucumis melo L.) belongs to the Cucurbitaceae family and is a diploid species having 2n = 2x = 24 chromosomes [1]. Detailed karyotype analysis in the Cucumis genus, in melon, has been difficult to achieve because of their small chromosome sizes and poor stainability [2, 3]. The identification of secondary constrictions and the procurement of more detailed chromatin images are difficult, even when using properly dispersed metaphase chromosomes, because of their. Other methods to accumulate metaphase and prometaphase chromosomes, such as with ice water (ice) treatment for 24 h [11] or 0. 002 M 8-hydroxyquinoline (8-Hq) [12, 13] have
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