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Fulltext An improved method for inducing prometaphase chromosomes in plants

Setiawan AB, Teo CH, Kikuchi S, Sassa H, Koba T

Mol Cytogenet, 2018;11:32.
PMID: 29760782 DOI: 10.1186/s13039-018-0380-6

Background: Detailed 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.
Results: In 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.
Conclusion: The 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.
Chromosomal Locations of a Non-LTR Retrotransposon, Menolird18, in Cucumis melo and Cucumis sativus, and Its Implication on Genome Evolution of Cucumis Species

Setiawan AB, Teo CH, Kikuchi S, Sassa H, Kato K, Koba T

Cytogenet Genome Res, 2020;160(9):554-564.
PMID: 33171461 DOI: 10.1159/000511119

Mobile elements are major regulators of genome evolution through their effects on genome size and chromosome structure in higher organisms. Non-long terminal repeat (non-LTR) retrotransposons, one of the subclasses of transposons, are specifically inserted into repetitive DNA sequences. While studies on the insertion of non-LTR retrotransposons into ribosomal RNA genes and other repetitive DNA sequences have been reported in the animal kingdom, studies in the plant kingdom are limited. Here, using FISH, we confirmed that Menolird18, a member of LINE (long interspersed nuclear element) in non-LTR retrotransposons and found in Cucumis melo, was inserted into ITS and ETS (internal and external transcribed spacers) regions of 18S rDNA in melon and cucumber. Beside the 18S rDNA regions, Menolird18 was also detected in all centromeric regions of melon, while it was located at pericentromeric and sub-telomeric regions in cucumber. The fact that FISH signals of Menolird18 were found in centromeric and rDNA regions of mitotic chromosomes suggests that Menolird18 is a rDNA and centromere-specific non-LTR retrotransposon in melon. Our findings are the first report on a non-LTR retrotransposon that is highly conserved in 2 different plant species, melon and cucumber. The clear distinction of chromosomal localization of Menolird18 in melon and cucumber implies that it might have been involved in the evolutionary processes of the melon (C. melo) and cucumber (C. sativus) genomes.
Fulltext Centromeres of Cucumis melo L. comprise Cmcent and two novel repeats, CmSat162 and CmSat189

Setiawan AB, Teo CH, Kikuchi S, Sassa H, Kato K, Koba T

PLoS One, 2020;15(1):e0227578.
PMID: 31945109 DOI: 10.1371/journal.pone.0227578

Centromeres are prerequisite for accurate segregation and are landmarks of primary constrictions of metaphase chromosomes in eukaryotes. In melon, high-copy-number satellite DNAs (SatDNAs) were found at various chromosomal locations such as centromeric, pericentromeric, and subtelomeric regions. In the present study, utilizing the published draft genome sequence of melon, two new SatDNAs (CmSat162 and CmSat189) of melon were identified and their chromosomal distributions were confirmed using fluorescence in situ hybridization. DNA probes prepared from these SatDNAs were successfully hybridized to melon somatic and meiotic chromosomes. CmSat162 was located on 12 pairs of melon chromosomes and co-localized with the centromeric repeat, Cmcent, at the centromeric regions. In contrast, CmSat189 was found to be located not only on centromeric regions but also on specific regions of the chromosomes, allowing the characterization of individual chromosomes of melon. It was also shown that these SatDNAs were transcribed in melon. These results suggest that CmSat162 and CmSat189 might have some functions at the centromeric regions.

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