The Divergence of Chromosome Structures and 45S Ribosomal DNA Organization in Cucumis debilis Inferred by Comparative Molecular Cytogenetic Mapping.

Cucumis debilis W.J.de Wilde & Duyfjes is an annual and monoecious plant. This species is endemic to Southeast Asia, particularly Vietnam. However, C. debilis is rarely studied, and no detailed information is available regarding its basic chromosome number, 45S ribosomal DNA (rDNA) status, and divergence among other Cucumis species. In this study, we characterized the morphological characters and determined and investigated the basic chromosome number and chromosomal distribution of 45S rDNA of C. debilis using the fluorescent in situ hybridization (FISH) technique. A maximum likelihood tree was constructed by combining the chloroplast and internal transcribed spacer of 45S rDNAs to infer its relationship within Cucumis. C. debilis had an oval fruit shape, green fruit peel, and protrusion-like white spots during the immature fruit stage. FISH analysis using 45S rDNA probe showed three pairs of 45S rDNA loci located at the terminal region in C. debilis, similar to C. hystrix. Meanwhile, two, two, and five pairs of 45S rDNA loci were observed for C. melo, C. metuliferus, and C. sativus, respectively. One melon (P90) and cucumber accessions exhibited different chromosomal localizations compared with other members of Cucumis. The majority of Cucumis species showed the terminal location of 45S rDNA, but melon P90 and cucumber exhibited terminal-interstitial and all interstitial orientations of 45S rDNA loci. Based on molecular cytogenetics and phylogenetic evidence, C. debilis is more closely related to cucumber than melon. Therefore, C. debilis may serve as a potential parental accession for genetic improvement of cucumber through interspecific hybridization.

Centromeres of Cucumis melo L. comprise Cmcent and two novel repeats, CmSat162 and CmSat189.

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.

An improved method for inducing prometaphase chromosomes in plants.

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.