Comparative karyotype analysis of eight Cucurbitaceae crops using fluorochrome banding and 45S rDNA-FISH

Abstract

To have an insight into the karyotype variation of eight Cucurbitaceae crops including Cucumis sativus Linnaeus, 1753, Cucumis melo Linnaeus, 1753, Citrullus lanatus (Thunberg, 1794) Matsumura et Nakai, 1916, Benincasa hispida (Thunberg, 1784) Cogniaux, 1881, Momordica charantia Linnaeus, 1753, Luffa cylindrica (Linnaeus, 1753) Roemer, 1846, Lagenaria siceraria var. hispida (Thunberg, 1783) Hara, 1948 and Cucurbita moschata Duchesne ex Poiret, 1819, well morphologically differentiated mitotic metaphase chromosomes were prepared using the enzymatic maceration and flame-drying method, and the chromosomal distribution of heterochromatin and 18S-5.8S-26S rRNA genes (45S rDNA) was investigated using sequential combined PI and DAPI (CPD) staining and fluorescence in situ hybridization (FISH) with 45S rDNA probe. Detailed karyotypes were established using the dataset of chromosome measurements, fluorochrome bands and rDNA FISH signals. Four karyotype asymmetry indices, CVCI, CVCL, MCA and Stebbins’ category, were measured to elucidate the karyological relationships among species. All the species studied had symmetrical karyotypes composed of metacentric and submetacentric or only metacentric chromosomes, but their karyotype structure can be discriminated by the scatter plot of MCA vs. CVCL. The karyological relationships among these species revealed by PCoA based on x, 2n, TCL, MCA, CVCL and CVCI was basically in agreement with the phylogenetic relationships revealed by DNA sequences. CPD staining revealed all 45S rDNA sites in all species, (peri)centromeric GC-rich heterochromatin in C. sativus, C. melo, C. lanatus, M. charantia and L. cylindrica, terminal GC-rich heterochromatin in C. sativus. DAPI counterstaining after FISH revealed pericentromeric DAPI+ heterochromatin in C. moschata. rDNA FISH detected two 45S loci in five species and five 45S loci in three species. Among these 45S loci, most were located at the terminals of chromosome arms, and a few in the proximal regions. In C. sativus, individual chromosomes can be precisely distinguished by the CPD band and 45S rDNA signal patterns, providing an easy method for chromosome identification of cucumber. The genome differentiation among these species was discussed in terms of genome size, heterochromatin, 45S rDNA site, and karyotype asymmetry based on the data of this study and previous reports.