CHROMOSOME OBSERVATION METHODS IN THE GENUS FRAGARIA

Distinguishing strawberry chromosomes using light microscopy is extremely difficult, not only because of their small size and large number, but also because the current observation methods warrant improvement. To date, karyotype analysis in strawberry plants has not been completely examined. The objective of the present research was to improve the chromosome staining methods, to obtain clear images for counting, and to conduct karyotype analysis. For improved staining methods, the root tips of diploid, F. vesca L. and octoploid F. ×ananassa Duchesne ex Rozier and F. chiloensis (L.) Miller were collected, pretreated in a 2 mM 8-hydroxyquinoline solution for 1 h at room temperatures (around 23°C), and stored at 4°C for 16 h. Subsequently, they were fixed in a fresh solution of pure alcohol:glacial acetic acid (3:1) for 1 h, hydrolyzed in 1 mol/L HCl solution at 23°C for 1 h, then at 60°C for 11.5 min and stained separately with 1.5% lacto-propionic orcein (LPO), 60% acetic acid (AA) solution, and the two-step staining method (60% AA and 1.5% LPO). For 4’,6-diamidino-2-phenylindole (DAPI) staining, the root tips were macerated using an enzyme solution for 25 min at 42°C, and soaked in 60% AA for 5 min before staining. Clear digital images of the F. vesca, F. ×ananassa and F. chiloensis were obtained using light and fluorescent microscopy. Fluorescent microscopy displayed clear chromosome images at the pro-metaphase in F. vesca, F. ×ananassa and F. chiloensis samples. The clearest chromosome images were obtained with the two-step staining method in both F. ×ananassa and F. chiloensis. However, clear chromosome images can also be obtained by staining with 1.5% LPO alone. Results of genomic in situ hybridization (GISH) analysis of F. ×ananassa with F. iinumae, F. nipponica and F. vesca strongly support a proposed allo-polyploid origin of F. ×ananassa (2n = 8x = 56). The GISH results also confirmed that both F. iinumae and F. vesca have similar genomes to the diploid progenitors of F. ×ananassa ‘Umenoka’.