The Development of a Primed In Situ Hybridization Technique for Chromosome Labeling in Cultivated Strawberry (Fragaria×ananassa)

Few studies have aimed to develop physical chromosome mapping methods for cultivated strawberry (Fragaria×ananassa). Recently, linkage maps have been developed in this species using many single sequence repeat (SSR) markers. However, the accuracy of these needs to be evaluated because cultivated strawberries are allo-octoploids. This could be achieved by fluorescently labeling chromosomes that contain SSR markers on the same linkage group and observing whether other fluorescent signals can also be detected on the same chromosomes. The aim of this study was to develop a direct cycling primed in situ (C-PRINS) labeling technique for chromosome mapping of cultivated strawberry. To achieve this, we investigated the effect of the 1) maceration time for softening the root tips, 2) concentration of acetic acid for spreading chromosomes on the glass slide, 3) incubation time after slide preparation and before PRINS labeling, 4) number of PCR cycles for obtaining fluorescent signals, and 5) temperature accuracy for PCR. All of these factors were considered important for developing a chromosome labeling method. We found that a maceration time of 25 min was appropriate for obtaining many somatic cells with 56 chromosomes and that 45% acetic acid was effective for reducing the amount of damage to the chromosomes and obtaining clear chromosome images. A 72-h incubation time was appropriate for detecting chromosomes that had fluorescent signals, and the number of signals on the chromosomes increased with an increase in the number of PCR cycles. Finally, an aluminum tape treatment was effective for maintaining the PCR temperatures.