High resolution FISH to delineate contiguous and small DNA sequences

Somatic and meiotic metaphase, and pachytene chromosomes were subjected to DNA: DNA in situ hybridization to elucidate relative resolution of FISH signals for weak/contiguous hybridization sites. Hybridization with a `350 family' rye repetitive DNA probe pSc 200 characteristically differentiated the rye chromosome 5 from the rest of the complement on account of two small terminal homologous sites in the long arm, resolution of which is substantially improved using pachytene. Higher resolution of the two weak hybridization sites; a very small distal and a small proximal, is unequivocally demonstrated in the FISH painted 5RL examined at pachytene in the 5AS/5RL wheat background. Additionally this probe exhibits a large block of distal telomeric hybridization site in 5RS, followed by a more prominent proximal site homologous to `610 family' rye repetitive probe pSc 250. Precise denaturation – hybridization incubation and post hybridization stringency washing facilitates spatial resolution of contiguous repetitive rye probes pSc 200 and pSc 250, and physical localisation of small RFLP probe xpr 115 of wheat on barley chromosomes.