Chromosomal in situ DNA hybridization (ISH) entails the hybridization of a single-stranded DNA (probe) to homologous sequences of partially denatured chromatin, followed by processing to allow detection of the sites of hybridization. Advent of this technique has led to the development of a new genetic discipline, molecular cytogenetics. We previously reported seminal development of ISH procedures for cotton (Gossypium hirsutum L.) meiotic chromosomes, based on non-radioactive detection. Subsequently, refinements in technique were reported, along with the localization of one of the three NOR sites to chromosome arm 9L. Evidence that meiotic ISH allowed physical mapping was thus gained. We report herein on some additional technical improvements, interpretive molecular cytogenetics, and mapping information on additional molecular cytogenetic markers.

Among the technical improvements made was altered fixation; acetone:glacial acetic acid (:1) with 1% PVP (MW 40,000) rendered specimens far more amenable to ISH than did fixation in 95% ethanol:glacial acetic acid (3:1). Squashing of microsporocytes on a 70 C slide warmer improved squashing by some persons. single-layer amplification with biotinylated anti-avidin, followed by streptavidin-peroxidase facilitated detection of smaller ISH sites. Inadequate penetration of anti-avidin seems to limit success. Smaller types of anti-avidin and prolongued incubation in anti-avidin improved results; anti-avidin treatment included 1 hr at 5 C, followed by an extra 40 min to allow for ample diffusion.

We describe interpretive procedures to deduce the positions of ISH sites, based on relative positions of ISH sites on multivalents. Sites can thus be assigned not only to chromosome, but also the arm and, where applicable, proximal vs. distal segment, relative to translocation breakpoints.

We have determined the locations of all three nucleolar organizing regions of G. hirsutum. They are chromosomes 9 in the distal region of the "left"/"short" arm; 16, in the "left"/"short" arm; and 3, in the distal region of the "left" arm. We also found two 5S RDNA sites exist in G. hirsutum. These were located in chromosome 9, in the proximal region of the "left"/"short" arm; and 3, in the proximal region of the "left" arm. The results strongly suggest that chromosomes 9 and 3 are homeologous, or, at least, that the "left" arms are homeologous. The TT5-9 breakpoint seems to lie between the 5S and 18S RDNA sites in short arm of chromosome 9. The results illustrate refinements in technology and interpretation. However, ISH is still a very underdeveloped technology, so marked improvements are anticipated. With continued technological improvements, numerous potential genetics and breeding-related applications exist for ISH to cotton chromosomes. The research was supported by USDA CRGO grants 88376-390 and 90-37140-56 and the Texas Agricultural Experiment Station.