USE OF REPEATED DNA SEQUENCES AS CYTOLOGICAL MARKERS

The majority of DNA that is found in most of the flowering plants appears to be non-coding DNA. Much of this excess DNA consists of nucleotide sequences which exist as multiple copies throughout the genome and are designated as repetitive sequences. Those sequences which are found in moderately high to high numbers of copies are observed to be of the greatest value as cytological markers. Moderately high copies may exist as sequences which are dispersed throughout the chromosomes of some species and not dispersed in other more distantly related species. By taking advantage of this characteristic and the technique of in situ hybridization with biotinylated probes, breakpoints of chromosomal translocations may be observed between species such as wheat and rye. Many of the high copy number repetitive sequences are organized in a tandem fashion in specific loci in the chromosome. Chromosomal identification may be accomplished by using the in situ hybridization technique. Upon in situ hybridization with a repetitive sequence isolated from Aegilops squarrosa, the patterns of the sites of hybridization allowed the D-genome chromosomes to be identified. The sequence was also observed only on the D-genome chromosomes of several polyploid species indicating its usefulness as a genome specific marker. Using this genome specificity, assessment of the orientation of the D-genome chromosomal segments ofhexaploid wheat carrying the sequence during interphase and prophase of mitotic root tip cells was possible. Repetitive DNA sequences, therefore, provide cytological markers necessary for studies of chromosomal identification, genome allocation, and genome orientation. The use of biotin-labeled DNA probes allows the technique of in situ hybridization to be performed much more rapidly and with a greater degree of safety and reliability. IN THE EARLY 1960's, studies were underway in which the DNA duplex of various organisms was disassociated into two strands and then restored by incubation under proper conditions. Before these studies were undertaken in higher organisms, the speculation was that the large dilution ofnucleotides in the vast amounts of DNA per cell would require restoration (renaturation) to proceed for months. However, certain classes ofDNA which rapidly renatured were observed in eukaryotic organisms (Britten and Kohne, 1968). These classes of DNA represent sequences which are reiterated throughout the genome and are designated as repeated DNA sequences. The most highly repeated DNA sequences may consist of simple sequence or satellite DNA. Satellite DNA is defined as DNA which can be separated from the bulk of the main DNA by ultracentrifiguation in a neutral cesium chloride density gradient. 'Received for publication 29 June 1986; revision accepted 24 November 1986. 2 Current address is Department of Agronomy, Oklahoma State University, Stillwater, OK 74078. In addition, species containing cryptic or hidden satellite sequences have been observed (Comeo, Ginelli and Polli 1970; Bendich and Anderson, 1974; Peacock et al., 1981). These cryptic sequences require changes in the conditions of centrifugation, yet still represent highly repeated DNA sequences. Triticum aestivum L. has been reported to have a 2C DNA content of approximately 34.6 picogranis (pg) (Bennett, Smith and HeslopHarrison, 1982). A genome size of this magnitude would require less than 1% of the total DNA to carry out coding and other sequencerelated functions (Flavell, 1980). Much of the excess DNA appears to be composed of repeated DNA sequences. Estimates of the amount of repeated DNA sequences in plants have ranged from 35% to 95% (Flavell et al., 1974; Murray et al., 1979; Nagl et al., 1983). In T. aestivum, the proportion of repeated DNA sequences has been estimated at 83% and has been speculated to be much higher (Flavell et al., 1974; Flavell, 1980). Repeated sequences in plants appear to be undergoing constant amplification, deletion, and sequence divergence