CYTOMOLECULAR MAPPING
Cytomolecular mapping unites molecular, genetic, genomic, and cytological data in a way that provides unique insight into how genomes function and evolve within the context of chromosomes.
OVERVIEW: We are currently involved in exploring relationships between meiotic chromosome structure, DNA sequence, recombination, and genome evolution using cytomolecular mapping. In cytomolecular mapping single-copy molecular markers are localized on synaptonemal complex (SC) spreads via fluorescence in situ hybridization. The chromosomal positions of the markers are then characterized with respect to each other and to cytological features such as centromeres, telomeres, heterochromatin, euchromatin, and chromomeres. Because the DNA markers used as probes in cytomolecular mapping are part of molecular maps (usually RFLP maps in plants), cytomolecular mapping allows molecular maps to be directly superimposed onto their corresponding chromosomes. In species such as sorghum where physical maps are being constructed (or in species in which genome sequencing is completed), cytomolecular mapping presumably can be used to position a complete (or nearly complete) chromosomal DNA molecule directly onto its corresponding chromosome (i.e., cytophysical mapping). In such a case, each cytomolecular marker serves as a point at which the DNA molecule is "anchored" onto the framework of the chromosome (see FIGURE 1 below).
FISH is usually performed on mitotic metaphase chromosomes, but SC spreads (= hypotonically-spread pachytene chromosomes/bivalents) appear to be better substrates for cytomolecular mapping (see Meiotic Cytology for review). Because each bivalent is composed of synapsed homologues (each containing two sister chromatids), there are four closely associated copies of each locus available for hybridization on a bivalent. In comparison, there are only two nearby copies of each locus available for FISH on a metaphase chromosome. However, the primary advantage of using pachytene chromosomes is that they are 5-15 times longer than corresponding metaphase chromosomes. Consequently, many closely associated loci that are not resolvable by FISH on metaphase chromosomes can be resolved on pachytene chromosomes.
Click to open PDF.
FIGURE 1: Cytophysical mapping.
TOMATO: During his Ph.D. work, Dr. Peterson used cytomolecular mapping to study the chromosomes/genome of tomato. In brief, tomato SC spreads were probed with two single-copy sequences and one single/low-copy sequence containing RFLP markers associated with SC 11. Individual SCs were identified based on relative length, arm ratio, and differential DNA staining patterns. Each of the three genomic probes hybridized exclusively to a different, highly-localized region on SC 11 (e.g., FIGURE 2). Based on the relative locations of hybridization sites, the three probes were unambiguously positioned in relationship to the centromere and to one another. In this first report of single-copy FISH to SC spreads, it was demonstrated that plant SCs are well suited for the rigorous requirements of in situ detection of single-copy sequences. Additionally, a discrepancy between the cytomolecular map and the molecular genetic map was discovered, the distance between molecular markers (in base pairs) was estimated, and the cytomolecular map was integrated with the pre-existing recombination nodule map for tomato. For further details see Peterson et al. (1999).
Click to enlarge image
FIGURE 2: Localization of two single/low-copy sequences (yellow foci) on tomato SC 11. Sites of hybridization are marked by yellow foci (FITC fluorescence). The chromosomes have been counterstained with propidium iodide (red).
SORGHUM: Sorghum is a staple in the diets of millions of people around the world. It is extremely drought resistant and can grow in soil with relatively high salt content. Consequently, its use as a human food source is most common in Africa where water is scarce and soil quality is poor. In terms of economic value, sorghum is the fourth most important cereal species in the United States (after ) where it is grown almost exclusively as animal feed.
Sorghum is closely related to maize and sugarcane although the sorghum genome is less than one-third the size of the maize and sugarcane genomes. Consequently, the relatively small sorghum genome is being used as a gateway into the larger, less tractable genomes of maize and sugarcane, and large-scale physical and molecular mapping projects are well underway.
We are involved in initiating cytomolecular mapping in sorghum. The sorghum cytomolecular mapping project has proven especially challenging as it has been difficult to consistently prepare quality SC spreads from sorghum (a species in which SC spreads had never been prepared prior to our work). To date, we have constructed a rudimentary karyotype of the sorghum SCs (Draye et al. 2001), performed a Cot analysis for sorghum (Peterson et al. 2002a), isolated repetitive sequences, and prepared BAC clones for FISH. We are now working on using FISH to localize single-copy sequences on sorghum SC spreads (see FIGURE 3 for example of FISH to sorghum SCs). The information gained will be useful in evaluating syntenic relationships between chromosomes of different grass species and will provide insight into the evolutionary mechanisms involved in the divergence of these species. Likewise, it will generate basic information on how genomes function and evolve within the context of chromosomes.
Click to enlarge image
FIGURE 3: Localization of the pSau3A10 centromeric sequence on two sorghum SC spreads. All ten sorghum centromeres are recognized by the probe. Sites of hybridization (pink foci) have been identified using an antibody conjugated to rhodamine. The chromosomes have been counterstained with DAPI (blue).
Visit the Image Gallery for more pictures of meiotic chromosomes and SCs.
