In the past, genes or markers, were intra- chromosomally mapped exclusively through analysis of co-segregation in a testcross (also known as a backcross) or other type of segregating progeny. Co-segregation of genes in the progeny indicated that they were tightly linked. In contrast, the frequency of meiotic recombinantion between the genes of interest in the progeny provided a measure of the genetic distance between them. In the absence of polymorphism between the parents, meiotic mapping of genes, is not possible.
In an article published in the December 2002 issue of Trends in Plant Science (vol. 7:521-23), Robbie Waugh and colleagues at Scottish Crop Research Institute, Dundee and Paul Dear at Mol. Biology Lab, Cambridge, discuss the advantages of physical mapping over conventional ones. In their introductory remarks, they describe several limitations of conventional methods. These include: (a) inability to map identical alleles or markers, (b) lack of resolution because of the near-absence of recombinants in a population when the genes are in close proximity (c) failure to determine the actual physical distance between different genes (d) problems in mapping multi-gene families. The authors point out that these difficulties make it attractive to resort to new technologies based on physical mapping.
Advantages of physical mapping: One major advantage of physical mapping is that it does not require markers to be polymorphic. Physical mapping assists (a) in the isolation of genes and (b) identifying candidate genes linked to a particular phenotype. The great advantage of the techniques, they describe over BAC- or YAC- based physical mapping, is that they help avoid distortions in the map that are caused by the occurrence of (a) gaps in libraries, (b) rearranged or chimaeric fragments, or (c) repeated regions larger than the size of the clones. Because of these advantages, the construction of clone-based physical maps on an independently STS (sequence tagged sites) scaffold has become the obvious route to ensure both long-range accuracy and map integrity.
Radiation hybrid (RH): Following the successful application of this technique to decipher a part of the human genome by building up a physical map based on 40,000 STS, RH technology is being used more and more in vertebrate and invertebrate species. The RH procedure consists of irradiating cells of a donor
species with lethal dosages, with a intent to cause chromosome breakages. Fragmented chromosomes are thereafter recovered by fusing them with a suitable recipient cell line from a different species. In other words, donor chromosome fragments are integrated into the recipient genome. If a good number of hybrid lines are generated using this procedure, there is a good chance that the entire donor genome would be contained as chromosome mosaics in the synthetic lines. Furthermore, two genes in close proximity on the same chromosomal fragment would frequently be transmitted together and maintained in the same cell lines. Scoring of co-retention frequencies aided by STS-PCR allows calculation of physical distance based on the use of appropriate statistical methods. The RH technology has thus proved to be a powerful tool in determining high-resolution physical mapping.
RH mapping in plants: RH mapping in plants became easier when a polyploid host species such as a monosomic oat-maize chromosome-9- addition line was used to rescue fragmented maize chromosomes. Being hexaploid with three sets of homeologous chromosomes, the oat genome acted as a genetic buffer to give protection to potentially lethal chromosomal mutations that occurred in gamma-irradiated maize cells. The buffering action of the hexaploid host species also allowed regeneration of fertile ‘RH’ plants. Analyses of chromosome-9-specific markers and FISH fluorescence in situ hybridization, revealed the presence of maize chromosome-9-fragments in the plants. From one to as many as ten radiation-induced breakages were observed per line.
Oscar Riera-Lizarazu and associates, who carried out the above experiment, described the technique as powerful enough to permit physical mapping to a resolution of 0.5.0 Mb, assuming (a) availability of a collection of 100 RH lines, (b) the presence of three breaks per chromosome, each being on an average 191 Mbp long and (c) the availability of ~400 evenly spaced markers. The authors reiterated that the same procedure may be applied with panels of the remaining nine oat-maize chromosome addition lines for mapping maize ESTs at high physical resolution. The authors are also of the opinion that this procedure can be applied successfully for chromosome mapping of other species that have similar aneuploid stocks (e.g. the Triticeae).
The authors describe the work of Julie Wardrop and associates. They produced a whole genome RH panel for barley using Nicotiana tabacum cv. Xanthi as the host. As in oat-maize combinations, polyploid tobacco mesophyll protoplasts were used as the host species and electro-fused with irradiated bialaphos-resistant (i.e. bar gene transgenic) barley cell suspension protoplasts, containing chromosome fragments. Hybrid cells were cultured and PCR was performed to verify their hybrid nature i.e., whether or not they contained the bar gene. About 20% of the putative hybrids were found to contain barley chromosome segments. Suitable tests revealed an overall donor DNA retention frequency of 26% (on average) with representation of all regions of the genome that were tested.
HAPPY mapping: Happy mapping is an in vitro mapping technique. It has been successfully applied to map human chromosome number 14 and the genomes of Cryptosporidium parvum and Dictyostelium discoideum. HAPPY mapping consists of the following steps: First, naked genomic DNA is fragmented at random. Iin the second step, the fragments are separated into aliquots, called the ‘mapping panel’, the quantity of DNA in each aliquot amounting to less than the amount of genomic DNA found in an individual cell. In the third step, the frequency of co-segregation of markers is measured in a manner similar to RH mapping. Pre-amplification by PCR allows the members of a HAPPY panel to be used to do thousands of marker-typings. The requirement of small quantities of DNA is an additional advantage. Constructing and screening a HAPPY mapping panel is straightforward.
In this connection, the authors cite the commendable work recently completed by Madan Thangavelu and associates. They constructed a high-resolution HAPPY map of a part of chromosome 4 of Arabidopsis thaliana.This section of chromosome 4 was characterized by an average marker spacing of 16 Kbp. Using the above procedure, they were able to estimate and calibrate the relative order of genes and the distance between two markers. A comparison of the above data to the DNA sequence showed a remarkably good correspondence. Their success with Arabidopsis chromosome No. 4 has prompted them to turn their attention to crop plants. Besides being a versatile, powerful and ‘comparative physical mapping’ tool, the HAPPY mapping technique has other advantages such as the procedure can be applied in species with large genomes. The procedure also does not require construction of large insert DNA libraries.
Future Prospects: The authors conclude that RH and HAPPY mapping technologies would bridge the resolution gap between meiotic and BAC-based STS-content mapping. Both the techniques, described in the article, will greatly contribute to constructing physical maps, eliminating the need to look for polymorphism. Because of these advantages, the authors consider the two technologies will prove extremely helpful in deciphering large or intractable crop plant genomes, whose complete base sequences have not yet been obtained.
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