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Map-based Cloning Approach – A Powerful Tool for Citrus Genetic Improvement






MBC, also called positional cloning, is a very powerful approach used to isolate genes without prior knowledge of the gene product or even the mechanism of function, as long as the gene is associated with a seg- regating natural or mutant phenotype (Jander et al., 2002). MBC consists of four basic steps; Fig. 13.1 shows the schematic diagram of the entire process. The fi rst step is to develop a local genetic map where at least two closely linked or co-segregating

 

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First-pass mapping (about 10 cM resolution)

 

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Initial physical mapping

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Fine-scale mapping (about 200 kb resolution)

 

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Local fine physical mapping

 

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Sequencing

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Candidate gene prediction

 

 

Transformation and complementary test

 

Fig. 13.1. A schematic diagram of the map-based cloning process.


 


molecular markers fl anking the gene have been identifi ed. The genetically closer to the gene of interest the markers are, the less diffi cult the subsequent screening will be. A larger mapping population is used after the initial marker screening of a smaller population so that the recombination between those markers and the gene is evaluated more precisely. The second step is to screen a large insert genomic library such as BAC (bacterial artifi cial chromo- some) with the markers and conduct chro- mosome walking to construct a physical contig covering the fl anked region. This step also includes the essential task of fi nd- ing additional co-segregating (no recombi- nation) markers to narrow the gene further into a short physical region, facilitating the selection of the BAC clones to sequence from the contig. The third step is to sequence the selected large insert clones and to predict the candidate gene(s). This step relies on the application of various programmes for gene prediction. The last step generally is to perform genetic comple- mentation tests through transformation of the candidate gene(s) into a selection with- out the known phenotype to confi rm the gene function. If the transgenic selection expresses the expected phenotype such as a disease resistance, the gene of interest should be enclosed within that transformed fragment. Once the gene is successfully iso- lated, an in-depth molecular and biochemi- cal analysis can follow.

The tremendous advantage of the MBC process is the ability to tag and clone any gene associated with a distinct and scorable phenotype. Such resources, either natural or mutant, appear nearly unlimited. One of the most interesting applications of MBC is the isolation of resistance genes to the dis- eases and pests that cause tremendous pro- duction losses in food species, as well as resulting in environmental pollution due to use of pesticides for control. The process is likely to become more routine as the so- called post-genome era of most model organisms is coming. Already for some organisms there exist powerful and publi- cally available resources, including high


density genetic and physical maps, and even full genome sequences, which greatly speeds up the whole process of MBC. For example, in Arabidopsis, it took a total effort of 3–5 person-years to isolate a gene in 1995, while now less than 1 person-year is required (Jander et al., 2002). For citrus, an intensive effort in genomic tool develop- ment and MBC will enable greater under- standing of citrus genetics and cloning of important genes. Application of the improved tools for genetic improvement is essential to keep the world’s citrus indus- tries functioning as profi table enterprises into the future.

 

 


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