Cytological Analysis of Reciprocal Translocation Hypothesis in Chestnut Using BAC-FISH

The American chestnut (Castanea dentata, 2n = 2x = 24), once known as the king of the eastern US hardwood forests, has been completely decimated by chestnut blight caused by an introduced fungal pathogen (Cryphonectria parasitica).  Scientists at the American Chestnut Foundation (TACF) and other research organizations have been working since early 1980s to transfer blight resistance genes into American chestnut from Chinese chestnut (C. mollissima) through backcross breeding and genetic engineering.  The Chinese chestnut has been mapped and is currently being sequenced to further facilitate these improvement approaches.  It is generally accepted that plant genomes contain high amounts of repetitive DNA that hinder accurate construction of genetic and physical maps and the proper ordering of sequence scaffolds during genome sequencing.  Specific DNA sequences (gene and/or molecular markers) can be physically localized onto chromosomes using fluorescence in situ hybridization (FISH) and thereby facilitating the correct ordering of maps and sequence scaffolds.   A full structural characterization of the linkage group B (LG_B) chromosome of Chinese chestnut has been developed using FISH with 20 BAC clones.  The same analysis is being completed for LG_E to study the possibility of a reciprocal translocation involving these chromosomes.  BAC clones with distinct FISH signals, 2 to 4 from each arm of these two chromosomes, are being localized on F₁ pachytene chromosomes to confirm whether these two chromosomes are involved in a reciprocal translocation as previously hypothesized.  Furthermore, the centromeric positions of all 12 chromosomes are being determined and cyto-molecular maps are being developed for each chromosome using 16 to 20 BACs from each LG.  The accurate positioning of the centromeres and the resulting cyto-molecular maps of the chestnut chromosomes should help improve the understanding of the chestnut genome allowing for powerful comparative studies that will inform gene discovery and mapping, interspecies breeding and genetic engineering.