Chestnut (Castanea spp.) Cytogenetics for Genomics and Breeding

The American chestnut (Castanea dentata, 2n = 2x = 24), once thriving on 80 million ha in eastern North America and known as the “King of the Forest”, was destroyed in the first half of the 20^thcentury by chestnut blight caused by Cryphonectria parasitica.  Chinese chestnut (C. mollissima) is relatively resistant to the blight, and efforts are being made to transfer this trait into American chestnut through backcross breeding and genetic engineering.  The Chinese chestnut genome has been mapped and is currently being sequenced to further facilitate these improvement approaches.  However, essentially no cytological data are available to aid backcross breeding, genome mapping, gene discovery or sequence assembly.  Fluorescence in situ hybridization (FISH), a modern cytogenetic technique, has been proven to play an important role in revealing the structural organization of a genome and locating molecular markers and/or genes to specific chromosome(s).  We have been using FISH and various genomic and genetic resources to fill this knowledge void for chestnut. The chestnut genome is relatively small (780 Mb/1C) for a forest tree and its 12 chromosomes are metacentric and sub-metacentric.  We have identified two 18S-28S rDNA (one major and the other minor) and one 5S rDNA (minor) sites in both American and Chinese chestnuts.  A second minor 5S rDNA site was also identified in Chinese chestnut.  Each of the 12 linkage groups (LG) has been assigned to each of the 12 chromosomes using genetically and physically mapped bacterial artificial chromosomes (BACs).  A full structural characterization of the chromosome corresponding to LG_B of Chinese chestnut has been developed using FISH with 20 BACs.  The same analysis is being completed for LG_E to study the possibility of a reciprocal translocation involving these chromosomes.  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.