Submitted to: Theoretical and Applied Genetics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/16/2005
Publication Date: 7/26/2005
Citation: Jiang, S.M., Hu, J., Yin, W.B., Chen, Y.H., Wang, R., Hu, Z. 2005. Cloning of resistance gene analogs located on the alien chromosome in an addition line of wheat-thinopyrum intermedium. Theoretical and Applied Genetics 111:923-931 Interpretive Summary: Homology based gene/gene-analog cloning method has been extensively applied in isolation of resistance gene analogs (RGAs) in various plant species. However serious interference of homology sequences in complicated plant genome usually occurred when RGAs to be cloned are located in a specific chromosome. To overcome this problem we successfully cloned seven RGAs that belong to NBS-LRR type of resistance genes by combining chromosome microdissection technique with homology-based cloning. We demonstrated that one of the RGAs, ACR3, is a single-copy gene that expressed higher in leaves than in roots. This is a new way to clone RGAs located on a known chromosome.
Technical Abstract: The techniques of chromosome microdissection combined with homology-based cloning were used to clone RGAs from a specific chromosome of Wheat-Thinopyrum alien addition line TAI-27, which has a pair of chromosomes from Th. intermedium. The alien chromosomes carry genes for resistance to BYDV. The alien chromosome in TAI-27 was isolated by a glass needle and digested with proteinase K. The DNA of the alien chromosome was amplified by two rounds of Sau3A linker adaptor-mediated PCR. RGAs were amplified by PCR with the degenerated primers designed based on conserved domains of published resistance genes (R genes) using the alien chromosome DNA, genomic DNA and cDNA of Thinopyrum intermedium, TAI-27 and 3B-2 (a parent of TAI-27) as templates. A total of seven RGAs were obtained and sequenced. Of which, ACR3 was amplified from the dissected alien chromosome of TAI-27, TcDR2 and TcDR3 were from cDNA of Th. intermedium, AcDR3 was from cDNA of TAI-27, FcDR2 was from cDNA of 3B-2, AR2 was from genomic DNA of TAI-27 and TR2 was from genomic DNA of Th. intermedium. Sequence homology analyses showed that the above RGAs were highly homologous with known resistance genes or resistance genes analogs and belonged to NBS-LRR type of R genes. ACR3 was recovered by PCR from genomic DNA and cDNA of Thinopyrum intermedium and TAI-27, but not from 3B-2. Southern hybridization using the digested genomic DNA of Thinopyrum intermedium, TAI-27 and 3B-2 as the template and ACR3 as the probe showed that there is only one copy of ACR3 in the genome of Thinopyrum intermedium and TAI-27, but it is absent in 3B-2. The ACR3 could be used as a specific probe of the R gene on the alien chromosome of TAI-27. Results of Northern hybridization suggested that ACR3 was constitutively expressed in Th. intermedium and TAI-27, but not 3B-2, and expressed higher in leaves than in roots. This research demonstrated a new way to clone RGAs located on a specific chromosome. The information reported here should be useful to understand the resistance mechanism of, and to clone resistant genes from, the alien chromosome in TAI-27 and Thinopyrum intermedium.