Homology-based gene/gene-analog cloning method has been extensively applied in isolation of RGAs (resistance gene analogs) in various plant species. However, serious interference of sequences on homoeologous chromosomes in @entity5700 species usually occurred when cloning RGAs in a specific chromosome. In this research, the techniques of chromosome microdissection combined with homology-based cloning were used to clone RGAs from a specific chromosome of @entity3505 -Thinopyrum alien addition line TAi-27, which was derived from @entity3505 and Thinopyrum intermedium with 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) by using the alien chromosome DNA, genomic DNA and cDNA of Th. intermedium, @entity68147 (a parent of TAi-27) as templates. A total of seven RGAs were obtained and sequenced. Of which, a constitutively expressed single-copy NBS-LRR type RGA ACR 3 was amplified from the dissected alien chromosome of TAi-27, TcDR 2 and TcDR 3 were from cDNA of Th. intermedium, AcDR 3 was from cDNA of TAi-27, @entity68147 was from cDNA of @entity68147 , AR 2 was from genomic DNA of TAi-27 and TR 2 was from genomic DNA of Th. intermedium. Sequence homology analyses showed that the above RGAs were highly homologous with known resistance genes or resistance gene analogs and belonged to NBS-LRR type of R genes. ACR 3 was recovered by PCR from genomic DNA and cDNA of Th. intermedium and TAi-27, but not from @entity68147 . Southern hybridization using the digested genomic DNA of Th. intermedium, @entity68147 as the template and ACR 3 as the probe showed that there is only one copy of ACR 3 in the genome of Th. intermedium and TAi-27, but it is absent in @entity68147 . The ACR 3 could be used as a specific probe of the R gene on the alien chromosome of TAi-27. Results of Northern hybridization suggested that ACR 3 was constitutively expressed in Th. intermedium and TAi-27, but not @entity68147 , 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.

Cloning of resistance gene analogs located on the alien chromosome in an addition line of XXXX -Thinopyrum intermedium.

@entity3505 :: ('4565', 'Species') :: ['Wheat', 'common wheat'] @entity68147 :: ('543099(Tax:4565)', 'Gene') :: ['3B-2', 'TAi-27 and 3B-2', 'FcDR 2'] @entity5700 :: ('MESH:D011123', 'Disease') :: ['polyploidy']
@entity3505 :: (4565,Species) :: ['wheat']
