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

Abstract

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 polyploidy 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 Wheat-Thinopyrum alien addition line TAi-27, which was derived from common wheat 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, TAi-27 and 3B-2 (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, FcDR 2 was from cDNA of 3B-2, 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 3B-2. Southern hybridization using the digested genomic DNA of Th. intermedium, TAi-27 and 3B-2 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 3B-2. 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 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.

Isolation, characterization and evolution of NBS-LRR encoding disease-resistance gene analogs in eggplant against bacterial wilt

“…This unequal genetic variation within resistance genes is because of unequal crossingover, recombination, gene conversion and mutations (Michelmore and Meyers 1998;Zhuang et al 2012). According to Tarr and Alexander (2009); early land plants had TIR-NBS-LRR sequences but these have been diminished significantly in monocots, and, the only reported TIR-NBS-LRR in monocots is from Triticum thinopyrum line (Jiang et al 2005). However, further analysis of eggplant NBS analogs is required to gain a better knowledge and confirm this result to draw certain conclusions.…”

Isolation of expressed sequences from a specific chromosome ofThinopyrum intermediuminfected by BYDV

“…Research on molecular markers and special probes of alien addition lines has been carried out since the wheat-Thinopyrum alien addition lines were created (Zhang et al 1991Jiang et al 2004Jiang et al , 2005. Jiang et al (2004) obtained nine ESTs from a subtractive library of TAi-27 infested by S. graminum carrying GAV strain of BYDV of which seven were located on the alien chromosome.…”

“…The chromosome microdissection and DNA amplification of the alien chromosome in TAi-27 were performed according to the procedures described by Jiang et al (2004Jiang et al ( , 2005. In brief, the alien chromosome in TAi-27 was identified by its smallest size ( Fig.…”

“…Therefore, it appears that the BYDV resistance bearing chromosome in TAi-27 is the same group 2 chromosome as in addition line Z1 (Larkin et al 1995;Han et al 2003). We previously obtained several resistancerelated ESTs from the alien chromosome of TAi-27 by combining the techniques of chromosome microdissection, homology-based cloning, and subtractive library screening (Jiang et al 2004(Jiang et al , 2005.…”

The Development of Chromosome Microdissection and Microcloning Technique and its Applications in Genomic Research

“…However, work has been limited to chromosomes that are easy to identify, such as the satellite chromosomes [11,33,[80][81][82], the largest or the smallest chromosome, and B chromosome [27]. Chromosomes in cytogenetic stocks that can be easily distinguished *Address correspondence to this author at the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Chaoyang District, Beijing 100101, P. R. China; Tel: 86-10-64854561; Fax: 86-10-64854561; E-mail: zmhu@genetics.ac.cn were used for chromosome microdissection, such as telochromosomes [43,44], addition lines [37,65,22,34,35], and reconstructed translocation chromosomes.…”