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

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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 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 gene analogs and belonged to NBS-LRR type of R genes. ACR3 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 ACR3 as the probe showed that there is only one copy of ACR3 in the genome of Th. 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.

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Acknowledgements

This research was supported by National Natural Science Foundation of China (30270708), Chinese Academy of Sciences (No.KZCX1-SW-19) and Ministry of Science and Technology of China (No. Z2002-B-004, JY03-B-23).

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Authors and Affiliations

  1. Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Datun Road, Beijing, 100101, China

    Shu-Mei Jiang, Jun Hu, Wei-Bo Yin, Yu-Hong Chen & Zan-Min Hu

  2. USDA-ARS, FRRL, Utah State University, Logan, UT, 84322-6300, USA

    Richard R. -C. Wang

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  1. Shu-Mei Jiang
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  2. Jun Hu
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  4. Yu-Hong Chen
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Correspondence to Zan-Min Hu.

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Communicated by D. A. Hoisington

Shu-Mei Jiang and Jun Hu contributed equally to this work

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Jiang, SM., Hu, J., Yin, WB. et al. Cloning of resistance gene analogs located on the alien chromosome in an addition line of wheat-Thinopyrum intermedium . Theor Appl Genet 111, 923–931 (2005). https://doi.org/10.1007/s00122-005-0022-3

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  • DOI: https://doi.org/10.1007/s00122-005-0022-3

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