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Isolation of TIR and nonTIR NBS–LRR resistance gene analogues and identification of molecular markers linked to a powdery mildew resistance locus in chestnut rose (Rosa roxburghii Tratt)

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Abstract

Toll and interleukin-1 receptor (TIR) and nonTIR nucleotide binding site–leucine rich repeat (NBS–LRR) resistance gene analogues (RGAs) were obtained from chestnut rose (Rosa roxburghii Tratt) by two PCR-based amplification strategies (direct amplification and overlap extension amplification) with degenerate primers designed to the conserved P-loop, kinase-2, and Gly-Leu-Pro-Leu (GLPL) motifs within the NBS domain of plant resistance gene (R gene) products. Thirty-four of 65 cloned PCR fragments contained a continuous open reading frame (ORF) and their predicted protein products showed homology to the NBS–LRR class R proteins in the GenBank database. These 34 predicted protein sequences exhibited a wide range (19.5–99.4%) of sequence identity among them and were classified into two distinct groups by phylogenetic analysis. The first group consisted of 23 sequences and seemed to belong to the nonTIR NBS–LRR RGAs, since they contained group specific motifs (RNBS-A-nonTIR motif) that are often present in the coiled-coil domain of the nonTIR NBS–LRR class R genes. The second group comprised 11 sequences that contained motifs found in the TIR domain of TIR NBS–LRR class R genes. Restriction fragment length polymorphic (RFLP) markers were developed from some of the RGAs and used for mapping powdery mildew resistance genes in chestnut rose. Three markers, RGA22C, RGA4A, and RGA7B, were identified to be linked to a resistance gene locus, designated CRPM1 for chestnut rose powdery mildew resistance 1, which accounted for 72% of the variation in powdery mildew resistance phenotype in an F1 segregating population. To our knowledge, this is the first report on isolation, phylogenetic analysis and potential utilization as genetic markers of RGAs in chestnut rose.

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Acknowledgements

This project was supported by the National Natural Science Foundation of China (NSFC) (Nos.30260070 30123001 and 30471201), the 863 Project of China, and Natural Science Foundation of Guizhou Province. The authors are grateful to Dr. S. Xiao from Center for Biosystems Research of University of Maryland Biotechnology institute, Dr. Z. Deng from Gulf Coast Research and Education Center of University of Florida, and Dr. W.W. Guo from our laboratory for critical review and helpful advice, and to Prof. K. Watanabe from Plant Genetic Diversity Gene Research Center of Tsukuba University (Japan) for his reading the manuscript. Our appreciations also extend to Mr. Y.Z. Liu, J.K. Song, X.D. Cai and S.H. Zeng in our laboratory for their assistance with the experiments.

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  1. National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, P. R. China

    Qiang Xu & Xiuxin Deng

  2. Guizhou Key Laboratory of Agricultural Bioengineering, Guizhou Univeristy, Guiyang, 550025, P. R. China

    Xiaopeng Wen

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Correspondence to Xiuxin Deng.

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Communicated by H. Nybom

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Xu, Q., Wen, X. & Deng, X. Isolation of TIR and nonTIR NBS–LRR resistance gene analogues and identification of molecular markers linked to a powdery mildew resistance locus in chestnut rose (Rosa roxburghii Tratt). Theor Appl Genet 111, 819–830 (2005). https://doi.org/10.1007/s00122-005-0002-7

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