| Powdery mildew and scab are serious wheat diseases occurring worldwide. The translocation line 6VS/6AL of Triticum aestivum-Haynaldia villosa, developed by Cytogenetics Institute of Nanjing Agricultural University, has been proved to be highly resistant to powdery mildew. A resistant gene locus for powdery mildew, Pm21, has been identified in the chromosome arm 6VS in this translocation line. Efforts in studying and cloning of resistance(R) genes of wheat are of great importance both in control of wheat diseases and understanding of the molecular mechanism of disease resistance in wheat. The objects of this study are cloning resistant genes by~ the R gene homology strategy, based on the finding that plant resistant genes contain conserved sequences such as nucleotide binding site (NBS), leucine-rich repeats (LRR), and serine/threonine kinase domain. Three degenerate primers were designed based on NI3S, LRR or serine/threonine kinase domain and used to perform PCR with genomic DNA or cDNA from 6VS/6AL or genomic DNA from Haynaldiu villosa. Amplified products were cloned in pGEM-T vector and then sequenced. Eighteen clones characterized with NBS, three with LRR and one with serine/threonine kinase domain were obtained by sequencing analysis. These clones were classified to 8 groups according to their nucleotide sequence identities (90% or higher). These resistance gene analogs (RGAs) all have open reading frames (ORFs), and show high homology to Yr]O in wheat, Mlal and M1a6 in barley, RPS2 in Arabiadopsis. (f 2, Cf.-4, Cf 5 and Cf-9 in tomato and other cloned R genes with conserved motifs. They were preliminarily mapped to homeologous groups 1, 2 or 5 of the chromosomes of common wheat by nulli- tetrasomic analysis. TAIL-PCR was performed to amplify sequences flanking a 296-bp LRR fragment obtained from the 6VS/6AL line, and a total of 800-bp sequence containing this LRR was determined. A pair of specific primers was designed from the sequence and was used to amplify the genomic DNAs of Yangmai 5 (susceptible check), 6VS/6AL line, substitution line of 6V and amphidiploid of Triticum durum-Haynaldia 3 villosa. The results showed that a 730-bp PCR product was amplified in all lines with Pm21 but Yangrnai5. The same results were observed with other 6VS/6AL translocation lines and other susceptible cultivars. The LRR sequence specific primers were used to screen a genomic TAC library of 6VSI6AL consisting of 2x I 0~ clones. The library was stored in twenty-two 96-well plates, each well containing approximately 1000 TAC colonies. By using a pooled PCR screening procedure, a positive TAC clone was obtained. The clone plasmid DNA was digested with various 6-base restriction enzymes, then transferred to membrane, and conducted southern hybridization with the LRR fragment as a probe. A 4-kb Sea I fragment was identified and subcloned. Part of the sequence (2731 bp) was determined by specific primer walking. The sequence encodes a predicted protein with 753 amino acids, designated as TaLRR1. The nucleotide sequence was compared with the GeneBank database with software Blast2.0. No high scored homologous sequence was found, indicating TaLRRI is a novel gene. When the amino acid sequence of TaLRRI was compared with the GeneBank database with software Blast2.0, the result show that it has strong alignments with the amino acid sequence of Cf 2, Cf-4, Qf-5 and Zf 9 in tomato and othe...
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