Robert Brueggeman1, Arnis Druka1, David Kudrna1 and Andris Kleinhofs1,2
1 Depts. of Crop and Soil Sciences, Washington State University, Pullman, WA, 99164-6420
2 Depts. of Molecular Biosciences, Washington State University, Pullman, WA, 99164-6420
In recent years disease resistance genes (R-genes) have been cloned and characterized from a variety of plants including both mono- and dicotyledon species. This growing list of R-genes has revealed that a majority belongs to the NBS-LRR class of genes which are characterized by a nucleotide-binding site (NBS) near the N-terminus and a leucine-rich repeat (LRR) region near the C-terminus (Fig 1). An increasing number of NBS-LRR encoding sequences are also being identified through PCR amplification with degenerate primers, EST sequencing, BAC end sequencing and genome sequencing projects. The Arabidopsis Genome Initiative reported that the Arabidopsis genome is estimated to contain approximately 200 genes encoding NBS-LRR related motifs. This represents close to 1% of all Arabidopsis genes (The Arabidopsis Genome Initiative, 2000). In rice it has been predicted that NBS-LRR encoding sequences may be even more prevalent than in Arabidopsis (Meyers et al., 1999). The wide distribution of these sequences in the plant kingdom and their prevalence in the Arabidopsis and rice genomes indicates that they are ancient, diverse and common in plants. Their abundance in plant genomes and the belief that NBS-LRR encoding genes confer pathogen recognition triggering defense mechanisms suggests that they play a significant role in plant disease resistance. Identifying these resistance gene analog (RGA) sequences in barley and mapping them could prove to be important in the identification of actual R-genes as well as areas of the genome were R-genes may reside. These cloned RGAs will be a valuable resource as markers for molecular breeding programs as well as excellent probes for the identification of RGAs or R-genes in other cereal crops.
Overall sequence homology among R-genes of the NBS-LRR class is low, but the NBS contains several sequence motifs, P-loop, kinase2, kinase3 and GLPLAL, that are highly conserved among R-genes even in distantly related plants (Hammond-Kosack and Jones, 1997). Synthetic oligonucleotides designed from these highly conserved motifs have been used to amplify and clone RGAs from plant genomic DNA. However, in large genome species this procedure also generates large numbers of DNA fragments with no apparent homology to R-genes. In order to improve barley RGA cloning efficiency, we performed three methods: 1) Hybridization of NBS derived oligonucleotides to arrayed BAC and cDNA libraries, 2) PCR amplification of RGAs from BAC DNA pools, and 3) PCR amplification of RGAs from cDNA libraries. All methods yielded confirmed RGA clones, but experiments involving hybridization yielded many artifacts or non-RGA genes containing NBS-like motifs. Hybridization to either the BAC or cDNA libraries recovered RGA containing clones with only a 3% efficiency. Experiments involving PCR amplification from BAC DNA pools were more successful giving 44% efficiency, but the best results were obtained by PCR amplification from cDNA libraries which gave 52% efficiency. All methods were more efficient than PCR from genomic DNA, which had an efficiency of about 1%. All confirmed RGAs were genetically mapped and BAC clones isolated. Subcloning weakly hybridizing bands identified in BAC clones discovered additional new RGAs.
Two previously undescribed RGAs, ABG331 and RSB001, were identified by labeling NBS derived oligonucleotides and utilizing them as hybridization probes on the BAC library (Yu et al., 2000). BACs identified were further screened by PCR and the resulting fragments of the expected size were cloned and sequenced. From this hybridization procedure 68 clones have been sequenced and two were RGAs, giving a RGA cloning efficiency of about 3%. RSB001 identified a gene family that mapped to loci on chromosomes 1,2,4,6 and 7(Fig 2) and possibly other yet undetermined loci. ABG331 mapped to eight loci on chromosome 1 and one locus on chromosome 7(Fig 2). One RGA was identified by NBS oligo hybridization to an arrayed cDNA library. Thirty-three cDNAs identified were sequenced and one was a RGA, giving an efficiency of 3%. This RGA, ABC1024, had been previously identified by PCR amplification using a cDNA library as template.
Five new RGAs, ABG1001, ABG1006, ABG1019, ABG1014 and ABG1032, were identified by PCR amplification with the NBS derived oligonucleotides using BAC clone pools as template. From this amplification procedure 68 clones of the expected size have been sequenced and 28 were RGAs, giving an efficiency of about 44%. ABG1014 mapped to loci on chromosomes 5 and 7(Fig 2). ABG1001, ABG1006, ABG1019 and ABG1032 each mapped to a single locus (Fig 2).
Six new RGAs, ABC1022, ABC1023, ABC1024, ABC1025, ABC1027, ABC1029, and ABC1035 were identified by PCR amplification from cDNA libraries. From this procedure 61 clones were sequenced and 32 were RGAs, giving an efficiency of 52%. ABC1023 mapped to two loci on chromosome 1 (Fig 2). ABC1029 mapped to two loci on chromosome 6 and one locus on chromosome 7(Fig 2). ABC1022, ABC1024, ABC1027 and ABC1035 each mapped to a single locus (Fig 2). ABC 1025 is the cDNA equivalent of ABG1014. BAC clones corresponding to the mapped loci were identified and the RGA-related fragments subcloned and analyzed by sequence comparison with known NBS-LRR genes. These methods allowed us to identify new RGA clones, for example ABG1034, which had not been mapped to date. These RGAs could have utility in the isolation of new RGAs and perhaps actual disease resistance genes.
References
The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 408, 796-815.
B.C. Meyers, A.W. Dickerman, R.W. Michelmore, S. Sivaramakrishnan, B.W. Sobral and N.D. Young (1999) Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide binding superfamily. The Plant Journal, 20, 317-332.
K.E. Hammond-Kosack and J.D.G. Jones (1997) Plant disease resistance genes. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48, 575-607.
Y. Yu, J.P. Tomkins, R. Waugh, D.A. Frisch, D. Kudrna, A. Kleinhofs, R.S Brueggeman, G.J. Muehlbauer, R.P. Wise, R.A. Wing (2000) A bacterial artificial chromosome library for barley (Hordeum vulgare L.) and identification of clones containing putative resistance genes. Theor. Appl. Genet., 101, 1093-1099.