N. Rostoks1, J.M. Zale2, J. Soule2, R. Brueggeman1, A. Druka1,
D. Kudrna1, and A. Kleinhofs1
1 Department of Crop and Soil Sciences, Washington State University,
Pullman, WA 99164-6420, USA
2 Present address: USDA Wheat Genetics, Quality, Physiology, and Disease Research Unit, 209 Johnson Hall, Washington State University, Pullman, WA 99164-6420, USA
Reported plant disease resistance genes encode proteins, which have conserved motifs in nucleotide binding site (NBS). Conservation extends across different species, therefore resistance genes from one species can be used to isolate homologous regions from another employing DNA sequences encoding conserved protein motifs as probes. Despite similarity in sequence, function of such homologues is often unknown, hence they are usually referred to as resistance gene analogs (RGAs).
Here we report isolation and characterization of barley RGA family homologous to the maize rust resistance gene Rp1-D. Family members were isolated using the maize pic20 probe (Collins et al., 1998; received from Dr. A. Pryor), which is 98% homologous to maize Rp1-D gene NBS region. Pic20 probe was of particular interest to us, because it was reported to co-segregate with a barley stem rust resistance gene Rpg1 (Ayliffe et al., 2000). Our results indicate that, although structurally similar to plant disease resistance genes, barley pic20 homologues co-segregate neither with Rpg1, nor with any other known disease resistance gene in barley.
Mapping and subcloning of barley pic20 homologues
Pic20 probe hybridized to 10 cv. Morex genomic DNA HindIII bands designated A to I and was used to screen a 6.3x barley cv. Morex BAC library. 60 BAC clones were selected and Southern analysis of their HindIII restriction digests with pic20 probe confirmed that all bands are present. Representatives of all bands were subcloned and mapped on Steptoe x. Morex RFLP Bin map (http://barleygenomics.wsu.edu/databases/databases.html). G band represents two different HindIII fragments of the same size. H and I bands represent a contiguous DNA fragment cleaved by a HindIII restriction site. Map locations of barley pic20 homologues are listed in Table 1.
A recombinant genetic map of Pic20A, B, C, D and F bands and Rpg1 region, as well as corresponding BAC contig is shown in Figure 1. Genetic distances are based on recombinant mapping of 3000 gametes. Pic20A locus, which is the closest to Rpg1 and has been reported to co-segregate with it (Ayliffe et al., 2000; pic20c1 locus from M1-13 clone), is 0.27 cM away from Rpg1 gene in our experiments.
Barley BAC clone contig encompassing Pic20A, B, C, D and F bands spans approximately 700 kb and extends ca. 200 kb from Pic20A towards Rpg1.
Sequence analysis of barley pic20 homologues
Representative of every band was sequenced in the pic20 homology region. All but one (C band) represent contiguous open reading frame in the pic20 homology part, although some of them have non-sense mutations and retrotransposon insertions upstream or downstream. DNA and deduced amino acid sequences of barley pic20 homologues were aligned with pic20 sequence. High degree of homology was observed among all sequences ranging from 50 - 87% identity at DNA level and from 60 - 84% identity at amino acid level.
cDNA library screening
We screened several arrayed barley EST libraries representing 200,000 clones (Close et al., 2001)) with maize pic20 probe and several barley pic20 homologues. Positive clones were not identified suggesting low expression levels of barley pic20 homologues. However, 2 different cDNAs were PCR-amplified from the bulk cDNA libraries. Both were identical to previously identified barley pic20 genomic clones. cDNA corresponding to A band was amplified from cv. Morex embryo cDNA library, whereas cDNA corresponding to C band was amplified from cv. Morex seedling green leaf cDNA library. The presence of cDNA corresponding to C band is somewhat surprising, since C band has a non-sense mutation in NBS region. It suggests a rather recent mutation event, since no other mutations have accumulated after removal of selective pressure.
Barley pic20 homologues represent a family of genes and pseudogenes homologous to NBS-LRR plant disease resistance genes, but not co-segregating genetically with any known barley resistance gene. Although presently their function remains unknown, structure conservation suggests their involvement in barley resistance responses and probably in evolution of disease resistance genes.
Table 1. Location of barley pic20 homologues on Steptoe x Morex RFLP Bin map.
|Chromosome||Bin||Flanking Bin Markers|
|Pic20A||1(7H)||1||ABG704 - ABG320|
|Pic20B||1(7H)||1||ABG704 - ABG320|
|Pic20C||1(7H)||1||ABG704 - ABG320|
|Pic20D||1(7H)||1||ABG704 - ABG320|
|Pic20E||3(3H)||8||ABG377 - ABG453|
|Pic20F||1(7H)||1||ABG704 - ABG320|
|Pic20G1||7(5H)||14||ABG390 - ABG463|
|Pic20G2||6(6H)||5||ABG387B - Ldh1|
|Pic20H,I||5(1H)||13||BCD1930 - ABC261|
Figure 1. Genetic map of barley Rpg1 and Pic20A, B, C, D and F region (top) and BAC clone contig of Pic20A, B, C, D and F region (bottom). Genetic distances are based on recombinant mapping in 3000 gamete population.
Ayliffe, M.A., Collins, N.C., Ellis, J.G. and Pryor, A. (2000). The maize rp1 rust resistance gene identifies homologues in barley that have been subjected to diversifying selection. Theor Appl Genet 100, 1144-1154.
Collins, N.C., Webb, C.A., Seah, S., Ellis, J.G., Hulbert, S.H. and Pryor, A. (1998). The isolation and mapping of disease resistance gene analogs in maize. MPMI 11, 968-978.
Close, T.J., Wing, R., Kleinhofs, A. and Wise, R.P. (2001). Genetically and physically anchored EST resources for barley genomics. Plant and Animal Genome IX Conference W28, San Diego, USA.