Hordeum bulbosum - A New Source of Disease and Pest Resistance Genes

for Use in Barley Breeding Programmes

R. Pickering, P.A. Johnston, G.M. Timmerman-Vaughan, M.G. Cromey and E.M. Forbes
New Zealand Institute for Crop & Food Research Limited
Private Bag 4704, Christchurch, New Zealand

B.J. Steffenson, T.G. Fetch Jr. and R Effertz
North Dakota State University, Dept of Plant Pathology
PO Box 5012, Fargo, ND 58105-5012, USA

L. Zhang and B.G. Murray
School of Biological Sciences, The University of Auckland
Private Bag 92019, Auckland, New Zealand

G. Proeseler, A. Habekuß and D. Kopahnke
Bundesanstalt für Züchtungsforschung an Kulturpflanzen, Institut für Epidemiologie
und Resistenz, Postfach 1505, D-06435 Aschersleben, Germany

I. Schubert
Institut für Pflanzengenetik und Kulturpflanzenforschung
Corrensstr. 3, D-06466 Gatersleben, Germany

New Zealand's contribution to world barley production is minor (ca. 400 000 tonnes p.a.) but the crop is important for the domestic malting and animal feed industries. Factors that limit production in New Zealand are similar to those worldwide and include diseases and pests, which reduce yield and adversely affect quality. To address these problems, breeders have successfully introduced resistance genes from within the primary genepool of cultivated barley, which comprises Hordeum vulgare itself and H. vulgare ssp. spontaneum. Within the secondary genepool there is only one species (H. bulbosum), which has been used primarily as a means to obtain doubled haploids (Kasha and Kao 1970). However, despite its excellent disease and pest resistance (Zeller 1998) there have been few reports describing the transfer of resistance genes from H. bulbosum into cultivated barley (Xu and Kasha 1992; Michel et al. 1994; Pickering et al. 1995; Pickering et al. 2000). This limited success is mainly ascribed to pre- and post-fertilisation interspecific crossability barriers. These include pollen tube-stylar incompatibility, endosperm degeneration, chromosome instability, low chromosome pairing and crossing-over, hybrid infertility and certation effects (Pickering 1991; Zhang et al. 1999). By carefully selecting parental genotype and crossing environment we have solved several of these problems and developed partially fertile triploid hybrids (VBB) combining seven chromosomes from H. vulgare and 14 from H. bulbosum (Pickering 1988). VBBs are backcrossed to H. vulgare (cvs 'Emir', 'Golden Promise' and 'Morex') and after selecting amongst progeny we have identified 43 chromosome substitution lines and 37 recombinants (recombinant lines - RLs), containing one or more introgressions from H. bulbosum. The commonest substitution lines involve the substitution of H. vulgare chromosomes 7H (49%) and 6H (28%) with their H. bulbosum homoeologues. Among the RLs, those most frequently found contain introgressions of H. bulbosum chromatin on chromosomes 2HS, 2HL, 4HL, 6HS and 7HS (Table 1). Several of the RLs have improved resistance to pathogens such as powdery mildew (Pickering et al. 1995), leaf rust (Pickering et al. 2000), scald and barley mild mosaic virus as well as morphological traits transferred from H. bulbosum (Table 2). In addition to using VBB triploid hybrids, we recently selected a sterile diploid hybrid (VB) with high intergenomic chromosome pairing (Zhang et al. 1999). Its fertility was restored with colchicine treatment to double the chromosome number to 28 chromosomes, 14 from each parent. Eight out of the 159 selfed progeny contain introgressed chromatin from H. bulbosum and are partially resistant to powdery mildew and/or leaf rust, or exhibit a morphological trait from H. bulbosum (e.g. pubescent leaf sheath). The advantage of using a tetraploid hybrid is that no backcrossing is needed since selfed seeds are readily formed (36% seed setting).

Characterisation of progeny Backcross and selfed progeny are screened in the glasshouse for powdery mildew, and plant phenotype is compared with the backcross parent. Seeds harvested from each plant are field-sown in the autumn and spring to obtain information on their response to scald and leaf rust. We also perform glasshouse inoculations for these pathogens as well as for spot blotch and net blotch. Screening for diseases and pests not present in New Zealand are also carried out at North Dakota State University, (USA) and Institut für Epidemiologie und Resistenz (Aschersleben, Germany) as well as by breeders in many parts of the world. Any interesting lines are analysed in more detail cytologically and with molecular methods (e.g. RFLP and AFLP), and in situ hybridisation (Pickering et al. 1995, 1997, 2000).

To improve the efficiency of our preliminary screen, we have developed the use of a rye repetitive sequence (pSc119), which hybridises strongly to H. bulbosum but not to H. vulgare (Gupta et al. 1989). A subclone (pSc119.1; McIntyre et al. 1990) used with fluorescence in situ hybridisation, revealed weak and dispersed signals across the whole H. bulbosum genome, but little or no hybridisation to the chromosomes of H. vulgare (Xu et al. 1990; Pickering and Johnston, unpublished). In further experiments, we used pSc119.1 to probe Southern blots and found that fragments diagnostic for H. bulbosum DNA were present even in small H. vulgare - H. bulbosum introgressions. PCR primers were designed from the rye pSc119.1 sequence (McIntyre et al. 1990) and were tested against the H. vulgare and H. bulbosum parents, resulting in the amplification of different length fragments for H. vulgare (675bp) and H. bulbosum (720bp). After optimising the experimental conditions, we detected semi-quantitatively, the presence of varying amounts of H. bulbosum chromatin using DNA from H. vulgare - H. bulbosum whole chromosome substitution lines down to RLs with very small distal and interstitial introgressions. This PCR-based assay will enable us to identify RLs faster, cheaper and more efficiently than our previous procedures. AFLPs have been used as a complementary tool to identify RLs also at an early stage in the screening process. We are developing H. bulbosum chromosome-specific markers by excising amplified fragments from AFLP gels that are present in H. bulbosum and RLs or substitution lines but absent from the H. vulgare parent. After cloning, the fragments are sequenced, and primers developed for PCR analysis with a tester set of H. vulgare - H. bulbosum RLs to assess their specificity. So far, two markers specific for the H. bulbosum homoeologue of H. vulgare chromosome 6HS have been developed.

In conclusion, we have developed triploid and tetraploid hybrids between H. vulgare and H. bulbosum that have proved valuable for introgressing desirable genes into cultivated barley. We have also improved our analytical methods for detecting introgressions at an early stage in the screening process. In the future we will look more closely at H. bulbosum to determine whether it is a source of other useful traits that will improve the performance and quality of H. vulgare.

Acknowledgements:We thank the Foundation for Research, Science and Technology, New Zealand and the North American Barley Genome Mapping Project for financial support.

Table 1. Frequencies of introgressions and homoeologous chromosome substitutions

among progeny derived from H. vulgare x H. bulbosum crosses

Table 2. Chromosomal location of traits transferred from Hordeum bulbosum into H. vulgare. Locations in parenthesis are tentative

References:

Gupta, P.K., G. Fedak, S.J. Molnar and R. Wheatcroft. 1989. Distribution of a Secale cereale DNA repeat sequence among 25 Hordeum species. Genome 32:383-388.

Kasha, K.J. and K.N. Kao. 1970. High frequency haploid production in barley (Hordeum vulgare L.). Nature 225:874-876.

McIntyre, C.L., S. Pereira, L.B. Moran and R. Appels. 1990. New Secale cereale (rye) DNA derivatives for the detection of rye chromosome segments in wheat. Genome 33:635-640.

Michel, M., G. Proeseler, M. Scholz, R. Pickering and G. Melz. 1994. Transfer von H. bulbosum - Genen in die Kulturgerste. Vortr. Pflanzenzüchtung 28:187-189.

Pickering, R.A. 1988. The production of fertile triploid hybrids between Hordeum vulgare L. (2n = 2x = 14) and H. bulbosum L. (2n = 4x = 28). Barley Genetics Newsletter 18:25-29.

Pickering, R.A. 1991. Comparison of crossover frequencies in barley (Hordeum vulgare L.) and H. vulgare x H. bulbosum hybrids using a paracentric inversion. Genome 34:666-673.

Pickering, R.A., A.M. Hill and R.G. Kynast. 1997. Characterization by RFLP analysis and genomic in situ hybridization of a recombinant and a monosomic substitution plant derived from Hordeum vulgare L. x H. bulbosum L. crosses. Genome 40:195-200.

Pickering, R.A., A.M. Hill, M. Michel and G.M. Timmerman-Vaughan. 1995. The transfer of a powdery mildew resistance gene from Hordeum bulbosum L. to barley (H. vulgare L.) chromosome 2 (2I). Theor. Appl. Genet. 91:1288-1292.

Pickering, R.A., S. Malyshev, G. Künzel, P.A. Johnston, V. Korzun, M. Menke and I. Schubert. 2000. Locating introgressions of Hordeum bulbosum chromatin within the H. vulgare genome. Theor. Appl. Genet. 100:27-31.

Xu, J. and K.J. Kasha. 1992. Transfer of a dominant gene for powdery mildew resistance and DNA from Hordeum bulbosum into cultivated barley (H. vulgare). Theor. Appl. Genet. 84:771-777.

Xu, J., J.D. Procunier and K.J. Kasha. 1990. Species-specific in situ hybridization of Hordeum bulbosum chromosomes. Genome 33:628-634.

Zeller, F.J. 1998. Nutzung des genetischen Potentials der Hordeum-Wildarten zur Verbesserung der Kulturgerste (Hordeum vulgare L.). Angewandte Botanik 72:162-167.

Zhang, L., R. Pickering and B.G.Murray. 1999. Direct measurement of recombination frequency in interspecific hybrids between Hordeum vulgare and H. bulbosum using in situ hybridization. Heredity 83:304-309.