GrainGenes

ITEMS FROM SPAIN

UNIVERSIDAD POLITECNICA DE MADRID
Departamento de Biotecnologia, E.T.S. Ing. Agronomos.- C. Universitaria, 28040, Madrid, Spain.

A. Delibes, I. López-Braña, M. J. Montes, and C. González-Belinchón.

CONSEJO SUPERIOR DE INVESTIGACIONES CIENTIFICAS
Serrano, 115, 28006, Madrid, Spain.

D. Romero and M.F. Andres.

UNIVERSITY OF LLEIDA

Institut de Recerca i Tecnologia Agroalimentaries (UdL-IRTA).
Rovira Roure, 191-25198 Lleida, Spain.

J.A. Martín-Sánchez, G. Briceño-Félix, E. Sin, C. Martínez, A. Michelena, and L. Torres.

Characterization of resistance to cereal cyst nematode (Heterodera avenae) in Triticum aestivum/Aegilops introgression lines. [p. 123-125]

The gene Cre2 that confers resistance to the cereal cyst nematode H. avenae has been transferred from Ae. ventricosa to the T. aestivum subsp. aestivum introgression line H-93-8. We combined different strategies to characterize peroxidase expression in different tissues and times of lines, H-93-8 and H-10-15 (susceptible parent) in presence or absence of the nematode. Northern analysis using peroxidase-specific probes showed that root tissue taken from line H-93-8, at the nematode feeding site, seven days after infection, contained significantly more peroxidase transcripts than any other tissue sample. We cloned and sequenced RT-PCR products using primers based on conserved sequences among wheat peroxidases. Some of the resulting transcripts have homology with pox2, a gene preferentially expressed in roots in response to different stresses. Some of these transcripts could be specific of the nematode feeding site in the root. All these rapid changes in PER activity might be associated to defence mechanisms. A similar study is being carried out with line TR-3531, which carries the Cre7 gene transferred from Ae. triuncialis. The Cre2 and Cre7 genes have been introgressed into commercial wheat cultivars with high yield and/or good quality. To assist the selection of resistance conferred by Cre genes we are currently searching markers based on LRR sequences, which are a common motif on resistance genes in plants. In parallel, we are investigating relationships between mobile elements and resistance genes in wheat.

Material and methods. The resistant line H-93-8, carrying the Cre2 gene, was obtained from the cross 'T. turgidum cv. Rubroatrum, H-1-1/Ae. ventricosa AP-1//T. aestivum cv. Almatense H-10-15' (Delibes et al. 1993). The resistant line TR-3531, carrying the Cre7 gene, was derived from the same bridge and recipient Triticum species, but using Ae. triuncialis as donor species (Romero et al. 1998). Seedlings of H-93-8, cultivated in the laboratory under controlled conditions, were inoculated with the Spanish pathotype Ha71 of H. avenae (100 individuals J2/plant). Susceptible parent H-10-15 and uninfected H- 93-8 were used as controls. Root sections and leaves taken four, seven and fifteen days after infection were excised and used for analysis of mRNA. Peroxidase expression was analyzed by Northern using a peroxidase specific probe (Båga et al. 1995). Obtaining of cDNAs and cloning of RT-PCR peroxidase products were carried out as described by suppliers of reverse transcriptase (Amersham Pharmacia Biotech) and of pGEM-T Easy cloning vector (Promega), respectively. Sequencing was performed on Applied Biosystem ABIPRISM 3100 sequencer and the sequences analysis was done using BLAST and CLUSTALW. Markers for Cre2 were searched using a PCR approach that combine in a single reaction a primer annealing regions typically conserved in plant resistance genes (Yu et al. 1996), and a primer complementary to conserved motifs within mobile elements naturally present in monocots and characterized by a high copy number and preferential insertion to gene-rich regions (Moreno-Vázquez et al. 2005; Sabot et al. 2004). The search was based on a F2:3 population generated from the cross 'H-93-8 x H-10-15'.

Resistance genes Cre2 and/or Cre7 were incorporated into the genetic background of commercial cultivars with suitable agronomic traits by backcrossing. Commercial wheat cultivars, Anza, Rinconada, Cartaya, Betres, Recital, Alcotán, and Osona, were used as recurrent parents, whereas H-93-8 and TR-3531 were the donor lines for Cre2 and Cre7, respectively. Isoelectrofocusing patterns from advanced lines were obtained as described in Andrés et al. 2001.

Results and conclusions. The introgression line H-93-8 showed increased mRNA peroxidase levels on roots at the nematode feeding site in response to H. avenae infection, reaching a maximum 7 days postinoculation. Consistent results were obtained analyzing peroxidase activity by spectrophotometry and IEF (Andres et al. 2001; Delibes et al. 2004). No significant rise in the peroxidase levels was observed in leaves in any case. 3'mRNA sequences for leaf and root peroxidases from both infected and uninfected H-93-8 plants were obtained by RT-PCR. Cluster analysis separated in two clear-cut groups leaf and root sequences. The group containing root sequences exhibited higher variability and some of them shown less homology to wheat peroxidases from the Genebank than the group containing leaf sequences. We are currently investigating if these root peroxidases could have been introgressed from Aegilops ventricosa in H -93-8 and if some of them could be nematode-induced. The results obtained with TR-3531 line are still preliminary.

A PCR marker for Cre2 has been found. For the generation of this marker a primer annealing the LTR region of a typical monocot retro-element and a primer annealing the NBS region of published NBS-LRR disease-resistance genes in monocots, were combined in the same PCR. We are currently evaluating the performance of this marker in different segregating populations and breeding lines.

Advanced bread wheat lines carrying Cre2 and/or Cre7 resistance genes, evaluated under field conditions, showed tolerance as well as a lower number of cysts than their susceptible controls. Evaluation of grain yield over 2 years of field testing across four locations in Spain, showed a good performance for our advanced lines compared to commercial wheat varieties (Table 1). Peroxidase patterns of these advanced lines obtained by IEF, revealed an early response in infected roots, indicative of the presence of Cre2 (in ID-2150) and Cre7 (in ID-2181 and T-2003).

Financial support. This work was supported by grant AGL2004-06791-CO4 from the Comisión Interministerial de Ciencia y Tecnología of Spain.

References.

• Andrés MF, Melillo MT, Delibes A, Romero MD, and Bleve-Zacheo T. 2001. Changes in wheat root enzymes correlated with resistance to cereal cyst nematodes. New Phytol 152:343-354.
• Båga M, Chibbar RN, and Kartha KK. 1995. Molecular clonig and expression analysis of peroxidase genes from wheat. Plant Mol Biol 29:647-662.
• Delibes A, Romero D, Aguaded S, Duce A, Mena M, López-Braña I, Andrés MF, Martín-Sánchez JA, and García-Olmedo F. 1993. Resistance to the cereal cyst nematode (Heterodera avenae Woll.) transferred from the wild grass Aegilops ventricosa to hexaploid wheat by a "stepping stone" procedure. Theor Appl Genet 87:402-408.
• Delibes A, López-Braña I, Moreno-Vázquez S, Gonzalez-Belinchón CM, Romero MD, Andres MF, Martín-Sánchez JA, Sin E, Martínez C, and Michelena A. 2004. Changes in peroxidase gene expression in response to Heterodera avenae infection in a wheat/Aegilops ventricosa introgression line carrying the resistance gene Cre2. Ann Wheat Newslet 50:166-168.
• Moreno-Vázquez S, Ning J, and Meyers BC. 2005. hATpin, a family of MITE-like hAT mobile elements conserved in diverse plant species that forms highly stable secondary structures. Plant Mol Biol 58:869-886.
• Romero MD, Montes MJ, Sin E, Lopez-Braña I, Duce A, Martín-Sánchez JA, Andrés MF, and Delibes A. 1998. A cereal cyst nematode (Heterodera avenae Woll.) resistance gene transferred from Aegilops triuncialis to hexaploid wheat. Theor Appl Genet 96:1135-1140 .
• Sabot F, Simon D, and Bernard, M. 2004. Plant transposable elements, with an emphasis on grass species. Euphytica 139:227-247.
• Yu YG, Buss GR, and Saghai Maroof M. 1996. Isolation of a superfamily of candidate disease-resistance genes in soybean based on a conserved nucleotide-binding site. Proc Natl Acad Sci USA 93:11751-11756.

Publications. [p. 125]

• Delibes A, López-Braña I, Montes MJ, González-Belinchón CM, Martín-Sánchez JA, Sin E, Martinez C, Michelena A, del Moral J, Perez-Rojas F, Espinal FJ, and Senero M. 2003. Studies in relation to the Hessian fly resistance gene (H30) transferred from the wild grass Aegilops triuncialis to hexaploid wheat. Ann Wheat Newslet 49:142-144.
• Delibes A, López-Braña I, Moreno-Vázquez S, and González-Belinchón CM. 2005. Selección y caracterización molecular y agronómica de trigos hexaploides portadores de genes de resistencia a Heterodera avenae y/o Mayetiola destructor transferidos desde Aegilops. PHYTOMA-España 169:72-75 (In Spanish).
• Delibes A, López-Braña I, Moreno-Vázquez S, Gonzalez-Belinchón CM, Romero MD, Andres MF, Martín-Sánchez JA, Briceño G, Sin E, Martínez C, Michelena A, Del Moral J, Pérez Rojas F, and Senero M. 2005. Resistance of bread wheat advanced lines to nematodes and Hessian fly. Progress update. Ann Wheat Newslet 51:161-163.
• Martín-Sánchez JA, Gómez-Colmenarejo M, Del Moral J, Sin E, Montes MJ, González-Belinchón C, López-Braña I, and Delibes A. 2003. A new Hessian fly resistance gene (H30) transferred from the wild grass Aegilops triuncialis to hexaploid wheat. Theor Appl Genet 106:1248-1255.
• Martín-Sánchez JA, Montes MJ, López-Braña I, Romero MD, Sin E, Martínez C, Andrés MF, Gómez-Colmenarejo M, González-Belinchón CM, and and Delibes A. 2003. Differential induction of defence-enzymes and chromosomal location in wheat/Aegilops ventricosa introgression lines of Cre2 and Cre5 Heterodera avenae resistance genes. A progress report. In: Breeding strategies for Small Grains Cereals in the Third Millennium. EUCARPIA". Editors C. Marè, P. Faccioli and A.M. Stanca. Pp. 307-309
• Martín-Sánchez JA., Sin E., Delibes A., López-Braña I., Romero MD., Andres MF., Del Moral J., Torres L., Briceño-Felix G. (2005). Advanced bread wheat lines with Hessian fly and Cereal Cyst Nematode resistance genes transferred from Ae. ventricosa and Ae. triuncialis. 7th International Wheat Conference, Mar del Plata, Argentina. Abstracts pp. 146.
• Montes MJ, Martín-Sánchez JA, González-Belinchón CM, López-Braña I, Delibes A, Sin E, and Martinez C. 2003. Characterisation by isozyme analysis of a wheat/Aegilops triuncialis introgression line carrying resistance to cereal cyst nematode and Hessian fly. In: Biotic and Abiotic Stresses (Pogna N et al. Eds). Editorial, SIMMI, Roma, Italy. Pp. 1211-1213.
• Montes MJ, López-Braña I, Romero MD, Sin E, Andrés MF, Martín-Sánchez JA, and Delibes A. 2003. Biochemical and genetic studies of two Heterodera avenae resistance genes transferred from Aegilops ventricosa to wheat. Theor Appl Genet 107:611-618.
• Montes MJ, López-Braña I, and Delibes A. 2004. Root enzyme activities associated with resistance to Heterodera avenae conferred by gene Cre7 in a wheat/ Aegilops triuncialis introgression line. J Plant Physiol 161:493-495.
• Moreno-Vázquez S, Ning J, and Meyers BC. 2005. hATpin, a family of MITE-like hAT mobile elements conserved in diverse plant species that forms highly stable secondary structures. Plant Mol Biol 58:869-886.
• Moreno S, López-Braña I, González-Belinchón CM, Simonetti E, Delibes A, Romero MD, Andrés MF, and Martín-Sánchez JA. 2005. Peroxidase induction in resistant hexaploid wheat in response to cereal cyst nematode (Heterodera avenae) infection. In: Plant genomics and Environment (abiotic and biotic). Plant Genomics European Meeting, Amsterdam, the Netherlands. Abstract P 8-030, p. 212.