Occurrence of the rust resistance gene Lr37 from Aegilops ventricosa in Argentine cultivars of wheat
Leaf rust of wheat (Triticum aestivum L.) caused by the fungus Puccinia triticina (formerly P. recondita f. sp. tritici), is one of the most important foliar diseases of this crop. Lr37 rust resistance gene, which confers resistance in wheat against leaf rust, was introgressed into cultivated wheat from Aegilops ventricosa Tausch. Rust races with virulence to Lr37 have been identified in different countries, but it still provides resistance to a wide range of races and is useful in combination with other resistance genes. There are no reports about the presence, frequency and origin of Lr37 in Argentinean wheat cultivars. In this work, we analyzed 88 registered Argentinean wheat cultivars developed by different breeding companies and institutions during the last 15 years by means of a molecular marker which is diagnostic of the 2NS-2AS translocation which carries Lr37. Only 4 cultivars showed the amplification product associated with this chromosome fragment. These four cultivars which carry the translocated 2NS-2AS chromosome were registered by the same breeding company during the last seven years and all of them have European germplasm in their genealogy. To the best of our knowledge this is the first report of the presence of Lr37 in registered South American cultivars.
Leaf rust of wheat (Triticum aestivum L.) caused by the fungus Puccinia triticina (formerly P. recondita f. sp. tritici), is one of the most important foliar diseases of this crop. Breeding wheat cultivars with resistance to leaf rust is the most effective, economical and environmentally friendly method of disease control and was used in numerous wheat breeding programs worldwide (Kolmer, 1996). However, a gene-for-gene interaction exists between host resistance genes and pathogen avirulence genes in the wheat-P. triticina pathosystem and virulence shifts in the pathogen populations have reduced the effectiveness of a number of leaf rust resistance genes (Johnson, 2000), which increased the search of new resistant genes, not only in the cultivated gene-pool, but also in the wild relatives of wheat (Friebe et al.1996; Valkoun, 2001).
The wild wheat species, Aegilops ventricosa Tausch (syn. Triticum ventricosum Ces), is an allotetraploid with the genome designation DvDvNvNv derived from the hybridization of the D genome from Ae. tauschii (coss.) Schmal and the N genome from T. uniaristatum (Vis.) Richter (Kimber and Zhao, 1983). Ae. ventricosa is the source of several disease resistance genes that are of agronomic importance and have been successfully introgressed into wheat. These genes include Lr37, which confers resistance in wheat against leaf rust. This gene, located in a 2NS-2AS translocation (Bariana and McIntosh, 1993), was initially introgressed in the winter bread wheat VPM1 (Maia, 1967) and was used by breeders in different parts of the world (Robert et al. 1999; Mesterházy et al. 2000; Seah et al. 2000; Park et al. 2001; Stepien et al. 2003). Rust races with virulence to Lr37 have been identified in different countries (Mesterházy et al. 2000; Winzeler et al. 2000), but it still provides resistance to a wide range of races and is useful in combination with other resistance genes (Park and McIntosh, 1994; Park et al. 2001; Kolmer et al. 2005).
Greater knowledge on the identity of rust resistance genes present in cultivars that can be used as donors of resistance in wheat breeding programs could greatly improve the efficiency of developing resistant cultivars by using these genes per se or by stacking different resistant genes in a given cultivar (Sawhney and Joshi, 1996). In this context, there are no reports about the presence, frequency and origin of Lr37 in Argentinean wheat cultivars. Resistance gene postulation is a rapid method to hypothesize which resistance genes are present in a host genotype (Loegering et al. 1971). It is based on the gene-for-gene specificity between host resistance genes and pathogen avirulence genes. Host genotypes are evaluated with a well-characterized collection of pathogen isolates with different avirulence gene combinations. However, gene postulation can be complicated by interactions between resistance genes and it is best suited for resistance genes that are clearly expressed at the seedling stage (Kolmer, 1996). Resistance genes could also be postulated by testing host genotypes with DNA-based markers linked to resistant genes. This alternative approach overcome some of the problems associated with traditional gene postulation, such as gene interactions and the plant stage of gene expression (McCartney et al. 2005). For this reason, the objective of this work was to determine the presence of Lr37 resistance gene in a sample of 88 wheat cultivars registered in Argentina during the last 15 years by means of a molecular marker which is diagnostic of it.
Eighty eight cultivars of bread wheat registered in Argentina from 1990 to 2005 were used. These materials represent more than 80% of the commercialized cultivars in Argentina during the last 15 years by eight different companies and institutions (Table 1). The cultivars Thatcher and Soisson were used as negative controls and VPM1 and Balthazar as positive controls, since both of them carry Lr37 (Park et al. 2001). Seeds of these genetics materials were supplied by G. Vrdoljak and P. Paulucci from the Wheat Breeding Program of Nidera S.A.
Leaf material was collected from at least 20 different plants of each genotype. DNA was extracted from these bulked samples using a standard SDS protocol (Dellaporta et al. 1983).
Two pairs of primers were included in each PCR reaction. The primers VENTRIUP-LN2 developed by Helguera et al. (2003) were used to detect the 2NS fragment from Ae. ventricosa, that yield a 259-bp band. The primers for the microsatellite marker GWM400 (Röder et al. 1998) were used to evaluate the quality of the DNA and the presence of PCR reaction inhibitors. These last primers amplify a fragment of about 150-bp.
PCR reaction mixes contained 80 ng of wheat genomic DNA, 0.2 µM of each primer (Table 2), 200 µM of each dNTP, 1X PCR Buffer containing 50 mM KCl, 10 mM Tris-HCl pH 9.0, 0.1% Triton X-100, 1.5 mM MgCl2; 1 unit Taq DNA Polymerase (Biotools) in a total volume of 25 µL. Cycling conditions consisted of 3 min denaturation at 95ºC follow by 30 cycles of 45 sec denaturation at 94ºC, annealing at 65º C for 30 sec, and 1 min extension at 72ºC, and finally 7 min extension at 72ºC on a Biorad thermocycler. Amplification products (10 µL/lane) were separated on a standard 2% agarose gel in 1X TBE buffer, at constant voltage power for about 2 hrs. A 25 bp ladder (Promega) was used to estimate the size of each amplified DNA fragment. After electrophoresis, the gel was stained with ethidium bromide and bands were visualized with UV light.
Robert et al. (1999) and Seah et al.(2001) developed PCR markers for the identification of the cluster of resistance genes Sr38-Yr17-Lr37, but these markers were not publicly available. Helguera et al. (2003) developed the first public PCR marker for this chromosome fragment which was used in this work. Since the long chromosomal fragment (25-38 cM) from Ae. ventricosa does not recombine with the bread wheat chromosomes, the resistance genes located in the 2NS segment are transferred together and are completely linked to markers developed within this segment (Robert et al. 1999; Helguera et al. 2003).
The 259-bp PCR product from primers VENTRIUP-LN2, which is diagnostic for the 2N chromosome fragment, was observed in 4 out of 88 cultivars tested and in the positive checks VPM1 and Balthazar (Table 1). The other cultivars and the two negative controls, on the other hand, did not amplify this diagnostic fragment. In fact, all of them presented only one PCR amplification product corresponding to different alleles of the microsatellite locus Xgwm400, used as an internal control of the PCR reaction.
The four cultivars which carry the translocated 2NS-2AS chromosome (Baguette 10, Baguette 12, Baguette 5 Sur and Baguette 11 Premium) were registered by the same breeding company (Nidera S.A.) in 1999, 2001 and 2004, and all of them have European germplasm in their genealogy. This is not surprisingly because VPM1 (the original line which carry the 2NS chromosome segment) was developed and used intensively in France and other European countries. As a matter of fact, the Lr37 resistant gene was identified, by means of gene postulation or molecular markers, in different cultivars registered in UK, the Czech Republic, France and Poland (Winzeler et al. 2000; Singh et al. 2001; Blaszczyk et al. 2004). The frequency of this gene in UK cultivars, for example, reached 26.6% by 2001 (Park et al. 2001). Cultivars registered by other companies and institutions traced their origin to Argentinean, Uruguayan, Brazilian and Mexican wheat germplasm, where the deployment of the gene Lr37 was not reported.
Research efforts to breed cereals for resistance to rust diseases have identified resistance genes expressed throughout the entire growth cycle of the plant (seedling resistance genes), and genes which confer resistance to avirulent pathotypes only in post-seedling growth stages (adult plant resistance, APR; Park and McIntosh, 1994). Lr37 is an APR gene which shows certain level of resistance in seedlings at temperatures below 20ºC (Park and McIntosh, 1994; Kolmer, 1996). At temperatures above 20ºC Lr37 is ineffective at seedling growth stages, acting as classical APR genes in becoming effective at post-seedling growth stages (Park and McIntosh, 1994). It has been reported that this gene shows a better response than the more common APR genes Lr12 or Lr13 when tested with 20 isolates belonging to 13 different races of P. triticina from Uruguay and Argentina (Germán, 2003), with 49 isolates representing the most common virulence phenotypes of this pathogen in USA (Kolmer et al. 2005) and with 15 isolates from Canada (Kolmer, 1997).
Wheat lines released by the CIMMYT program are selected for high levels of resistance to leaf rust (Braun et al. 1996; Sayre et al. 1998). Gene postulation studies of CIMMYT lines indicated the presence of Lr1, Lr3, Lr3bg, Lr10, Lr14a, Lr17, Lr19, Lr23, Lr26, Lr27 and Lr31 (Kolmer, 1996). Since this germplasm is intensively used by Argentinean breeding programs, all of these genes may be present in Argentinean cultivars. In addition, many spring wheat developed in South America possibly have Lr13 and/or Lr34 (Kolmer, 1996). Other wheat developed in South America have been valuable sources of leaf rust resistance. Dyck and co-workers (cited by Kolmer, 1996) isolated Lr3 from Sinvalocho, Lr3ka from Klein Aniversario, Lr3bg from Bage, Lr11 from El Gaucho, Lr14b from Rafaela, Lr17 from Klein Lucero and Lr30 and Lr34 from Terenzio. Antonelli (2003) reported the presence of Lr3a, Lr10, Lr23 and Lr26 in ProINTA Oasis, Lr14a in ProINTA Federal and Lr24 in Trigal 800 and derivatives.
To the best of our knowledge this is the first report of the presence of Lr37 in registered South American cultivars. Its identification in high yielding and adapted cultivars contribute to enrich an already broad genetic base for resistance to leaf rust in Argentinean wheat germplasm. Moreover, taking into account that Lr37 is linked to the genes Sr38, Yr17 and Cre5 (Bariana and McIntosh, 1993; Jahier et al. 1996; Seah et al. 2000) which confer resistance to stripe rust (Puccinia striiformis West. F. sp. tritici), stem rust (Puccinia graminis Pers. f.sp. tritici Eriks and E. Henn.) and cereal cyst nematode (Heterodera avenae Woll.) respectively, indicates that this introgressed chromosome fragment can be highly useful in developing new wheat varieties.
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