MOLECULAR MARKERS IDENTIFICATION OF LEAF RUST RESISTANT GENES LR19, LR21, LR24, LR47 AND LR51 IN SELECTED EGYPTIAN WHEAT CULTIVARS

Leaf rust, caused by Puccinia triticina is a common and widespread disease of bread wheat (Triticum aestivum L.), in Egypt. Host resistance is the most economical, effective and ecologically sustainable method for controlling the disease. Molecular markers help to determine leaf rust resistance genes (Lr genes) that may be present in a large group of wheat germplasm. The objective of this study was to evaluate and detect leaf rust resistance genes in Egyptian wheat cultivars. Ten out of fifteen cultivars were resistance to leaf rust disease in four locations i.e., Dakahlia, Kafr el-Sheikh, Beheira and Sharqia during seasons 2011/2012 and 2012/2013. As for, using specific SSR primers proved that Lr19 was present in five cultivars i.e., Sakha-95, Gemmeiza-9, Gemmeiza-10, Misr-1 and Misr-2. Lr21. Lr24, Lr47, and Lr51 were detected in all tested cultivars. These genes should be taken into consideration in wheat breeding programs for successful rust resistance. Furthermore these materials can be used as a parent for plant breeders to add new effective resistance genes to their breeding materials because of the dynamic change of leaf rust races which can breakdown the resistance.


INTRODUCTION
Wheat leaf rust is one of the most important diseases resulting in high yield losses and reduced grain quality (Cloutieret et al., 2007). Resulting in the use of resistant cultivars offers the most effective and ecologically sustainable method of control of the disease; therefore, incorporating genetic resistance to this pathogen into adapted germplasm is a major goal in most wheat breeding programs. Plant disease resistance can be classified into two categories: qualitative resistance, conferred by a single resistance gene (also termed as major, seedling, or race specific resistance) and quantitative resistance, mediated by multiple genes or quantitative trait loci (QTLs) (also termed as adult plant, race non-specific or slow rusting resistance) with each providing a partial increase in resistance (Kou and Wang, 2010). More than sixty genes for leaf rust resistance (Lr), most of them major, seedling or race specific genes, have been catalogued to date in wheat (McIntosh et al., 2008 andSamsampour et al., 2010).
However, the gene-for-gene interaction between host resistance genes and pathogen virulence genes combined by virulence shifts in pathogen populations have reduced the effectiveness of a significant number of major leaf rust resistance genes (Johnson, 2000;Bulos et al., 2006). Replacement of highly variable land races by higher yielding, pure-line varieties in many parts of the world has further reduced the wheat gene pool and favored virulence shifts events in pathogen populations. In this context, a better knowledge on the identity of effective Lr genes present in adapted 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 or by stacking different resistant genes in a given cultivar, a process also known as gene pyramiding (Messmer et al., 2000); thereby helping to avoid the release of cultivars that are genetically uniform (Mebrate et al., 2008).
Before gene pyramiding is practiced, it is advisable to identify effective and genetically different sources of resistance.
Alternatively to gene postulation, presence of Lr genes can be determined by testing host cultivars with molecular markers linked to resistance genes. This approach overcomes some of the problems associated with traditional gene postulation, such as gene interactions and plant stage of gene expression. Recently there have been advances in the mapping and development of molecular markers of several leaf rust resistance genes (Helguera et al., 2000;Prins et al., 2001;Helguera et al., 2003, Helguera et al., 2005Gupta et al., 2006;Lagudah et al., 2006;Bansal et al., 2008;Mebrate et al., 2008;Kuraparthy et al., 2009;Sun et al., 2009;Samsampour et al., 2010). Once these genetic factors are mapped, they can be controlled by molecular markers and the corresponding genotypes of individuals can be assessed easily. As a consequence, the identification of cultivars carrying favourable alleles at these loci will provide valuable genetic material for the development of new improved varieties. The objective of this study was to identify leaf rust resistance in ten bread Egyptian wheat cultivars using molecular markers.

Molecular Markers
Laboratory studies: This part of the investigation was carried out at the molecular biology laboratory, Faculty of Agriculture Research Park (FARP), Faculty of Agriculture, Cairo University.

DISCUSSION
Leaf rust of wheat was the cause of eliminating many cultivars i.e., Giza 139, Super X, Mexipak 69 and Chenab 70 because of their susceptibility under field conditions. Moreover, some wheat genotypes were discarded very shortly after their release such as Giza 139. The failure of such cultivars was mainly due to the dynamic nature, in population, of the causal organism, which produces new virulence having the ability to breakdown their resistance. Thus, we evaluated 15 Egyptian wheat commercial cultivars under field condition in four locations: Dakahlia, Kafr el-Sheikh, Beheira and Sharqia during two seasons 2011/12 and 2012/13 for leaf rust resistance. We found ten out of fiteen cultivars: Sakha-94, Sakha-95, Gemmeiza-7, Gemmeiza-9, Gemmeiza-10, Gemmeiza-11, Sids-12, Sids-13, Giza-168, Misr-1 and Misr-2 showed high level of resistance against leaf rust in four locations during the two seasons. These results supported by Nazim et al. (1990) and Boulot (2007) showed that final rust severity (%) and area under disease progress curve (AUDPC) of wheat varieties Giza 168, Sakha 94, Gemmeiza 9 and Gemmeiza 10 were low compared to susceptible varieties. These results are agree with Liatukas 2003;Tariq et al., 2003;Masar et al., 2004;Martinez et al., 2005 andHanzalova andBartos 2006;Hanzalová 2010;Hanzalová et al., 2010and Hanzalová et al., 2012. Elyasi-Gomari (2010 showed that no leaf rust damage occurred on Lr9, Lr25, Lr28 and Lr29 in the field, and lines with Lr19, Lr16, Lr18, Lr35, Lr36, Lr37 and the combination Lr27 + Lr31 showed less than 15% severity. In this context, gene pyramiding of effective Lr genes is probably the faster strategy to develop leaf rust resistant wheat cultivars. Gene pyramiding can be greatly facilitated with associated markers through marker assisted selection programs (MAS), this is particularly true in the field of wheat breeding for leaf rust resistance where PCR-based markers are already available for almost half of the 80 or more designated resistance genes and alleles (Samsampour et al., 2010and McIntosh et al., 2012. Many authors conclude there is a greater predictive ability of molecular markers than pedigree data (Błaszczyk et al., 2008 andStępień et al., 2003). Our results clearly indicate the advantage of molecular markers for evaluating the presence of Lr genes in wheat cultivars compared to pedigree data and are in accordance with numerous studies and reviews (Stępień et al., 2003;Ordon et al. 2004). We selected 10 out of 15 cultivars for molecular markers identification and explained their resistance to leaf rust resistance. The Results obtained proved that resistance in the tested cultivars was due to the presence of resistance genes i.e., Lr19, Lr21, Lr24, Lr47, and Lr51. On a global scale, Lr19 is probably the most widely distributed gene for resistance to P. triticina (McIntosh et al., 1995 andWinzeler et al., 2000). Therefore, it is still considered important gene because it is present in several bred cultivars in CIMMYT in combination with other adult plant resistance genes which continue to give excellent leaf rust protection . In Egypt, this gene is important gene for resistance and detected in five cultivars, Sakha-95, Gemmeiza-9, Gemmeiza-10, Misr-1 and Misr-2 (present study). We advise especially the planting cultivars Misr-1 and Misr-2 because they carry many resistant genes (Lr19, Lr21, Lr24, Lr47, and Lr51) for leaf rust and resistant genes Sr2 and Sr25 for stem rust (Singh et al., 2011). Therefore, they considered very resistance to leaf and stem rusts (especially Ug99 race) and they can be grown in different environmental conditions. In addition the results showed that the genes Lr21, Lr24, Lr47 and Lr51 were identified by molecular 224 bp markers in the ten tested cultivars. Thus, the good resistance of these cultivars is due to the complementary effect between these major genes which enhance the response of a variety and give its higher levels of resistance. Finally, in the future studies we recommend the genes pyramiding as a method to achieve more durable resistance against pathogens with low genetic diversity, high gene flow and asexual mating systems (McDonald & Linde, 2002;Hysing et al., 2006). The combination of several effective resistance genes into a single cultivar should extend the period of resistance and this is called horizontal resistance.