Identifying leaf rust resistance gene Lr 19 in durum wheat using simple sequence repeat ( SSR ) marker

Leaf rust, caused by Puccinia triticina Eriks., is an important disease affecting durum wheat (Triticum turgidum ssp. durum) worldwide, particularly in the Mediterranean region. The disease can be controlled through the use of plant host resistance. Based on seedling resistance tests of 103 durum genotypes against a bulk of P. triticina, urediniospores were previously collected from Syria and Lebanon during 2007/08 and 2008/09 growing seasons. Percentage of resistance in the durum set tested was up to 52%. The resistant genotypes might have one of the following resistance gene(s): Lr15, Lr 19, Lr 24, Lr 25, Lr 27 +31, Lr 28 and Lr 29. Results revealed that the Gb/130-bp polymorphic band was linked to Lr19 and Sr25. In this study, twelve genotypes carrying resistance to Lr19 and Sr25 have been identified (Azeghar2, Rutucha1, Ammar9/Azeghar2, Ammar9/Terbol97-4, T.polonicum9/Ch1//Icamor-TA04-68/3/Icamor-TA04-69//(Lahn/ Ch1)2519, Arislahn5//Icamor-TA0463/Icasyr1, T.dicoccum1/Ch1//Ammar8/3/Bonadur/Icamor-TA04-63, Mrb3/ T. urartu500651/4/IcamorTA04-63/3/Bcr/Gro1// Mgnl1, SwAlg/Gd1-81//Ch1-48, Icamor-TA04-1//Mgnl3/Ainzen1, 319-ADDO/5/D68-193A-1A//Ruff/Fg/3/Mtl5/4/Lahn, and Mrf1/Stj2/3/1718/BT24//Karim). Promising results on Gb/130-bp and genotypes carrying Lr19 and Sr25 will be used in a marker assisted selection of the durum breeding programs in the Mediterranean region.


INTRODUCTION
The leaf rust fungus, Puccinia triticina Eriks., is considered to be one of the most important pathogens in wheat production worldwide (Dehne and Oerke, 1998) where it causes significant yield losses every year.The disease can reduce yield by 1% for every 1% increase in the infection level (Khan et al., 1997).It also reduces the number of kernels per spike and kernel quality (Bremenkamp-Barrett et al., 2008).The level of damage inflicted by leaf rust varies with the degree of infection and host plant resistance.Under severe infection, *Corresponding author.E-mail: M.Nachit@CGIAR.ORG.Tel: (963-21) 26912402.Fax: (963-21) 2213490 Abbreviations: STS, Sequence-tagged-site; RFLP, restriction fragment length polymorphism; AFLP, amplified fragment length polymorphism; RH, relative humidity; SDS, sodiumdodecyl-sulfate; EtOH, ethanol.
susceptible wheat varieties can suffer yield losses (up to 40%) (Marasas et al., 2004).Fungicides can be used to control leaf rust to some extent.Using resistant cultivars is an economic and environmental friendly way of minimizing losses caused by the disease.Fifty-six leaf rust resistance genes have been designated and 51 of them have been mapped to corresponding chromosomes (Yang and Liu, 2004).Lr19 is one of the few widely effective genes conferring resistance to leaf rust in wheat.Lr19 still provides effective resistance against all leaf rust races in Syria, Lebanon and Turkey (Kassem et al., 2010).Ayala-Navarrete et al. (2007) had developed sequence-tagged-site (STS) markers for Lr19 and Sr25 from wheat ESTs and mapped them to chromosome 7DL (Qi et al., 2004), which was also found to be in the translocated segment of wild relative Thinopyrum ponticum in durum wheat (Gennaro et al., 2009).Leaf rust resistance gene Lr19 has been developed with biochemical markers (Winzeler et al., 1995), restriction fragment length poly-morphism (RFLP) (Autrique et al., 1995) and amplified fragment length polymorphism (AFLP), and were converted to STS (Prins et al., 2001).The aim of the present work was to identify a DNA marker for the leaf rust resistance gene lr19 in resistant durum wheat genotypes.

Sources of pathogen isolates
Wheat leaves samples infected by leaf rust (P.triticina Eriks.) were collected from the International Center for Agricultural Research in the Dry Areas (ICARDA) summer nursery at Terbol research station, Lebanon in 2007; and from farmers' wheat fields in Syria during the growing season 2007/08 and 2008/09.Urediniospores from each collection were used to inoculate 7-day-old seedlings of the susceptible variety (Morocco).After increasing all isolates, a bulk of urediniospores from each isolate was diluted with talcum powder.The mixture was sprayed on 7-day-old seedlings leaves of 103 durum wheat genotypes from ICARDA according to Xing et al. (2006).The inoculated seedlings were placed in plastic-covered cages as described by Wang et al. (2010).Following the inoculation, plants were transferred to mist chambers and incubated for 16 h in darkness at 18°C and approximately 100% relative humidity (RH).After misting period, plants were allowed to dry slowly for 4 h before being placed in a growth chamber at 18 to 20°C with a 16-h photoperiod according to Kolmer et al. (2007).
Scoring of leaf rust symptoms was performed 12 to 14 days after inoculation (Kolmer et al., 2009).Infection types (ITs) of P. triticina were quantified using a standard 0 to 4 scale, where 0 = no symptoms, 1 = small uredinia surrounded by necrosis, 2 = small uredinia surrounded by chlorosis, 3 = moderate size uredinia without chlorosis and 4 = large uredinia without chlorosis as described by Stakman et al. (1962) and Long and Kolmer (1989).Designations of + and -were used with the 0 to 4 scale to indicate larger and smaller than normal uredinia, respectively (Mebrate et al., 2008).Category 0 and 1 were considered as resistant, that of 2 and 3 were moderately resistant and moderately susceptible, respectively and that of 4 was considered highly susceptible (Chu et al., 2009).

DNA extraction
The method used to extract DNA is a sodium-dodecyl-sulfate (SDS) as described by Nachit et al. (2001) and Elouafi and Nachit (2004).Young leaves were collected at seedling stage (10 to 14 days old) of 55 resistant durum genotypes from ICARDA and the differential line TC*7/Tr (RL6040) carrying Lr19 resistance used as control, were quick frozen in liquid nitrogen and grinded.The powdered leaves were transferred to tubes and 20 to 25 ml of heated extraction buffer (65°C) (500 mM NaCl; 100 mM Tris-HCl pH 8.0; 50 mM EDTA; 0.84 SDS sodium bisulfate) at pH 8.0 was added to each tube.This mixture was incubated for 30 min at 65°C, with shaking after every 5 min.20 ml of chloroform-isomyl alcohol (24:1) was added and the whole content was shaken vigorously to produce an emulsion.After centrifugation for 15 min at 2,800 rpm, the supernatant was recuperated, and 2 volumes of cold 95% ethanol (EtOH) (-20°C) was added.The DNA was precipitated and kept at -20°C for 30 to 60 min or for overnight.The precipitated DNA was afterward recuperated, and washed twice with cold ethanol 70% and dissolved in 500 µl of TE (10 mM Tris pH 8; 1 mM EDTA pH 8).The DNA was then treated by 2 µg/ml stock solution of RNAse for 30 min at 37°C and stored at -20°C until used.The concentration of DNA extract was determined by spectrophotometer (Amersham Biosciences-Gene Quant pro).Kassem et al. 8717

Electrophoresis
The resulting mixtures were denatured and loaded on a 5% denaturing polyacrylamide gel.After electrophoresis at 65 V constant voltages, the gels were silver-stained according to Nachit et al. (2001) and Elouafi and Nachit (2004).

RESULTS AND DISCUSSION
Results revealed a number of genotypes with resistance against the mixture of urediniospores (bulk) and the physiological races of P. triticina identified in Syria and Lebanon during 2007, 2008 and 2009 growing seasons.The bulk was highly virulent, with 81% of the available resistance gene(s) in the differential set being susceptible, including the resistance gene Lr9 which was broken in 2007/2008 season (Kassem et al., 2010).Whereas, only seven resistant gene(s) were found to still be effective against all races in Syria and Lebanon (Lr15, Lr19, Lr24, Lr25, Lr27+31, Lr28 and Lr29) at seedling and adult plant stages.Percentage of resistance in the ICARDA 103 durum set was up to 52% against the Lr-bulk collected in Syria and Lebanon.As for percentages of moderate resistance, moderate-susceptible and susceptible genotypes, they were 17.47, 14.56 and 15.53%, respectively.Table 1 shows the genotypes resistant against the bulk of P. triticina.Analyzing the pedigree of these genotypes to identify the source of resistance revealed the following: Mtl5, Icamor-TA04, Icasyr1, Atlast1, Ruff and Bcr with probably major resistance genes, as the resistance was shown at seedlings and adult stage.These genotypes might have one of the resistance gene(s): Lr15, Lr 19, Lr 24, Lr 25, Lr 27 +31, Lr 28 and Lr 29.It is clear that some of these genotypes have more than one source of resis=tance referred to earlier, such as genotypes Bcr/Lks4// Mrf1/Stj2/3/Icasyr1, Atlast1/961081//Icasyr1 and Gsbl1/4/ D68-1-93A-1A//Ruff/Fg/3/Mtl5, indicating accumulation of more than one resistance genes in one genotype.The program at ICARDA uses pyramiding of resistance genes (Nachit, pers. comm.).Therefore, these results show the importance of using these crossing models.
Both Lr19 and Sr25 genes were previously mapped to chromosome 7DL and positioned within confidence intervals delineated by molecular markers (Prins et al., 2001).In durum wheat, they are probably located on the 7A or 7B (Zhang et al., 2005).Further, the Lr19 is closely linked to the yellow pigment which was found to be located on chromosme 7B (Elouafi et al., 2001).
Results showed that the Gb/130-bp polymorphic band was present in both Lr19 and Sr25 differential lines, and marker is tightly linked to gene Lr19 and Sr25.Resistant genotypes have both genes Lr19 and Sr25 at Gb/130-bp, but this band in the same size was absent in another resistant genotypes which did not have Lr19 and Sr25 (Figure 1).
Using the above-mentioned 12 genotypes as resistant sources in a breeding program is an economic and effecttive way to minimize losses caused by leaf rust.Identification of molecular markers closely linked to resistance genes can facilitate the accumulation of other minor/ major genes into a single genotype.