DEVELOPMENT OF SSR & STS MOLECULAR MARKERS ASSOCIATED WITH STEM RUST RESISTANCE IN BREAD WHEAT ( Triticum aestivum L . )

Wheat is an edible grain, one of the oldest and most important cereal crops in Egypt. The annual consumption of wheat grains in Egypt is about 12.4 million tons, while the annual local production is about 9.38 million tons/ 3.4 million faddan in 2014/2015 (Agric. Economics and Statistics Department, Ministry of Agriculture, Egypt, 2015). The required yield increase may be achieved by developing highyielding cultivars a long with implementing improved cultural practices. The new improved cultivars must be resistant to serious diseases such as wheat rusts, tolerant to abiotic stresses namely; drought, salinity and heat, and should be genetically stable in a broad spectrum of environments (having wide adaptability). Therefore, the efforts of wheat breeders and geneticists must continue to increase the productivity per unit area to narrow the gap between supply and consumption in Egypt.

Wheat is an edible grain, one of the oldest and most important cereal crops in Egypt.The annual consumption of wheat grains in Egypt is about 12.4 million tons, while the annual local production is about 9.38 million tons/ 3.4 million faddan in 2014/2015 (Agric.Economics and Statistics Department, Ministry of Agriculture, Egypt, 2015).The required yield increase may be achieved by developing highyielding cultivars a long with implementing improved cultural practices.The new improved cultivars must be resistant to serious diseases such as wheat rusts, tolerant to abiotic stresses namely; drought, salinity and heat, and should be genetically stable in a broad spectrum of environments (having wide adaptability).Therefore, the efforts of wheat breeders and geneticists must continue to increase the productivity per unit area to narrow the gap between supply and consumption in Egypt.
Stripe, leaf and stem rusts caused by Puccinia striiformis, Puccinia triticinea and Puccinia graminis, respectively, are globally important wheat fungal diseases that cause significant grain yield losses.Use of resistant wheat cultivars is the most economic and environmentally safe approach to reduce crop losses from rust diseases.However, understanding the genetic behavior of wheat resistance to these diseases is essential for deciding the breeding strategies that maximize the genetic improvement of these traits (Shehab El-Din et al., 1991).
Wheat resistance to rusts has been assumed to be a relatively simple inherited trait (Biffen, 1905) governed by one, two or few number of major genes (Dyck, 1991;Bai et al., 1997).Meanwhile, several investigators indicated that resistance is a quantitative character controlled by many genes as well as the prevailing environmental conditions (Shehab El-Din et al., 1991;Yadav et al., 1998;Nawar et al., 2010).Furthermore, resistance was dominant over susceptibility in most cases W (Shehab El-Din and Abd El-Latif, 1996;Bai et al., 1997;Patil et al., 2000) while others claimed an opposite concept (Singh et al., 1998;Ganeva et al., 2001).On the other hand, some reports fit a simple additive genetic model with no dominance or epistatic interactions, while dominance and/or epistasis were more pronounced and had important roles (Shehab El-Din and Abd El-Latif, 1996;Singh et al., 1998;Nawar et al., 2010).
Molecular markers are useful tools to study genetic variations, since the genetic variability among wheat varieties is narrow as in all self-pollinated crops (Röder et al., 2002).The applications of molecular markers in plant breeding programs facilitate the improvement of many crop species (Williams et al., 1990).It offers the simplest and fastest method for detecting a great number of genomic markers in a short period of time (Edwards et al., 1992).Michelmore et al. (1991) developed the F 2 plants population to the highest and the lowest extremes for the development of markers needed for marker-assisted selection.Marker-assisted selection was successfully practiced in several crop plants such as rice (Naqvi et al., 1995), wheat (Penner et al., 1996), durum wheat (Wang et al., 1995), rapeseed (Jourdren et al., 1996) and maize (Abdel-Tawab et al., 1998).
The objectives of this study were to screen the response of twelve bread wheat genotypes under infection condition with respect to their performances to select the most resistant and the most susceptible varieties or lines, test stem rust on the contrasting parents and their F 1 and F 2 plants by recording the rust reaction and some related traits to stem rust and detect some molecular genetic markers associated with stem rust using SSR & STS markers.

Materials
This study was carried out in the research farm and the laboratory of the Wheat Research Department, Field Crops Research Institute, Agricultural Research Center, Giza, Egypt.Department of Genetics, Faculty of Agriculture, Ain Shams university and laboratories of INRA, Rabat, Morocco, during the period from 2010 to 2015.Three bread wheat genotypes (Triticum aestivum L.) namely; Misr1 (resistant to stem rust), Line 37 (susceptible to stem rust) and Line 92 (susceptible to stem rust) were chosen from a preliminary screening trial for stem rust resistance which comprised twelve bread wheat genotypes according to their resistance to stem rust.The grains of 12 wheat genotypes were kindly obtained from the Wheat Research Department, Field Crops Research Institute, Agricultural Research Center, Giza, Egypt as listed in Table (1).

Methods
The pedigrees and origins of the three selected genotypes (Misr1, Line 37 and Line 92) are shown in Table (2), they were grown in the field and crossed (Misr1 x line 37, Misr1 x Line 92 and line 37 x line 92) to obtain the F 1 grains for these three crosses.Some of the F 1 grains for each cross were sown in the field and selfed to obtain the F 2 grains.

Evaluation of the parent, (F 1 ) plants and (F 2 ) individual plants for each hybrid under normal and infected conditions in the field
Parents, F 1 plants for the three crosses were grown at Gemmeiza Research Station in three replications in a randomized complete block design experiment and 200 F 2 individual plants for each cross were cultivated in the optimum planting date (normal conditions) and in the late planting date (infection conditions).
Data were recorded for all plants at the end of the experiment for the following yield-related traits related to stem rust; days to heading, days to maturity, plant height (cm), number of spikes/plant, spike length (cm), number of spikelets/spike, grain yield/plant (g) and rust reaction.

Statistical analysis
The collected data from the three crosses (parents and F 1 plants were statistically analyzed using analysis of variance (ANOVA) procedure according to Snedecor and Cochran (1969).
The F 2 plants, represented by 200 plants for each cross were classified ac-cording to their behavior under infection conditions.According to their performances (rust reaction), eight resistant F 2 individual plants and eight susceptible F 2 individual plants for Misr1 X Line 37 hybrid, seven resistant F 2 individual plants and five susceptible F 2 individual Plants for Misr1 X Line 92 hybrid and seven resistant F 2 individual plants and eight susceptible F 2 individual plants for Line 37 X Line 92 hybrid were chosen for further molecular analysis with their parents and F 1 plants.

Genomic DNA extraction
DNeasy TM Plant Mini Kit (Qiagen Inc.,cat. No. 69104) was used for DNA isolation as described by the manufacturer manual from plant samples, i.e., the three parents, their F 1 plants and the most resistant F 2 individual plants as well as the most susceptible F 2 individual plants for each cross (Dellaporta et al., 1983).

SSR & STS markers by PCR-based analysis
PCR reactions were performed according to Williams et al. (1990) using six SSR & STS specific primers (Operon Technology, USA) as shown in Table (3).

Analysis of gel images
All fragments resulting from polyacrylamide and agarose gels were detected on an UV-transilluminator filter.All gels were photographed under UV light with Polaroid film 667 and scanned with Bio-Rad video densitometer Model 620 at a wavelength of 577.Appropriate software was used for data analysis.

Response of the parents and F 1 plants
The means of stem rust-related traits of the three parents and F 1 plants for each one under normal and infected conditions are shown in Table (4).Infection condition (late planting date) caused reductions in the estimates of all traits except spike length and number of spikelets per spike traits.
Plant height trait values showed reductions in all genotypes under infection condition which were lower than all genotypes under normal condition (optimum planting date), except the F 1 plants for cross 3 (line 37 x line 92) which showed the same value under both conditions.Moreover, line 37 displayed the highest reduction value, while line 92 showed the lower reduction value.These results agreed with those reported by Khattab (2009) and Darwesh (2011).
With respect to number of spikes per plant trait, the resistant parent (Misr1) and the F 1 plants for cross 2 (Misr1 x Line 92) exhibited a higher number (elven spikes per plant) than the two susceptible parents (Line 37 and Line 92) under infection condition, the F 1 plants for cross 1 (Misr1 x Line 37) and the F 1 plants for cross 3 (Line37 x Line 92) which were 9, 10, 8 and 9, respectively, under infection condition.The F 1 plants for cross 2 (Misr1 x Line 92) showed the same value under normal and infection condition which was 11 spikes/ plant.Comparable results were reported by Talbert et al. (2001) and Hendawy et al. (2009).
For grain yield per plant trait, there were sharp decreases in the values of the F 1 plants for cross 3 (Line 37x Line 92), the susceptible parent Line 37, the susceptible parent Line 92, the F 1 plants for cross 2 (Misr1 x Line 92) and the F 1 plants for cross 1 (Misr1 x Line 37) under infected condition (23.32, 23.85, 25.78, 32.99 and 34.72, respectively) compared with the normal condition (47.54, 44.69, 49.5, 47.41 and 45.52, respectively).While the resistant parent (Misr1) recorded the high-est value in grain yield under infection condition (46.91) compared with normal condition (49.39), which indicated that this resistant parent could relatively resist to stem rust disease.These results are in agreements with those of Tammam (2005) and El-Hawary (2010).
Days to heading and days to maturity traits showed lower values in all genotypes under infection condition compared with all genotypes under normal condition, the susceptible parent (Line 37) showed the sharpest reduction between days to heading under normal and infection conditions (107 and 97, respectively), on the other hand the susceptible parent (Line 92) showed the highest reduction between days to maturity under normal and infection conditions (154 and 144, respectively).Similar results were obtained by Talbert et al. (2001), Akhter et al. (2003) and Hendawy et al. (2009).
Spike length trait (Table 4) showed a slight difference between the normal and infection conditions for all genotypes.The resistant parent (Misr1) under normal condition and the F 1 plants for cross 2 (Misr1 x Line 92) under normal and infection condition, value (12 cm) was longer than all other genotypes.The susceptible parent (line 92) showed the lowest value (10 cm) under normal and infection conditions compared with other parents and their F 1 plants.These results agreed with those reported by Khattab (2009) and El-Hawary (2010).
For number of spikelets per spike trait, there was a slight difference between the normal and infection conditions for all genotypes.These results are in agreement with Tammam (2005)

Performance of F 2 plants
F 2 plants, represented by 200 individuals for each cross, were classified into groups according to their performances under infection condition for each trait.Then, rust reaction, plant vigor and grain yield traits classified the F 2 plants into groups for the three crosses.The first group refers to the best growing F 2 plants that were resistant to stem rust and high yielding under infection condition and the last group refers to the worst ones that were susceptible to stem rust and low yielding under infection condition.The F 2 plants were arranged in descending order according to their frequency.Plants with high frequency in the first group were chosen as the most resistant F 2 plants.While, plants in the last group were taken to represent the most susceptible F 2 plants.
According to these classifications, eight F 2 individual plants were selected to represent the most resistant F 2 plants and eight plants were chosen as the most susceptible ones to stem rust in cross 1 as shown in Table (5).Seven F 2 individual plants were selected to represent the most resistant F 2 plants and five plants were chosen as the most susceptible ones to stem rust in cross 2 as shown in Table (6).Seven F 2 individual plants were selected to represent the most resistant F 2 plants (these plants were escaped from stem rust infection, because the two parents for this hybrid are susceptible to stem rust) and eight plants were chosen as the most susceptible ones to stem rust in cross 3 as shown in Table (7).
These F 2 resistant plants and F 2 susceptible plants were used as individual plants to obtain SSR and STS markers associated with stem rust.
Many authors evaluated contrasting parents and their segregated F 2 population plants to detect some molecular markers associated with abiotic and biotic stresses as well as yield component and quality traits in plants.However, their results reflected significant differences between parental genotypes for the studied trait(s) which indicated the presence of variability between these parents.Moreover, the segregated F 2 population plants were classified to the highest and the lowest groups based on the studied trait(s) to develop molecular markers.Thus the resulting resistance genes in the F 2 populations must be inherited from the resistant parent (Haley et al., 2008;Onweller, 2011).In this respect, Rashed et al. (2006) evaluated some salt tolerance-related traits in sorghum, Atta et al. (2006) recorded some iron deficiency-related traits in maize, McIntosh et al. (1995) found that many of the introgressed genes are also associated with undesirable effects on agronomic traits.Michelmore et al. (1991) identificatied some molecular markers linked to disease-resistance genes by bulked segregant analysis.Although, several markers were reported as tightly linked to target resistance genes in a specific population in previous studies, they were not diagnostic when in different backgrounds.

SSR and STS markers for stem rust resistance
DNA isolated from Misr1 as a stem rust resistant parent, Line 37 and Line 92 as susceptible parents, their subsequent F 1 plants, and the F 2 segregated population (the most resistant and the most susceptible individual plants) for the three crosses were tested against six preselected SSR & STS specific primers as shown in Figs.
Only two primers (Sr2 and Sr25) detected positive molecular markers for stem rust resistance with the studied genotypes in crosses 1 and 2, while the other four primers failed to develop molecular markers for stem rust resistance as shown in Tables (8 and 9).Sr2 primer exhibited a positive molecular marker with molecular size of 120 bp which was found only in the resistant parent Misr1, the F 1 plants and the most resistant F 2 individual plants, while they were absent in the susceptible parents (Line 37 for cross 1 and Line 92 for cross 2) and the most susceptible F 2 individual plants (five plants for cross1 and three plants for cross 2).
Sr25 primer detected a positive molecular marker with molecular size of 130 bp which was found only in the resistant parent Misr1, the F 1 plants and the most resistant F 2 individual plants, while they were absent in the susceptible parents (Line 37 for cross 1 and Line 92 for cross 2) and the most susceptible F 2 individual plants (six plants for cross 1 and three plants for cross 2).
Consequently, Sr2 and Sr25 loci at fragment sizes of 120 and 130 bp, respectively, were apparently associated with stem rust resistance according to the presence of them in cross 1 (RP x SP1) and cross 2 (RP x SP2) as Misr1 (the resistant parent) was included in each one of them.
Moreover, these two loci were present in the resistant F 2 groups, while only one locus of these two loci were observed in some individuals of the susceptible F 2 groups of these two crosses due to the contribution of the resistant parent (Misr1).On the other hand, these two loci were actually absent in cross 3 (SP1 x SP2) which included the two susceptible parents.In addition, Sr38 locus at fragment size of 262 bp was present in cross 1 (RP x SP1) due to the contribution of the susceptible parent Line 37 (SP1) as well as in cross 3 (SP1 x SP2), while it was absent in cross 2 (RP x SP2).Finally, Sr24 locus at fragment size of 200 bp was a common fragment in all crosses, while Sr36 and Sr39 loci at fragment sizes of 155 and 900 bp, respectively, were absent in all crosses but they appeared in monogenic lines Sr36 and Sr39, which means that these three loci were not associated with stem rust resistance.Therefore the resistant parent, their F 1 plants and most resistant F 2 individual plants which were resistant to stem rust exhibited both Sr2 and Sr25.
These two positive markers could be considered as reliable markers for stem rust resistance in bread wheat.These results agreed with many reports which detected molecular markers for biotic stresses resistance.Molecular markers are available for only few resistance genes such as Sr2 (Hayden et al., 2004), Sr24 (Mago et al., 2005), Sr36 (Bariana et al., 2001;Tsilo et al., 2008) and Sr39 (Gold et al., 1999).Abdel-Tawab et al. (2003) detected five positive and negative RAPD markers for drought tolerance in Egyptian bread wheat.Some of these markers have been used in MAS (Marker assisted selection).At the present time, the research of stem rust in wheat is focusing on identifying more resistance genes to stem rust.Moreover, our results were in agreement with those of Nachit et al. (2000) who associated yield-related traits as grain yield, yield components and stress physiological traits with some molecular markers in durum wheat.Several markers showed strong relationships with grain yield, yield components and stress physiological traits, indicating that there are potential markers for use in marker-assisted selection to improve biotic stresses resistance known as molecular breeding.

SUMMARY
Screening experiment was performed on twelve genotypes of bread wheat (Ttriticum aestivum L.) to select the most stem rust resistant genotype (Misr1) and the most stem rust susceptible genotypes (Line 37 and Line 92) according to stem rust reaction.Crosses were carried out between the resistant parent (Misr1) with each of the susceptible parents as well as between the two susceptible parents (Line 37 and Line 92) to obtain the F 1 kernels.Some of the F 1 kernels were sown in the field and selfed to obtain the F 2 kernels for each cross.These three selected parents, their F 1 and the most resistant and susceptible F 2 plant groups for the three crosses were evaluated for their response to stem rust resistance by recording some stem rust-related traits.However, infected condition caused a reduction in the values of all traits except spike length and number of spikelets per spike traits.The three parents, their F 1 plants and some individual plants of the two contrasting F 2 plant groups (the most resistant and the most susceptible F 2 groups) for the three crosses were used to develop some molecular genetic markers associated with stem rust resistance using SSR and STS markers.The results indicated the presence of two positive markers out of the three SSR and three STS primers which used in this study.Sr2 (SSR) and Sr25 (STS) primers gave positive markers at fragment sizes of 120 and 130 bp, respectively, for stem rust resistance that could be considered as reliable markers for stem rust resistance in bread wheat (Ttriticum aestivum L.).

Table ( 1
): Screening the responses of the twelve studied bread wheat genotypes under infected condition at the season of 2010-2011.

Table ( 2
): Name, pedigree and origin of the three selected parental genotypes.

Table ( 4
): Means of the recorded stem rust yield-related traits of the three contrasting parents and F 1 plants for each one.

Table ( 6
): The performances of the most resistant & most susceptible F 2 (individual plants) for cross 2 (Misr 1 x Line 92) under infection condition (late planting date).