Molecular detection of disease resistance genes to powdery mildew ( Blumeria graminis f . sp . tritici ) in wheat ( Triticum aestivum ) cultivars

A study was conducted to detect the presence of disease resistance genes to infection of wheat powdery mildew (Blumeria graminis f. sp. tritici) in selected wheat cultivars from China using molecular markers. Genomic DNA of sixty cultivars was extracted and tested for the presence of selected prominent resistance genes to the fungal disease using molecular markers linked to Pm genes. Results showed that 17 cultivars were detected with Pm2 gene, 24 cultivars were detected with Pm4b gene, two cultivars were detected with Pm6 gene while 24 wheat cultivars were detected with Pm8 gene. Multiple genes were also detected in the study. Cultivars Xinxuan2039, Lankao008 and Zhengmai366 were detected with possible multiple Pm2+Pm4b+Pm8 genes while Yumai368 was detected with possible multiple genes Pm2+Pm4b+Pm6. The results of this study provide a significant contribution to breeding for resistance to wheat powdery mildew disease since the identified cultivars detected with Pm genes will contribute to further studies on improving wheat resistance to the disease. Also, the continued resistance of cultivars with designated resistance genes demonstrates that the responsible Pm genes are still effective in overcoming powdery mildew infections.


INTRODUCTION
Wheat powdery mildew, caused by Blumeria graminis f. sp.tritici (Bgt), is one of the most damaging foliar diseases of wheat in most parts of the world, especially in cool and humid areas (Cao et al., 2013;Li et al., 2013; highly affected by the damage caused by the disease (Bennet, 1984;Alam et al., 2011).The disease causes devastating effects both in grain yield and quality (Everts et al., 2001;Conner and Kuzyk, 2003;Asad et al., 2014).The affected area for powdery mildew from 2001 to 2006 in China was 5.9 to 9.4 million hectares resulting in 0.26 to 0.32 billion kilograms reduction in grain yield (Xue et al., 2009).
Currently, the yield losses range from 13 to 34% under high infestation in the field but during epidemic seasons, the disease can cause up to 50% yield loss (Yao et al., 2007;Zhang et al., 2008;Li et al., 2011;Alam et al., 2013;Quijano et al., 2015;Yu et al., 2015).The shift in pathogenic virulence structures range from the southwest and the southeast coastal regions of China to almost all the wheat growing areas throughout the country and the damage exceeds any other known wheat disease (Liu and Shao, 1994;Luo et al., 2009;Cao et al., 2013;Zhao et al., 2013;Shen et al., 2015).
In an attempt to control the diseases, use of resistant or tolerant cultivars has been one of the effective methods widely used by scientists as it is economical as well as environmentally friendly (Song et al., 2009;Ben-David et al., 2010).Several wheat cultivars are being developed and tested for resistance to wheat powdery mildew but their resistance is easily broken down due to continuous development of new and more virulent strains through genetic recombination (Piarulli et al., 2012;Hurni et al., 2014).Some of the developed cultivars carrying resistance genes to wheat powdery mildew lose their resistance even before they are made available for commercial production (Hao et al., 2015;Ma et al., 2015).It is, therefore, important that the development of resistant cultivars should target multiple resistances to disease isolates so that effective and lasting control can be attained.Assessment of disease resistance on new wheat cultivars can be effective when the cultivars are subjected to existing as well as new strains of powdery mildew isolates over a considerable period of time (Li et al., 2012).
Sources of these genes have been the cultivated or wild relatives of Triticum species and then they got transferred to common wheat.For example, resistance gene Pm2 originated from the wild species Ae. tauschii that got introgressed into common wheat T. aestivum (Lutz et al., 1995).Powdery mildew resistance gene Pm4a originated from T. diccocum while Pm4b originated from T. carthlicum (Briggle, 1969;Law and Wolfe, 1966).Resistance gene Pm6 was transferred from T. timopheevii (Jorgensen and Jensen, 1972).Another distant wild cultivar, S. cereale, is where resistance gene Pm8 came from (Hsam and Zeller, 1997) while gene Pm21 and Pm30 came from wild relatives H. villosa and T. dicoccoides, respectively (Chen et al., 1995;Liu et al., 2002).These genes are common in cultivars grown in Asian region and gene Pm21 has been very effective against a broad-spectrum of wheat powdery mildew isolates found in China (Song et al., 2009).Currently, over 56 cultivars being grown in China possess Pm4b and Pm8 genes (Wang et al., 2005;Zeng et al., 2014), while most red soft winter cultivars carrying gene Pm8 are widely grown in southeastern USA (Cowger et al., 2009;Hao et al., 2012).
This study, therefore, was aimed at detecting the presence of some prominent wheat powdery mildew resistance genes in selected wheat cultivars using molecular markers linked to the genes.

MATERIALS AND METHODS
Sixty w heat cultivars from different parts of China w ere identified for the study (Table 1).Ten seeds of each cultivar w ere sow n on trays in greenhouse located at the Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.Around 15 days after sow ing, w hen three to four leaves had been developed, seedling leaves w ere detached and their DNA w as extracted follow ing cetyltrimethylammonium bromide (CTAB) extraction method (Zheng, 2010).Chancellor, a susceptible cultivar w ith no know n Bgt resistance genes, w as used as negative control.
Seven additional cultivars w ith know n w heat pow dery mildew resistant genes w ere selected and used as positive controls (Table 2).Hence, a total of 67 cultivars w ere selected for the molecular study using various molecular markers linked to w heat pow dery mildew resistance genes.To test their purity, the extracted DNA w as run on 2% Agarose gel of 1% TAE (Tris-Acetate-EDTA) buffer solution and the image w as captured using Gel Documentation and Image Analysis System after staining in ethidium bromide solution for 10 min.
Follow ing extraction and dilution to a concentration ranging betw een 50 and 80 ng/µl, the genomic DNA samples w ere amplified in a polymerase chain reaction (PCR) machine using the follow ing mixture: 5 µl containing PCR Master mix, 2 µl of double distilled w ater, 1 µl reverse primer and 1 µl of forw ard primer for SSR markers (or as described in Table 3 for non-SSR markers).Genomic DNA amounting to 1 µl w as added, making up a total of 10 µl of the PCR reaction mixture.A drop of paraffin oil w as, thereafter, added to prevent the reaction mixture from evaporation during the amplification.
PCR protocol w as done using Bio-Gener Technology, Gene explorer PCR machine as follow s: 94°C for 3 min, 35 cycles of 94°C for 40 s, a range of 55 to 61°C depending on primer annealing temperature (Table 3) for 30 s, 72°C for 40 s and a final extension of 72°C for 10 min before soaking at 4°C.The PCR products w ere, thereafter, run on 2% Agarose gel immersed in 1% TAE (Tris-Acetate-EDTA) buffer solution and the image w as captured using Gel Documentation and Image Analysis System after staining in

RESULTS
Among the sixty wheat cultivars, the results showed that 17 cultivars contained possible Pm2 gene (Figure 1).The cultivars included Tian0015, Yujiao0338, Tian00127, Zhengnong01059, Xinyumai836, Pu02056, Tian01-104, Lantian095, Punong1, Zhoumai19, Yumai368, Guomai301, Xinxuan2039, Zhongyu885, Lankao008, Zhengyumai9989 and Zhengmai366.Positive control wheat cultivar, Ulka/8Cc was used to determine the size of the gene and it showed that the resistance Pm2 gene was detected with a molecular weight of 480 bp (Figure  1).This was confirmed due to the absence of the alleles in the susceptible cultivar Chancellor.
Multiple genes were also observed in some wheat cultivars during the study.For example, four cultivars; Tian00127, Xinxuan2039, Lankao008 and Zhengmai366 were detected with three possible multiples genes for Pm2, Pm4b and Pm8 while one cultivar, Yumai368 had a possible combination of three multiple genes for Pm2,  Pm4b and Pm6.Sixteen cultivars were detected with two possible multiple wheat powdery mildew resistance genes (Table 4).
The molecular study showed that Pm4b and Pm8 were the powdery mildew genes present in the highest number of wheat cultivars.A total of 24 wheat cultivars carried these genes.Molecular marker detected possible Pm2 in 17 cultivars while Pm6 was the lowest detected in two cultivars.Three powdery mildew resistance genes Pm4a, Pm21 and Pm30 were not detected in any of the tested cultivars using the molecular markers (Figure 5).

DISCUSSION
In this study, 17 cultivars were detected with possible Pm2 gene.This resistance gene had been widely used and had been highly effective in different parts of Europe and China years ago (Liu et al., 2000).Though the growing number of virulent strains has been reported in some parts of China, the gene occurs in high frequency in so many commercial cultivars as it is easily transferred from resistant to susceptible cultivars (Parks et al., 2008;Gao et al., 2012).Recently, Ma et al. (2015) reported the presence of a new gene Pm2b in the cultivar KM2939, a Chinese breeding line, which exhibits high resistance to powdery mildew at both the seedling and adult stages.This gene was reported to carry a single dominant powdery mildew resistance allele of Pm2, designated as Pm2b and was mapped on chromosome 5DS, rendering the previous allelic designation Pm2 to be re-designated as Pm2a.
Resistance genes Pm4b and Pm8 were genes detected in highest frequency among the cultivars used in the present molecular study.This is consistent with the finding reported by Li et al. (2012) that these genes are the most widely distributed among wheat cultivars in China.Pm8 cultivars have been grown worldwide since the 1980s and due to wide use, their effectiveness has since declined (Parks et al., 2008;Tang et al., 2014;Zeng et al., 2014).Several virulent strains of powdery mildew have emerged rendering the gene ineffective.In fact, it is one of the major reasons that an epidemic boom of wheat powdery mildew was recorded in China in the early 1990s (Graybosch, 2001;Ryabchenko et al., 2003).
Among the 59 cultivars, only two cultivars Guomai301 and Yumai368 were detected with possible Pm6 gene apart from other multiple genes also suspected to be present in the cultivars.Pm6 has been widely and successfully used in breeding for wheat powdery mildew Pm4b +Pm8 Zheng366 Pm4b  resistance for long time.The gene usually exhibits its best expression from the third leaf stage of wheat growth and thereafter, it is moderately effective, but recognizable at the seedling stage (Bennett, 1984;Qin et al., 2011;Li et al., 2014).Virulence matching the Pm6 gene has occurred in many regions but still, the gene remains effective in some areas, especially when other genes such as Pm2 and Pm4b are combined during their use (Cai et al., 2005;Costamilan, 2005;Shi et al., 2009;Purnhauser et al., 2011).This could be one of the possible reasons why the two cultivars were resistant during the study (data not presented) as the genes such as Pm2, Pm4b and Pm8 were also possibly present in these cultivars.

Conclusion
This study detected 24 cultivars likely carrying Pm4b and Pm8 while two cultivars carried likely Pm6 gene.Despite the recent findings on these genes indicating that they are being overcome by emerging virulent isolates, their abundant availability in the currently produced wheat cultivars signifies their continued contribution to wheat production in China.The cultivars detected with these genes can still be useful in various breeding programs for disease resistance.The detection of likely multiple resistance genes in some wheat cultivars in this study showed that where multiple genes were involved in conferring resistance to powdery mildew disease, the cultivars could effectively resist infection from isolates that would have otherwise caused virulence if a single gene was involved.

Figure 1 .
Figure 1.Wheat pow dery mildew gene Pm2 as identified in the w heat cultivars at 480 bp.Lanes 1-59 represent cultivars designation.R is resistant cultivar Ulka/8Cc, S is susceptible cultivar Chancellor.M is a 100 bp DNA ladder.

Figure 2 .
Figure 2. Wheat pow dery mildew gene Pm4b as identified in the w heat cultivars at 1800 bp.Lanes 1-59 represent cultivars designation.R is resistant cultivar Armada, S is susceptible cultivar Chancellor.M is a D2000 DNA ladder.

Figure 3 .
Figure 3. Pow dery mildew gene Pm6 as identified in the tested cultivars at 140 bp.Lanes 23-42 represent cultivars designation.R is resistant cultivar Coker 747, S is susceptible cultivar Chancellor.M is a 100 bp DNA ladder.

Figure 4 .
Figure 4. Pow dery mildew gene Pm8 as identified in the tested cultivars at 1300 bp.Lanes 1-59 represent cultivars designation, R is resistant cultivar Kavkaz, S is susceptible cultivar Chancellor, M is a 100 bp DNA ladder.

Figure 5 .
Figure 5.Total number of w heat cultivars detected w ith w heat pow dery mildew resistance genes.

Table 1 .
Names of w heat cultivars, their pedigree information and origin.

Table 1 .
Contd.Cultivars w ith a dash (-) indicate that their information could not be traced. *

Table 2 .
Wheat cultivars used as positive control and their know n pow dery mildew resistance genes.

Table 3 .
Description of molecular markers used for Bgt resistance gene identification.

Table 4 .
Bgt resistance genes detected using molecular markers.