Evaluation and identification of stem rust resistance genes Sr2, Sr24, Sr25, Sr26, Sr31 and Sr38 in wheat lines from Gansu Province in China

Wheat stem rust, caused by Puccinia granimis f. sp. tritici, severely affects wheat production, but it has been effectively controlled in China since the 1970s. However, the appearance and spread of wheat stem rust races Ug99 (TTKSK, virulence to Sr31), TKTTF (virulence to SrTmp) and TTTTF (virulence to the cultivars carrying Sr9e and Sr13) have received attention. It is important to clarify the effectiveness of resistance genes in a timely manner, especially for the purpose of using new resistance genes in wheat cultivars for durable-resistance. However, little is known about the stem rust resistance genes present in widely used wheat cultivars from Gansu. This study aimed to determine the resistance level at the seedling stage of the main wheat cultivars in Gansu Province. A secondary objective was to assess the prevalence of Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 using molecular markers. The results of the present study indicated that 38 (50.7%) wheat varieties displayed resistance to all the tested races of Puccinia graminis f. sp. tritici. The molecular marker analysis showed that 13 out of 75 major wheat cultivars likely carried Sr2; 25 wheat cultivars likely carried Sr31; and nine wheat cultivars likely carried Sr38. No cultivar was found to have Sr25 and Sr26, as expected. Surprisingly, no wheat cultivars carried Sr24. The wheat lines with known stem rust resistance genes could be used as donor parent for further breeding programs.


INTRODUCTION
Puccinia graminis Pers. f. sp. tritici Eriks. and E. Henn (Pgt ) causes one of the most potentially destructive wheat diseases, seriously threatening world grain production (Pardey et al., 2013). Disease-resistance breeding to control wheat stem rust is economic, effective, and protective of the environment, and has been proved to be the best control method by repeated practice (Goutam et al., 2015). Wheat stem rust has been effectively controlled with the wide use of resistance gene Sr31 from a 1BL/1RS wheat-rye chromosome arm translocation (Rouse et al., 2012). However, a new race Ug99 virulent to Sr31 was identified in Uganda and classifed as TTKS by the North American Nomenclature System of Pgt in 1999 (Pretorius et al., 2000). Ug99 has broad virulence, and mutates and spreads quickly.
Since 1999, 13 variants of Ug99 have been found in 13 countries (FAO, 2017). Recently, Ug99 has been monitored in Egypt, which is the main wheat production area of the Middle East, revealing that its mode of spread is similar to that of a virulent stripe rust pathogen race to Yr9 predicted by Geographic Information System of CIMMYT (Singh et al., 2006). Following the identification and spread of the Ug99 race group, a new race TKTTF caused a wheat stem rust epidemic with an estimated 20,000 to 40,000 ha likely planted to 'Digalu' (with resistance to Ug99 race group) in Southern Ethiopia during 2013-2014 (Olivera et al., 2015). Currently it has been confirmed in 11 countries, and given the rapid and destructive nature of race TKTTF, close monitoring of this race is advised-especially in countries which have cultivars carrying the SrTmp resistance gene.
A new race TTTTF with virulence to Sr9e and Sr13 attacked thousands of hectares of durum wheat in Sicily, Italy in 2016, resulting in the largest burst of wheat stem rust in Europe since the 1950s (Bhattacharya, 2017). The large number of spores produced by TTTTF may continue the epidemic in 2017. Moreover, the researchers from the Global Rust Research Center shared a major concern in the warning report that TTTTF could infect not only durum wheat and bread wheat but also dozens of laboratory-grown strains of wheat (FAO, 2017). In view of this, in February 2017, Nature highlighted the potential threat to European wheat production of this race (Bhattacharya, 2017). Therefore, the spread of Ug99, TKTTF and TTTTF, and their variants, threaten the wheat production safety in China.
Gansu Province, located in the northwest of China, plays a significant role in the epidemic and spread of wheat stem rust in China (Cao, 1994). Resistance breeding for this disease has not been a primary objective because it has been effectively controlled in China since the 1970s (Wu et al., 2014). However, durable resistance to stem rust has been re-emphasized with the occurrence and spread of new races of Pgt. It is necessary to analyze the resistance genes in wheat cultivars (lines) from Gansu Province, and the information provided here will be important for developing potentially durable combinations of stem rust resistance genes in cultivars.

Wheat cultivars and near-isogenic lines
A total of 75 tested wheat cultivars in Gansu Province were provided by Dr. Fangping Yang from the Wheat Research Institute, Gansu Academy of Agricultural Sciences.
Molecular markers linked to six Sr genes were tested: Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38. Near-isogenic lines carrying 45 Sr genes were used to confirm the validity of these molecular markers. The near-isogenic lines carrying these resistance genes were provided by Dr. Yue Jin from USDA-ARS, Cereal Disease Laboratory, University of Minnesota, USA.
The tested Pgt races included the 21C3CTHTM, 21C3CFHQC, 34MKGQM, 34MKGSM, 34C3MTGQM and 34C3RTGQM (race 34C3MTGQM and 34C3RTGQM identified from the alternative host, Berberis). These races were named according to the methods described in a published study (Li et al., 2016b). The full names of the races and their virulence/avirulence patterns are shown in Table 1. They were isolated and identified by the Plant Immunity Institute, Shenyang Agricultural University, China.

Seedling resistance evaluation
The cultivars were planted in porcelain pots with a 12-cm-diameter. Seven days later, the leaves were moistened by water with 0.1% Tween 20 using an atomizer and then sprayed with 1 g of fresh urediniospores and dried talc in a ratio of 1:20 (v:v). The inoculated seedlings were transferred to a greenhouse with the temperature in a range of 18 to 22 ± 1 • C. Three biological replicates of the seedling assays were performed for each Pgt race. After 14 days of inoculation, the infection types (ITs) were recorded using the 0-4 IT scale (Stakman, Stewart & Loegering, 1962). ITs were then grouped into low ('0', ';', '1', '1+,'2 ,'2+, and X) and high ('3-', '3', '3+', and '4') infection types. The ITs used in this study are shown in Fig. 1.

DNA extraction
DNA was extracted from young leaves of 10-day-old seedlings using a genomic DNA extraction kit (http://www.sangon.com/, China). The DNA quality was examined by 1.2% (w/v) agarose gels and DNA quantification was performed using the NanoDrop-1000 version 3.3.1 spectrophotometer. Polymerase chain reaction (PCR)-specific primers were synthesized by Shanghai Biotech Biotech Co., Ltd, China (Table 2). PCR amplifications were carried out in 25 µL volume, including 0.5 µL of 10 mmol L −1 deoxyribonucleoside triphosphates, 2.5 µL of 10× buffer (Mg 2+ ), 0.2 µL of 5 U µL −1 Taq polymerase, 1 µL of 10 µmol L −1 of each primer, and 2 µL of 30 ng µL −1 DNA. De-ionized water was used to achieve 25 µL volume. Condition of PCR amplification were as follows: 94 • C for 4 min, 30 cycles of 94 • C for 45 s, 60 • C for 45 s, and 72 • C for 1 min, followed by the final extension at 72 • C for 8 min; other specific conditions were as described in previous studies (Table 1).

Validity of the markers
Six specific PCR markers closely linked with resistance genes Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were validated using 45 single differentials carrying known resistance genes. Table 5 shows that these ten markers amplified only specific bands in the expected wheat genetic stocks. For example, primer SCSS30.2 576 amplified only 576-bp specific bands in Siouxland, Sisson, Sr31/6*LMPG, and Federation*4/Kavl, while in other wheat lines without Sr31, no bands were amplified, indicating that these markers are able to be well applied for the molecular detection of the six resistance genes.

Sr2 screening
A DNA marker was developed to accurately predict Sr2 in diverse wheat germplasm for the partial resistance of Sr2 is very difficult to screen under field conditions (Mago et al., 2011). Two markers, Xgwm533 and csSr2, were used to detect Sr2 in wheat cultivars of Gansu Province. A specific PCR band with 120-bp in size was amplified with marker Xgwm533, but no PCR product was amplified using marker csSr2 in Hope with Sr2. In this study, a similar 120-bp band was detected in the 13 cultivars, indicating that these cultivars carried Sr2 (Table 6).

Sr24 screening
Two markers, Sr24#12 and Sr24#50, were developed to detect Sr24, located on chromosome 3DL in Agent-or 1BS in Amigo-derived lines (Mago et al., 2005). These two markers were applied to detect Sr24 existence in the 75 major wheat cultivars (lines) of Gansu Province in this study. The results showed that marker Sr24#12 amplified a 500-bp specific band and marker Sr24#50 amplified an approximately 200-bp specific band in the Sr24 control LcSr24Ag. No PCR fragment was amplified in Little Club (LC) and the tested cultivars, indicating that these cultivars lacked Sr24.

Notes.
a Symbol '+' indicates the cultivar (line) carry the tested genes; '-' indicates that the cultivar (line) does not carry the tested genes.

Notes.
a Symbol '+' indicates the cultivar (line) carry the tested genes; '-' indicates the cultivar (line) don't carry the tested genes.

Sr25 screening
Because of the resistance of Sr25 to the new race Ug99 and related strains, a dominant marker Gb was developed for haplotyping Sr25, (FAO, 2017;Liu et al., 2010;Pretorius et al., 2000). The presence of the marker was confirmed by detection of a 130-bp fragment.
The PCR results indicated that the 130-bp band was only amplified using the Sr25-positive line Agatha/9*LMPG (monogenic Sr25) genomic DNA (Liu et al., 2010;Yu et al., 2010), but not with other cultivar DNA samples, indicating that all 75 lines from Gansu Province examined lack Sr25.

Sr26 screening
Stem rust resistance gene Sr26 was transferred into the long arm of wheat chromosome 6A from Thinopyrum ponticum (Mago et al., 2005). Although the cultivars carrying Sr26 displayed resistance to all the dominant Pgt races in China, it is not utilized in wheat breeding. A dominant STS marker Sr26#43 was developed for detecting this wheat stem rust resistance gene and a 207-bp band was amplified in wheat lines with Sr26 (Mago et al., 2005). Marker Sr26#43 was used to detect this fragment in tested wheat cultivars. No any visible band was detected, suggesting that these varieties do not carry Sr26, as expected.

Sr31 screening
Two markers, SCSS30.2 576 and Iag95, linked to resistance gene Sr31 were used for detecting these locus. SCSS30.2 576 amplified a 576-bp fragment and marker Iag95 amplified an 1,100-bp PCR fragment in Sr31-carrying lines such as Sr31/6*LMPG and Siouxland (Fig. 2). No fragment was amplified in the negative control LC. These two markers were used to detect Sr31 in the tested cultivars. The result showed that these two fragments were detected in the 25 tested cultivars (Table 6).

Sr38 screening
The Lr37 -Sr38-Yr17 rust resistance gene cluster was transferred to the short arm of bread wheat chromosome 2AS from a segment of Triticum ventricosum (Tausch) Cess. chromosome 2NS (Helguera et al., 2003). The 2NS-specific primer VENTRIUP-LN2 and 2AS-specific primer URIC-LN2 were developed to detect this rust resistance gene cluster in commercial wheat cultivars and 262-bp and 285-bp PCR products were amplified in wheat line carrying Lr37 -Sr38-Yr17, whereas none of these amplification products were found in negative control LC (without Lr37 -Sr38-Yr17 ). In this study, both 262-bp and 285-bp PCR fragments were amplified in nine wheat cultivars, suggesting that these wheat cultivars carried Sr38 (Table 6).

DISCUSSION
The broad-spectrum wheat stem rust resistance gene Sr2 confers adult plant resistance to stem rust and is located on chromosome arm 3BS. It originated in tetraploid Yaroslav emmer (T. dicoccum) and later was transferred to the susceptible bread wheat 'Marquis' in the 1920s (McFadden, 1930). Several varieties with Sr2 were cultivated worldwide (Singh et al., 2011). Markers Xgwm533 and csSr2 were used to detect Sr2 in wheat cultivars from Gansu. However, marker csSr2 failed to predict Sr2. Only marker Xgwm533 amplified a 120-bp band in the positive control and 13 tested cultivars, but the 120-bp band also occurred in many North American and CIMMYT lines which are considered not to have Sr2. Therefore, it is difficult to conclude that all the accessions that showed a 120-bp fragment size for this marker carry Sr2. The stem rust resistance gene Sr24 is completely associated with leaf rust resistance gene Lr24. It has been widely used in wheat breeding programs worldwide, since it was introgressed into wheat lines (McIntosh, Wellings & Park, 1995). Gene Sr24 was ineffective to some variants of Ug99 but is effective to the new races TKTTF, TTTTF, and many Pgt races in China (Bhattacharya, 2017;Han, Cao & Sun, 2010). Therefore, two markers, Sr24# 12 and Sr24#50, developed by Mago et al. (2005) were used to detect the gene in Gansu wheat cultivars in this study. Surprisingly, no wheat cultivars carried this gene. However, it is reported that Chinese wheat cultivars in other provinces carry Sr24 Li et al., 2016b).
Wheat plants carrying stem rust resistance gene Sr25 were susceptible to several strains of Chinese Pgt races . Sr25 and its linked leaf rust resistance gene Lr19, were transferred into wheat from Thinopyrum ponticum to wheat chromosomes 7D and 7A (Friebe et al., 1994;Zhang et al., 2005). The use of Sr25-Lr19 was initially limited because of linkage with another Th. ponticum derived gene producing undesirably yellow flour. It has been further backcrossed into the Australian and CIMMYT wheat backgrounds with the mutant line (which contains Sr25-Lr19), but with white flour (Bariana et al., 2007;Knott, 1980). The use of this gene in wheat programs is increasing for its resistance to new races TTTTF and Ug99 race group, having potential yield increases under irrigated conditions (FAO, 2017;Liu et al., 2010;Monneveux et al., 2003;Singh et al., 1998). In this study, 75 wheat varieties from Gansu Province were examined for presence of marker Gb. The result showed that all 75 wheat varieties lack Sr25.
Lines containing the Sr26 fragment are resistant to new stem rust pathogen races such as Ug99 and its associated strains. None of the cultivars had Sr26 in the present study, as expected, and similar results were observed in our previous study (Li et al., 2016a).
The stem rust resistance gene Sr31 on 1BL/1RS was transferred into the bread wheat from 'Petkus' rye (Graybosch, 2001). Since then a higher number of wheat cultivars carrying Sr31 have been released in global wheat breeding (Das et al., 2006). It is reported that more than 60% (1.3 ×107 hm 2 ) of the total wheat planting areas carried this translocation in China (Jiang et al., 2007). Although the gene is ineffective to Ug99 and related variants, it is also an effective gene against all Pgt races in China and the new races TKTTF and TTTTF. Molecular marker detection showed that 25 wheat cultivars carried Sr31. All these cultivars (lines) produced resistance ITs (0, ;, ;1, 1+, and 2) to all tested Pgt races, as expected. Moreover, pedigree tracking indicated that resistant materials carrying the 1BL/1RS translocation such as 'Kavkaz' and 'Rye' were widely used in wheat breeding in Gansu Province (Cao et al., 2011), revealing the origin of Sr31 in these wheat varieties.
Rust resistance gene cluster Yr17-Lr37 -Sr38 was initially transferred into the winter bread wheat line 'VPM1' from T. ventricosum and was located in a 2NS/2AS translocation (Bariana & McIntosh, 1993;Cao et al., 2007;Maia, 1967). PCR assays using restriction fragment length marker cMWG682 were developed for selecting the 2NS/2AS translocation in wheat cultivars (Helguera et al., 2003). Sr38 became susceptible to new races related to Ug99 but no virulent Pgt race to Sr38 has been found in China. The results showed that nine wheat cultivars carried the gene cluster. The resistance of these cultivars against the tested Pgt races might be attributed to this gene.

CONCLUSION
Breeding resistant cultivars is an economic and effective way to protect wheat from disease. The development of molecular technology facilitated the identification and utilization of molecular markers for durable resistance breeding, leading to increased crop production. The molecular markers associated with Sr2, Sr24, Sr25, Sr26, Sr31, and Sr38 were used to detect the occurrence of these genes in 75 major wheat cultivars (lines) in Gansu Province in this study. The results showed that 35 tested cultivars might carry one of these genes. This information can be used in breeding for stem rust resistance in the future.