Seedling Resistance to Stem Rust and Molecular Marker Analysis of Resistance Genes in Wheat Cultivars of Yunnan, China

Stem rust is one of the most potentially harmful wheat diseases, but has been effectively controlled in China since 1970s. However, the interest in breeding wheat with durable resistance to stem rust has been renewed with the emergence of Ug99 (TTKSK) virulent to the widely used resistance gene Sr31, and by which the wheat stem rust was controlled for 40 years in wheat production area worldwide. Yunnan Province, located on the Southwest border of China, is one of the main wheat growing regions, playing a pivotal role in the wheat stem rust epidemic in China. This study investigated the levels of resistance in key wheat cultivars (lines) of Yunnan Province. In addition, the existence of Sr25, Sr26, Sr28, Sr31, Sr32, and Sr38 genes in 119 wheat cultivars was assessed using specific DNA markers. The results indicated that 77 (64.7%) tested wheat varieties showed different levels of resistance to all the tested races of Puccinia graminis f. sp. tritici. Using molecular markers, we identified the resistance gene Sr31 in 43 samples; Sr38 in 10 samples; Sr28 in 12 samples, and one sample which was resistant against Ug99 (avirulent to Sr32). No Sr25 or Sr26 (effective against Ug99) was identified in any cultivars tested. Furthermore, 5 out of 119 cultivars tested carried both Sr31 and Sr38 and eight contained both Sr31 and Sr28. The results enable the development of appropriate strategies to breed varieties resistant to stem rust.


Introduction
Stem rust (caused by Puccinia graminis Pers. f. sp. tritici Eriks. & E. Henn.) is one of the most serious diseases of wheat, worldwide [1,2]. In China, it has been effectively controlled through the development of resistant cultivars and deployment of effective resistance genes, especially 1B/1R translocation gene Sr31 in different epidemiological regions since 1970s [3,4]. However, in 1998, a new race of wheat stem rust pathogen designated as Ug99 (TTKSK), expressing virulence to Sr31, was first identified in Uganda [5,6]. It has spread throughout the major wheat growing regions of Africa such as Ethiopia, Zimbabwe, Mozambique, Kenya, Sudan, Yemen, Egypt, and Tanzania [7,8]. The variants exhibited stronger virulence and could rapidly spread Einkorn, and Vernal), five Chinese supplemental differentials (Mianzi 52, Huadong 6, Mini 2761, Orofen, and Rulofen), and five sets of 20 single Sr-gene lines (Sr5, Sr21, Sr9e, Sr7b, Sr11, Sr6, Sr8a, Sr9g, Sr36, Sr9b, Sr30, Sr17, Sr9a, Sr9d, Sr10, SrTmp,Sr24,Sr31,Sr38,and McN). At present, the races were identified and designated using three parts of differentials by Wheat Disease Laboratory, Shenyang Agricultural University, namely: part 1 uses four of Stakman's hosts and is given Arabic numerals, such as 21 or 34 etc.; the second part uses 5 Chinese supplemental lines, and was given a 'C' plus Arabic numerals, C1 or C2 or C3 etc.; and the third part uses 20 single Sr-gene lines and given five-letter-code as currently used in the United States and many other countries, for examples: HTTTM or RKGQM. The full names of the races and their virulence/avirulencepatterns are shown in Table 1 [3]. They were isolated and identified by Wheat Disease Laboratory, Shenyang Agricultural University, China.

DNA extraction and fragment analysis
DNA was extracted from young leaves of seedlings according to the method described by Lagudah et al. [22]. The DNA quality was determined using 1.2% (w/v) agarose gel. DNA quantification was performed using NanoDrop-1000 version 3.3.1 spectrophotometer. PCR primers were synthesized by Sangon Biotech (http://www.sangon.com/, China). PCR assays were performed according to the published protocols (Table 2). PCR amplifications were carried out in 25 μL volume, including 2.5 μL 10×buffer (Mg 2+ ), 0.5 μL 10 mmolÁL -1 dNTPs, 1 μL 10 μmolÁL -1 of each primer, 0.2 μL 5 UÁμL -1 Taq-polymerase, and 2 μL 30 ngÁμL -1 DNA. De-ionized water was used to obtain 25 μL. 1.2% (w/v) agarose gel was used to detect the fragments of target gene. The agarose gels were analyzed by Uvitec cambridge. The incubation time and voltage used for analysis of fragments were 200V and 30 min. All above reagents were provided by Sangon Biotech (http://www.sangon.com/, China). Further, the ddH 2 O and reaction system lacking the template DNA were used as controls. The specific reaction conditions were based on published studies.

Wheat seedling resistance
The reaction of 119 main wheat cultivars in Yunnan to the Pgt races are shown in Table 3. Seventy-seven (64.7%) of the tested wheat varieties showed varying levels of resistance to races 21C3CTHTM, 34MKGSM and 34C3RKGQM. The remaining 42 wheat cultivars (35.3%) showed varying levels of susceptibility. A total of 97 cultivars showed different levels of resistance to race 21C3CTHTM, accounting for 81.5% of all the tested cultivars. Ninety-five cultivars were resistant to race 34MKGSM, and 24 showed susceptibility. A total of 83 cultivars showed resistance to 34C3RKGQM, accounting for 69.7% of all the tested cultivars, which was relatively low compared to resistance to 34MKGSM and 21C3CTHTM.

Sr25 screening
LC was used as the negative control and the monogenic Sr25 served as the positive control. A 190 bp specific band was amplified in the positive control using the primer Xwmc221. This result showed that no amplification of the 190 bp band in the 119 wheat cultivars of Yunnan Province (Fig 1).

Sr26 screening
Mago et al. [24] developed a pair of RFLP markers for detection of wheat stem rust resistance gene Sr26, and the effectiveness of the specific marker Sr26#43 was evaluated. The results showed that the primer Sr26#43 amplified a 207 bp fragment, and was used to detect Sr26 in the cultivars (Fig 2). LC was used as a negative control and monogenic Sr26 was used as the positive control. Primer Sr26#43 amplified a 207 bp band in the positive control Sr26, while no

Tagged Sr genes Marker Size of markers (bp) Primer sequence References
Sr25

Sr28 screening
Rouse et al. [25] showed that the marker wPt-7004 amplified 194 bp and 166 bp fragments in the cultivars containing Sr28 resistance genes. Further analysis confirmed that the amplification of the 194 bp fragment represented the specific band for Sr28. Polyacrylamide gel electrophoresis was used for detection of Sr28. As shown in Fig (Table 4).

Sr31 screening
The SCSS30.2 576 marker is linked to stem rust resistance gene Sr31. The Sr31 genes transferred from rye into wheat (Triticum aestivum L.) contributes to resistance in all the virulent    [26] in the positive control Sr31 (Fig 4). It acted as a specific marker with no product generated in lines that do not carry Sr31. Among the tested cultivars, 43 cultivars tested positive for the Sr31 marker (Table 4).

Sr32 screening
Sr32 is located on the short arm of chromosome in Sears' wheat Aegilops speltoides 2D-2S#1 translocation line C82.2 [27]. A dominant marker, csSr32#1, derived from an AFLP fragment [27] amplified 184bp PCR product in wheat lines carrying Sr32. In this study, the marker csSr32#1 was used to determine the presence of Sr32 in 119 wheat materials. The band was amplified only in the positive control Sr32 and one wheat cultivar 91E001, but none was detected in the remaining cultivars ( Fig 5).

Discussion
Gene Sr25 transfer to wheat Thinpyrum ponticum, occurs on the long arm of chromosome 7DL. It is usually closely linked to leaf rust resistance gene Lr19. Another Th. ponticum-derived gene resulted in undesirable yellow flour [32], and improved the yield [21]. The Sr25 resistance is affected by growth periods and temperature. The resistance in seedling stage is higher than in adult stage and is more easily susceptible under high temperature conditions [33]. Due to its resistance to Ug99, the specific primer Xwmc221 was used for detection of 150 cultivars in China, but no Sr25 was identified [21]. In this study, the same primer pair was used for detection of Sr25 in119 wheat cultivars (lines) from Yunnan Province. The results also indicated that none of the tested wheat cultivars contained the gene. Therefore, the gene was targeted in the future breeding programs to improve the resistance level of wheat cultivars to Ug99 in China.
The wheat stem rust resistance gene Sr26 (derived from Agropyron elongatum) is derived from foreign chromosome translocated to wheat chromosomes [24]. This gene located on  Stem Rust Resistance Genes in Wheat Lines of Yunnan, China chromosome 6AL, confers resistance to all races of wheat stem rust worldwide [9] and is widely used in Australia [24]. It is reported that the Chinese wheat varieties may contain Sr26. Two out of 448 wheat cultivars contain Sr26 that was identified by researchers [34][35][36]. In this study, the Yunnan varieties were tested for presence of Sr26. However, the gene was not found in any of the materials tested, which indicated that Yunnan wheat varieties lack the Sr26.
Sr28 originates in T. aestivum L. Kota. Kota has been used as a differential host by Stakman since 1962. However, due to ineffectiveness to most races of Pgt, its application was limited in resistance breeding [25]. With the emergence of Ug99, researchers focused on the discovery of genes or gene combinations resistant to Ug99, to strengthen the resistant cultivars in the international wheat stem rust nursery of Kenya. SD1691 resistant to Ug99 (TTKSK) was studied by Rouse et al. [37], and found to harbor the Sr28 resistance gene [25]. In addition, Sr28 was ineffective to several popular American races of Pgt. However, it was combined with other resistance genes to yield effective resistance gene combinations, and has been used in breeding disease resistance [25]. Therefore, the diversity arrays technology (DArT) [38] marker closely linked to Sr28 was developed for more convenient screening and identification of the effective gene. Similarly, Sr28 was also ineffective to many Pgt races in China [39,40]. Chen et al. [35] reported that Chinese wheat cultivars carry Sr28. In this study, the DArTmarker wPt-7004 screened by Rouse was used to detect the gene in Yunnan wheat cultivars (lines). The results showed that 12 wheat varieties (lines) carried this gene. These were Nanyuan 1, Feng 0103, Jing07-2, Jing 05-1,088-16, E33, Linmai 15, Jing 0202, Jingmai 10, Yunmai 54, Yunmai 47, and Feng 615.
The gene Sr31, which confers a high degree of effectiveness to stem rust was introgressed into bread wheat from 'Petkus' rye as a 1BL/1RS translocation [26]. Sr31 has been widely used in Chinese and global wheat stem rust breeding programs [26,41,42]. However, wheat stem rust has re-emerged as a major threat to global wheat production following the emergence of the new race Ug99 virulent to Sr31 in 1999 [5]. To date, no virulent race against Sr31 has been reported in China [41]. In our study, 43 cultivars (lines) probably carried Sr31. Because Sr31 has been widely used in Chinese wheat breeding since 1970s, and identified that many cultivars carry this gene [41,42]. Our results were consistent with previous reports. For example, Wang et al. [43] and Zhang et al. [44] identified a 1BL/1RS translocation in 211 and 75 wheat cultivars (lines) using the co-dominant PCR marker. Their results showed that 81 and 25 wheat varieties carried this gene, respectively. Conversely, pedigree tracking indicated that resistant materials carrying Sr31 such as 'Kavkaz' and 'Luofu' lines were widely used in wheat breeding in Yunnan Province [45], suggesting the origin of resistance genes in these wheat varieties. In addition, a specific band was amplified in Yunmai 51 (91B-831/92B-84) and Fengmai 31 (918M40-1) using SCSS30.2 576 marker. However, the two cultivars were highly susceptible to Pgt in China and no race virulent to Sr31 was found, suggesting that the two cultivars do not contain the gene Sr31. This finding may be attributed to false-positive results, which are common challenges associated with MAS. For example, Yu et al. reported that the variety Thatcher does not carry Sr2 but tested positive [46].
Sr32 present on the short arm of chromosome 2S#1 was transferred to wheat from Aegilops speltoides Tausch [47]. Sr32 exhibited strong resistance against the predominant race group 21C3 in China [13,39]. In this study, we found that 91E001 carried Sr32. The wheat cultivar 91E001 used in breeding programs was introduced from Mexico to China, with strong resistance against wheat stripe rust [48]. Additionally, Sr32 was shown to be effective against seven race variants of Ug99 lineage [9], and was used in future resistance breeding. The durability of resistance genes is enhanced by deploying pyramids in cultivars.
The translocation line VPM1 of Triticum aestivum L. containing 2NS chromosome segments (25~38cM) from Ae. ventricosa was cultivated by Maia in 1967 [32]. The fragment carried three effective genes against rust: Lr37 against leaf rust, Sr38 against stem rust and Yr17 against stripe rust. These three genes have been widely used by breeders worldwide [32,41]. Sr38 is no longer resistant to new races related to Ug99. No virulent Pgt race to Sr38 has been found in China. PCR analysis was used for effective screening of resistance genes Lr37-Yr17-Sr38 established by Helguera [29], which played an important role in MAS [30], and was used in this study for identification of genes in wheat cultivars of Yunnan Province. The results showed 10 wheat cultivars harboring the gene. The resistance of these cultivars against the Chinese races 34MKGSM and 21C3CTHTM may be attributed to the Lr37-Yr17-Sr38 genes.
In this study, the molecular genetic markers of Sr25, Sr26, Sr28, Sr31, Sr32 and Sr38 in wheat cultivars against Pgt races were used to identify the 119 wheat cultivars (lines) in Yunnan province. Forty three and 10 wheat cultivars (lines) are likely to carry Sr31 and Sr38, respectively. Sr31 and Sr38 have been widely used in Chinese stem rust resistance breeding [41]. Although Sr31 and Sr38 are no longer resistant to new races related to Ug99, they are still useful when combined or used as pyramids with other genes effective against Ug99. Overall, wheat cultivars in Yunnan province lack the genes Sr25 and Sr26, which are effective against Ug99. Sr28 alone was detected in 12 wheat cultivars including Nanyuan 1, Feng 0103, Jing 07-2, Jing 05-1, 088-16, E33, Linmai 15, Jing 0202, Jingmai 10, Yunmai 54, Yunmai 47, and Feng 615. Sr32 was detected in one cultivar 91E001. Spread of Ug99 into China will most likely lead to colonization of the southwest area such as Yunnan and Sichuan provinces, where Pgt is already prevalent [41]. Therefore, the widespread cultivation of these wheat cultivars susceptible to Ug99 in Yunnan greatly increases the risk of its epidemic. Fortunately, the two genes Sr28 and Sr32 might play a role in the prevention of colonization and invasion of Ug99. The 12 cultivars containing Sr28 and the 91E001 carrying Sr32 identified in this study will be useful for resistance breeding and propagation of cultivars.