Physical Mapping of a Novel Locus Conferring Leaf Rust Resistance on the Long Arm of Agropyron cristatum Chromosome 2P

Wheat leaf rust is one of the most common wheat diseases worldwide and can cause up to 40% wheat yield loss. To combat the growth and spread of leaf rust disease, continual exploration and identification of new and effective resistance genes are needed. Here, we report for the first time a locus conferring leaf rust resistance located on the long arm of Agropyron cristatum chromosome 2P in Triticum aestivum–A. cristatum 2P translocation lines. This study used 50 leaf rust races, including two Chinese major dominant leaf rust races, named by THT and PHT, and other 48 different leaf rust races collected from 11 provinces, 1autonomous region and 1 municipality of China to test the resistance to T. aestivum–A. cristatum 2P chromosome translocation lines and their backcross populations, the results indicated that the novel leaf rust resistance locus was immune or nearly immune to all tested leaf rust races. Four long arm translocation lines with different breakpoints of A. cristatum chromosome 2PL and their backcross populations were tested with leaf rust race THT at the seedling and adult stages and genotyped with 2P-specific STS markers. The results showed that the novel leaf rust resistance locus of the T. aestivum–A. cristatum 2P translocation lines was located in the chromosomal bin FL 0.66–0.86 of 2PL. Therefore, T. aestivum–A. cristatum 2P chromosome translocation lines conferring leaf rust resistance locus could provide a novel disease-resistance resource for future wheat breeding programs.


INTRODUCTION
Common wheat (Triticum aestivum L., 2n = 6x = 42, genomes AABBDD) is one of the most important widely planted food crops worldwide (Su et al., 2013). Wheat production plays an important role in the development of the national economy (Costanzo and Barberi, 2014). Wheat leaf rust caused by Puccinia triticina Erikss. is one of the most common diseases of wheat throughout the world (Bolton et al., 2008;Kolmer, 2013), and serious cases can cause up to 40% wheat yield loss (Knott, 2012). Recent changes in climate conditions caused by global warming are more suitable for the spread of wheat leaf rust, increasing concern.
Wild relatives of common wheat contain a large number of desirable genes that can be exploited for wheat improvement . The discovery and utilization of the desirable genes from wild relatives is significant for the genetic improvement of wheat resistance. At present, 76 wheat leaf rust resistance genes have been named and identified in common wheat and its relatives (McIntosh et al., 2016;Bansal et al., 2017). Approximately half of the leaf rust resistance genes are derived from wild relatives of common wheat, including Aegilops L., Secale cereale L., and Elytrigia Desv.. Aegilops L. is the donor of 15 leaf rust resistance genes described in the Catalog of Wheat Gene Symbols, including Lr9, Lr21, Lr22a, Lr28, Lr29, Lr32, Lr35, Lr36, Lr37, Lr39, Lr42, Lr47, Lr51, Lr66, and Lr76 (Marais et al., 2010;Martynov et al., 2015;Bansal et al., 2017). Lr25 , Lr26 , and Lr45 (Friebe et al., 1996;Naik et al., 2015) were transferred from S. cereal L., and Lr19 (Sarma and Knott, 1966), Lr24 (Hart et al., 1976) and Lr38 were transferred from Elytrigia Desv. (Friebe et al., 1992(Friebe et al., , 1993Mebrate et al., 2007). These genes have played very important roles in wheat resistance breeding, but many of them have been defeated by new virulent races. Therefore, new and effective resistance genes constantly need to be explored and identified by breeders and researchers to combat the growth and spread of leaf rust disease.
Agropyron Gaertn., one of the important wild relatives of common wheat, is a useful genetic resource for wheat genetic improvement (Dewey, 1984;Dong et al., 1992;Lu et al., 2015). The exploitation of desirable genes from the P genome of Agropyron cristatum has mainly been reported by our laboratory. For example, A. cristatum chromosome 2P shows high resistance to powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) . A. cristatum chromosome 7P confers higher tolerance to drought compared with Fukuho . A. cristatum chromosome 6P contains genes conferring high numbers of kernels per spike genes (Wu et al., 2006;Han et al., 2014). Besides, it was also found that A. cristatum chromosome 6P carries leaf rust resistance gene(s). The leaf rust resistance gene(s) was mapped to the bin 6PS-0.81-1.00 using the BC 1 F 2 population of 6PS/6PL telosomics at the adult plant stage . Moreover, T. aestivum-A. cristatum 2P addition line was induced by 60 Co-γ ray and Aegilops cylindrical gametocidal chromosome 2C, leading to the creation of T. aestivum-A. cristatum 2P translocation lines and deletion lines with different chromosomal fragments and breakpoint positions . Then a physical map of A. cristatum chromosome 2P was constructed with specific STS (sequence-tagged site) markers . In addition, we previously showed that the T. aestivum-A. cristatum 2P addition lines are more resistant to leaf rust than the T. aestivum-A. cristatum 6P addition lines. We determined that T. aestivum-A. cristatum 2P addition line II-9-3 is not only highly resistant to powdery mildew but also shows good resistance to leaf rust.
In this study, T. aestivum-A. cristatum 2P disomic addition lines II-9-3 and translocation lines were inoculated with different races at the seedling stage to identify the resistance spectrum and availability of II-9-3 against leaf rust. Then, the BC 2 F 2 or BC 3 F 2 populations of T. aestivum-A. cristatum 2P translocation lines with different chromosomal segments were used to evaluate the resistance and genotype with molecular markers to map the novel locus to a specific region of chromosome 2P. These data provide a scientific basis for effectively using this novel leaf rust resistance locus in wheat improvement and basic research.

Collection of the Leaf Rust Isolates
This study used 50 leaf rust races, including two Chinese major dominant leaf rust races, named by THT and PHT, and other 48 different leaf rust races collected from different locations in Hebei, Henan, Shandong, Inner Mongolia, Jiangsu, Anhui, Hubei, Shaanxi, Xinjiang, Yunnan, Guizhou, Sichuan, and Chongqing of China in 2016 (Figure 1). The 50 races tested were identified with a total of 102 samples collected from the mentioned 11 provinces, 1 autonomous region and 1 municipality, and the detailed information was listed in Table 3. The 50 races were denominated using the Prt code system (Long and Kolmer, 1989) by Liu and Chen (2012

Identification of Leaf Rust Resistance at the Seedling Stage
Two Chinese major dominant races THT and PHT (Liu and Chen, 2012), and other 48 races were used for inoculation. The T. aestivum-A. cristatum 2P addition lines, the translocation lines and the backcross populations were infected as described by Liu and Chen (2012). The races were inoculated onto 7-day-old seedling leaves of wheat plants planted in pots with low-nutrient soils. After inoculation, the plants were placed in a dew chamber at 18 • C in the dark for 24 h. The inoculated wheat seedlings were then moved to a greenhouse at 24 • C. The infection types (ITs) on the first leaves were recorded when Zhengzhou5389 was fully rusted, 15 days after inoculation. A 0 to 4 scale was used for ITs, as described by Roelfs (1992). Compared to the normal IT, larger and smaller uredinia were indicated with plus and minus signs. Plants with ITs 0-2 were considered resistant to leaf rust, whereas plants with ITs 3-4 were considered susceptible to leaf rust. Leaf rust infections were carried out at the greenhouse of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

Identification of Leaf Rust Resistance at the Adult Stage
The procedures for leaf rust resistance at the adult stage in the field were carried out as described by Qi et al. (2016). In this study, T. aestivum cv. 'Zhengzhou5389' was used as the susceptible control. One row of Zhengzhou5389 was planted every tenth row of tested materials to aid the spread of the spores within the trial (Qi et al., 2016). Zhengzhou5389 was also planted in the spreader rows perpendicular and adjacent to the test rows. The race THT was used for inoculation. Leaf rust infections were established by the spraying method at the wheat jointing stage. The plants were sprayed with an aqueous suspension of uredinia spores of P. triticina containing 0.05% Tween 20. Then, the inoculated plants were covered with plastic film, and the film was removed after 16 h . ITs were recorded on the first leaf of each plant using a 0-4 scale when susceptible control Zhengzhou5389 were fully rusted. ITs were recorded in the same manner as the seedling stage.

Molecular Marker Analysis
Genomic DNA was extracted using a modified CTAB method (Kidwell and Osborn, 1992;Li et al., 2017). A total of 82 A. cristatum chromosome 2P-specific STS markers which were developed based on the A. cristatum transcriptome sequences  were mapped to 17 2P-specific bins, and the information of STS markers was described as Li et al. (2017). Of these, eight markers located on different 2P-specific bins were selected to detect the absence or presence of 2P chromosomal segments in the translocation lines and their backcross populations ( Table 4).
A. cristatum Z559 and II-9-3 were used as positive controls, whereas Fukuho and Zhengzhou5389 were used as negative controls.
The PCR amplification was performed as described by Li et al. (2017). The PCR amplification reactions were conducted in a total volume of 10 µl mixture containing 1.0 µl of template DNA (50 ng/µl), 1.0 µl of forward primer (2 µmol/l), 1.0 µl of reverse primer (2 µmol/l), 0.15 µl of 1 × Taq DNA polymerase (5 U/µl), 0.8 µl of MgCl 2 (25 mmol/l), 0.8 µl of dNTP (2.5 mmol/l), 1.0 µl of 10 × buffer, and 4.25 µl of ddH 2 O. The cycling conditions were as follows: 94 • C for 5 min followed by 38 cycles of 94 • C for 30 s, 59 • C for 30 s, and 72 • C for 30 s, followed by a final 10min extension at 72 • C. The amplified products were separated by electrophoresis on a polyacrylamide gel with an acrylamide concentration of 8% s and visualized by silver staining.

Leaf Rust Responses of the T. aestivum-A. cristatum 2P Addition Line and Translocation Lines in the Field
Triticum aestivum-A. cristatum 2P addition line II-9-3, the two backcross populations derived from the cross between 2PL translocation lines 2PT-3, 2PS translocation line 2PT-10 and Fukuho were planted in the nurseries in Xinxiang during 2014 and 2015. Fukuho was highly susceptible to leaf rust, whereas II-9-3 was highly resistant (Figure 2). According to the leaf rust resistance results and molecular marker analysis (Figure 3 and Table 1), in the 2PT-10 BC 1 F 2 populations, all progenies were susceptible to leaf rust, regardless of the presence or absence of 2P-specific STS markers, suggesting that the leaf rust resistance Frontiers in Plant Science | www.frontiersin.org Chongqing (   locus is not located on the 2PS arm. In the 2PT-3 BC 1 F 2 populations, all the progenies with 2P-specific STS markers were highly resistant to leaf rust, while other progenies of 2PT-3 without 2P-specific STS markers were as highly susceptible to leaf rust as Fukuho. The 2PT-3 translocation lines contained the whole 2PL arm, suggesting that the leaf rust resistance locus is located on the 2PL arm.

Evaluation of the Resistance Spectrum and Availability of the 2PL Arm Against Leaf Rust
During 2015 and 2016, the responses of the translocation lines 2PT-5(3BS.L-2PL(0.6-1)), 2PT-3(4DS.2PL), and T. aestivum-A. cristatum 2P addition line II-9-3 were 0;, and all plants were characterized as nearly immune to leaf rust. To evaluate the resistance spectrum of T. aestivum-A. cristatum 2P II-9-3 and 2PT-5, 102 leaf rust samples (Table 3), which collected from 11 provinces, 1 autonomous region and 1 municipality of China in 2016 were used to inoculate II-9-3, 2PT-5, Fukuho and Zhengzhou5389 at the seedling stage in the greenhouse. Fifty races were identified from 102 samples by the State Key Laboratory for Biology of Plant Diseases and Insect Pests. Among these races, PHK, PHT, PCH, PHJ, PHS, THT, and PCG were found in different provinces. II-9-3 and 2PT-5 were nearly immune (IT = 0) to all 50 races, whereas Fukuho and Zhengzhou5389 were highly susceptible (IT = 3 + ∼ 4) to all 50 races at the seedling stage (Figure 4 and Table 3). The disease  responses to duplicate races from different locations showed the same ITs. These results suggested that II-9-3 and 2PT-5 have broad-spectrum leaf rust resistance and may be useful for wheat disease resistance improvement.

Chromosomal Localization of Leaf Rust Resistance Locus on A. cristatum Chromosome 2P
To further pinpoint the chromosomal segment containing the leaf rust resistance locus, three BC 2 F 2 populations (2PT-5, 2PT-6, 2PT-8) and one BC 3 F 2 population (2PT-3) derived from four translocation lines with different chromosomal segments of 2PL were constructed ( Table 2). The major dominant races THT (Liu and Chen, 2012) was used to evaluate the leaf rust resistance of T. aestivum-A. cristatum 2P addition line II-9-3, Fukuho and the populations of 2PT-3, 2PT-5, 2PT-6, and 2PT-8 in the greenhouse and field, with Zhengzhou5389 used as the control. At the same time, the plants in each population were identified with the A. cristatum 2P-specific markers. According to the results of the molecular marker analysis (Figure 5) and the leaf rust responses of the four populations at the seedling stage (Figure 6 and Table 2), the responses of T. aestivum-A. cristatum 2P addition line II-9-3 was 0, and all plants were nearly immune to leaf rust. The responses of Fukuho was 4, and all plants were as highly susceptible as the susceptible control Zhengzhou5389. In the 2PT-3 BC 3 F 2 population, 142 positive plants were nearly immune (IT = 0) and 58 negative plants were highly susceptible (IT = 3 + ). In the 2PT-5 BC 2 F 2 population, 145 positive plants were nearly immune (IT = 0) and 55 negative plants were highly susceptible (IT = 3 + ). In the 2PT-6 BC 2 F 2 population, all of the 108 positive plants and 92 negative plants were highly susceptible (IT = 3 + ). In the 2PT-8 BC 2 F 2 population, 110 positive plants and 86 negative plants were also highly susceptible (IT = 3 + ). These results suggest that the novel leaf rust resistance locus of the T. aestivum-A. cristatum 2P translocation lines is located in the chromosomal bin FL 0.66-0.86 of 2PL. The four populations of translocation lines were assessed for leaf rust resistance at the adult plant stage in the field. The results for the disease response to the P. triticina at the adult stage (Figure 7 and Table 2), indicated that II-9-3 was nearly immune, whereas Fukuho was as highly susceptible as Zhengzhou5389 to leaf rust. In the 2PT-3 BC 3 F 2 population, 106 positive plants of the isolates of 2PT-3 were nearly immune (IT = 0) and 53 negative plants were highly susceptible (IT = 3 + ). In the 2PT-5 BC 2 F 2 population, 103 positive plants were nearly immune (IT = 0) and 50 negative plants were highly susceptible (IT = 3 + ). In the 2PT-6 BC 2 F 2 population, all of the 64 positive plants and 62 negative plants were highly susceptible (IT = 3 + ). In the 2PT-8 BC 2 F 2 population, 51 positive plants and 58 negative plants were also highly susceptible (IT = 3 + ). According to the evaluation of the leaf rust resistance of the BC 2 F 2 and BC 3 F 2 populations of the four T. aestivum-A. cristatum 2P translocation lines and their parents, II-9-3 and 2PT-5 were nearly immune to leaf rust at both the seedling and adult plant stages. Based on these results, the novel leaf rust resistance locus of T. aestivum-A. cristatum chromosome 2P is located in the chromosomal bin FL 0.66-0.86 of 2PL (Figure 8).
The leaf rust resistance locus is located in the same region as the powdery mildew resistance locus reported by Li et al. (2017). It is speculated that chromosomal bin FL 0.66-0.86 of 2PL carries two types of resistance loci: the leaf rust resistance locus and powdery mildew resistance locus.
Evaluation of the Resistance Locus in the Chromosomal Bin FL 0.66-0.86 of 2PL Recently, 2PT-3 was induced by 60 Co-γ ray, leading to the creation of thirty-seven 2PL translocation lines with different chromosomal fragments and breakpoint positions. According to the results of molecular marker analysis, leaf rust and powdery mildew responses, eleven 2PL translocation lines displays nearly immune to leaf rust and high susceptible to powdery mildew; ten 2PL translocation lines displays high susceptible to leaf rust and high resistance to powdery mildew. Based on these result, different locus are responsible for resistance to powdery mildew and leaf rust.

DISCUSSION
Wheat leaf rust generally occurs in the world's wheat-producing areas and is a major diseases in wheat affecting wheat production. Serious disease can significantly reduce yields. Recent increases  in temperature and precipitation have gradually increased the prevalence of wheat leaf rust in the main wheat-production areas in China, especially in southwestern and northwestern China, the middle and lower regions of the Yangtze River, and the southern parts of the Huang-Huai-Hai River regions (Huerta-Espino et al., 2011). Yield losses were recorded in the Henan, Sichuan, Anhui, Gansu, and Shaanxi provinces in China in the year 2012 (Zhou et al., 2013). Liu and Chen (2012) studied the race and virulence frequency of P. triticina in China, and found that THT, PHT, THT, PHJ, THJ, and PCG were the main races in the most provinces and wheat areas. They also found that only Lr9, Lr19, Lr24, Lr25, Lr28, and Lr29 were effective leaf rust resistance genes among the identified genes in China (Liu and Chen, 2012). Kolmer (2015) studied the leaf rust resistance genes and virulence of P. triticina from Sichuan, Hebei, Henan, Shandong, Anhui, Hubei, and Shanxi provinces of China. A total of 48 virulence phenotypes were detected and included FCBQQ, PCGLN, and PCGLL. Lr1, Lr2c, Lr3, Lr26, LrB, Lr10, Lr11, Lr3bg, Lr20, and Lr14b showed low disease resistance, indicating that the utilization value of these resistance genes was not significant. Most of the identified leaf rust resistance genes are race-specific resistance genes which easily to lose resistance in breeding (Kolmer, 2015). Because the disease continuously evolves and forms novel virulent races (Kolmer, 2005;Bolton et al., 2008), new varieties with different desirable genes are constantly needed to replace the varieties that have lost effectiveness (Herrera-Foessel et al., 2012). Therefore, mining novel resistance genes, breeding resistant cultivars by the utilization of leaf rust resistance genes, and using wheat cultivars with different resistance genes can extend the effective life of these genes and are of great significance to wheat breeding and production.
In this study, 50 races were collected from 11 provinces, 1 autonomous region and 1 municipality covering the main wheat-producing areas of the Huang-Huai-Hai River regions, the middle and lower regions of the Yangtze River, the northern and southwest parts of China. These races were used to inoculate plants to identify resistance locus. The novel leaf rust resistance locus were first confirmed in the T. aestivum-A. cristatum 2P addition line II-9-3 and 2PL translocation line 2PT-5 with broad-spectrum and strong availability. Lines of Thatcher wheat differing in the single leaf rust resistance genes Lr1, Lr2c, Lr3, Lr16, Lr26, Lr3ka, Lr11, Lr17, and Lr30 were resistant to19, 7, 12, 28, 5, 26, 25, 10, and 25 of the 50 tested races, respectively. Therefore, the novel leaf rust resistance locus exhibited a broader spectrum than the nine genes above. T. aestivum-A. cristatum chromosome 6P addition line 4844-12 was highly resistance to leaf rust at the adult plant stage and the resistance locus was finally located on chromosome arm 6PS . However, in the 6PS telosomic populations, the plants carrying A. cristatum chromosome 6P were not all resistance to leaf rust, in contrast to the resistance locus exploited in chromosome 2P in this study and this characteristic would affects the application of the gene from 6P in breeding. In conclusion, the novel leaf rust resistance locus have high utilization value and not only confer broad-spectrum resistance but also stronger and more stable than the resistance carried by A. cristatum chromosome 6P.
Many leaf rust resistance genes were found to have genetic linkages to genes conferring resistance to powdery mildew, stripe rust caused by P. striiformis f. sp. tritici (Pst) or stem rust caused by P. graminis Pers. f.sp. tritici. (Pgt) in wheat, such as Lr19/Sr25, Lr24/Sr24/Yr71 (McIntosh et al., 1977), Lr27/Sr2 (Mago et al., 2011), Lr34/Yr18/Pm38/Sr57 (Krattinger et al., 2009), Lr37/Yr17/Sr38 (Seah et al., 2001), Lr46/Yr29/Sr58/Pm39 (Rosewarne et al., 2006;Lillemo et al., 2008), Lr57/Yr40 (Kuraparthy et al., 2007), Lr62/Yr42 (Marais et al., 2009), and Lr67/Yr46/Sr55/Pm46 (Herrera-Foessel et al., 2014). The investigation and identification of leaf rust-resistance genes will allow these previously identified resistance genes to be used more effectively. Lr34/Yr18/Pm38/Sr57 is a single wheat gene that confers durable and partial adult plant resistance against leaf rust, stripe rust, powdery mildew, and stem rust (Krattinger et al., 2009). This gene has been widely used in wheat breeding for more than one hundred years, and no pathogen adaptation has been observed so far, indicating that wheat with durable resistance genes in wheat can effectively prolong the effectiveness of resistance genes for different diseases (Krattinger et al., 2009;Keller et al., 2015). In this study, leaf rust resistance locus was located in the chromosomal bin FL 0.66-0.86 of 2PL using translocation line backcross populations that also carried powdery mildew resistance locus . The leaf rust resistance locus, with broad-spectrum resistance, was high resistance to powdery mildew and nearly immune to leaf rust at both the seedling and adult stages. Chromosomal bin FL 0.66-0.86 of 2PL is probably an excellent gene segment with various disease resistance locus that are closely linked. T. aestivum-A. cristatum chromosome 2P translocation line 2PT-5, which is resistant to leaf rust and powdery mildew, is of great value for wheat resistance breeding.
Triticum aestivum-A. cristatum chromosome 2P translocation line 2PT-5 was not only showed nearly immune to wheat leaf rust and highly resistant to powdery mildew but also had many other desirable traits, such as a small and erect flag leaf, compact plant type, closely arranged spikelets, and long uppermost internode. T. aestivum-A. cristatum chromosome 2P translocation line 2PT-5 can be used in wheat breeding (submitted). Therefore, translocation line 2PT-5 is potentially useful for broadening the genetic basis of leaf rust resistance and provides valuable genetic material for wheat breeding. The combination of irradiation, backcrossing and other methods with the ph1b mutant will further delimit the chromosome 2P 0.66-0.86 segment smaller for the next step and even enable deeper study of leaf rust resistance gene(s) and powdery mildew gene(s).

AUTHOR CONTRIBUTIONS
WL conceived the research. BJ and TL performed the research. BJ wrote the paper. WL and HL created the translocation lines. LL created the T. aestivum-A. cristatum 2P addition line. HH, LL, JZ, XY, SZ, and XL participated in the preparation of the reagents and materials used in this study. All authors read and approved the manuscript.