Molecular mapping of leaf rust resistance genes in the wheat line Yu 356-9

Journal of Integrative Agriculture 2015, 14(7): 1223–1228 Available online at www.sciencedirect.com

ScienceDirect

RESEARCH ARTICLE

Molecular mapping of leaf rust resistance genes in the wheat line Yu 356-9 HAN Liu-sha1*, LI Zai-feng1*, WANG Jia-zhen1, SHI Ling-zhi1, ZHU Lin1, LI Xing1, LIU Da-qun1, Syed J A Shah2 1 2

College of Plant Protection, Agricultural University of Hebei, Baoding 071001, P.R.China Nuclear Institute for Food and Agricuture, Tarnab, Peshawar 25000, Pakistan

Abstract The Chinese wheat line Yu 356-9 exhibits a high level of resistance to leaf rust. In order to decipher the genetic base of resistance in Yu 356-9, gene postulation, inheritance analyses, and chromosome linkage mapping were carried out. Gene postulation completed using 15 leaf rust pathotypes and 36 isogenic lines indicated that Yu 356-9 was resistant to all pathotypes tested. F1 and F2 plants from the cross Yu 356-9 (resistant)/Zhengzhou 5389 (susceptible) were tested with leaf rust pathotype “FHNQ” in the greenhouse. Results indicated a 3:1 segregation ratio, indicative of the presence of a single dominant leaf rust resistance gene in Yu 356-9 which was temporarily designated as LrYu. Bulk segregant analysis and molecular marker assays were used to map LrYu. Five simple sequence repeat (SSR) markers on chromosome 2BS were found closely linked to LrYu. Among these markers, Xwmc770 is the most closely linked, with a genetic distance of 5.7 cM. Keywords: wheat, leaf rust, resistance gene, inheritance analyses, molecular mapping

1. Introduction Wheat is cultivated as a food crop throughout the world, and the wheat planting area in China is only slightly smaller than that of rice (Peng and He 2009). Among three rusts of wheat, leaf or brown rust caused by Puccinia triticina is the

Received 18 November, 2014 Accepted 4 February, 2015 HAN Liu-sha, Tel: +86-312-7528500, E-mail: [email protected]; Correspondence LI Xing, Mobile: +86-13513220265, Fax: +86312-7528500, E-mail: [email protected]; LIU Da-qun, Tel/Fax: +86-312-7528500, E-mail: [email protected] * These authors contributed equally to this study. © 2015, CAAS. All rights reserved. Published by Elsevier Ltd. doi: 10.1016/S2095-3119(14)60964-3

most widely distributed and can inflict 15% production losses and severe infection can result in losses up to 40% (Knott 1989; Kolmer 1996). Severe losses in wheat production were recorded due to leaf rust outbreaks during 1969, 1973, 1975 and 1979 in the Southwest China and some regions of the Yangtze River Basin, China (Dong 2001). During a recent leaf rust epidemic in 2012, large wheat acreage was affected in the Gansu Province of China. Development and deployment of resistant varieties has long been a preferred approach for preventing and controlling wheat leaf rust. More than 100 leaf rust resistance genes and alleles have been identified in wheat and 71 of these are officially named (Singh et al. 2013). However, a very limited number of these genes confer effective resistance to leaf rust in China. Therefore, it is essential to undertake regular screening for identifying novel rust resistance genes to breed and develop resistant wheat varieties. Various methods can be employed for the genetic analysis of wheat leaf rust resistance including gene postula-

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tion, and chromosome linkage mapping. The locations of several leaf rust resistance genes have been successfully determined using simple sequence repeat (SSR) markers including genes Lr13 (Messmer et al. 2000), Lr34 (Suenaga et al. 2001), Lr39 (Raupp et al. 2001), Lr46 (Suenaga et al. 2001), Lr50 (Brown et al. 2003), Lr37 (Blaszczyk et al. 2004), Lr19 (Li et al. 2005; Zhang et al. 2005), Lr52 (Hiebert et al. 2005), Lr45 (Zhang et al. 2007), Lr58 (Kuraparthy et al. 2007), and Lr61 (Herrera-Foessel et al. 2008). Two novel leaf rust resistance genes were identified and reported by our laboratory using SSR markers LrBi16 (in wheat variety Bimai16) and LrNJ97 (in Neijiang 977671) located on chromosomes 7B and 2B, respectively (Zhang et al. 2011; Zhou et al. 2013). The wheat genotype Yu 356-9 displays high resistance to leaf rust over sites and seasons but the genetics of resistance has not been reported. In the present study, the genetics of leaf rust resistance of Yu 356-9 was investigated and reported. The objectives of this study are to map the leaf rust resistance gene in Yu 356-9 using SSR markers.

2. Results 2.1. Gene postulation Results of leaf rust seedling screening are presented in Table 1. Yu 356-9 displayed resistance to pathotype PHJS, whereas leaf rust resistance genes including Lr1, Lr2C, Lr3, Lr16, Lr26, Lr11, Lr17, LrB, Lr10, Lr14a, Lr3bg, Lr13, Lr14b, Lr20, Lr21, Lr23, Lr33, Lr36, and Lr45 were susceptible to this pathotype. Thus, it was inferred that there is resistance in Yu 356-9 that is unique from these 19 known genes. Yu356-9 was also found resistant to pathotype TGTT which was virulent to Lr2a, Lr3ka, Lr30, Lr18, Lr2b, and Lr15. It was proposed that Yu 356-9 possesses resistance that is unique from these six known genes. Resistance in Yu 356-9 was also found to be different from Lr29 and Lr44 as Yu 356-9 was resistant to pathotype FHDS, whereas Lr29 and Lr44 were found susceptible. Leaf rust resistance genes including Lr9, Lr24, Lr19, Lr28, Lr39, Lr42, Lr47, Lr51, and Lr53 were found resistant to all tested pathotypes. Yu 356-9 displayed a reactive level of 2+ toward pathotype THJL; in contrast, the plants harboring Lr9, Lr28, and Lr47 had reactive levels of 0, and those with Lr9, Lr24, Lr39, Lr42, Lr51, and Lr53 exhibited reactive levels of 1. These infection types were lower than those observed for Yu 356-9, indicating that Yu 356-9 may possibly contain gene(s) different from the previously mentioned known genes or a widely effective gene combination.

2.2. Seedling resistance identification The resistant parent Yu 356-9, the susceptible parent

Zhengzhou 5389, and their F1 and F2 progeny seedling reactions following inoculation with P. triticina pathotype FHNQ are presented in Table 2. All 20 seedlings of the resistant parent Yu 356-9 were found resistant to pathotype FHNQ, while 20 seedlings of the susceptible parent displayed susceptible reactions to pathotype FHNQ. In the F2 generation, out of the tested 159 seedlings, 110 were found resistant while 49 were susceptible, which fit a single-locus segregation ratio (P=0.09) (Table 2). Thus, it was hypothesized that the leaf rust resistance of Yu 356-9 in response to pathotype FHNQ at the seedling stage is controlled by a single dominant gene.

2.3. Molecular marker screening and gene location A total of 1 082 SSR primers distributed across 21 chromosomes were screened between the two parents and the resistant and susceptible DNA pools. Five markers that were polymorphic between the parents and pools were selected (Xwmc25, Xbarc55, Xgwm148, Xgwm410, and Xwmc770); all five of these markers were distributed on chromosome 2BS. Thus, we preliminarily positioned the gene on chromosome 2BS and temporarily named it as LrYu.

2.4. Linkage analysis and molecular mapping Five pairs of primers that revealed polymorphisms between the parents and bulks were used for PCR amplification and electrophoresis detection of the same 159 F2 plants used in the seedling screening. Linkage with LrYu was evident, with genetic distances ranging from 5.7 to 28 cM; the most closely linked marker was Xwmc770, with a genetic distance of 5.7 cM (Fig. 1). A polyacrylamide gel image of Xwmc770 is shown in Fig. 2.

2.5. Temperature sensitivity test Genetic analysis and linkage mapping suggested that LrYu is positioned on chromosome 2BS near the Lr13 locus, which displays temperature sensitivity (Pretorius et al. 1984). Table 3 shows that LrYu also exhibited temperature-sensitive resistance and was resistant to all three strains. Yu 356-9 was most resistant at 18°C, whereas the plants harboring Lr13 were susceptible to all three of the pathotypes, suggesting that LrYu differs from Lr13.

3. Discussion 3.1. Preliminary positioning of LrYu The F2 progeny from crosses of Yu 356-9 and Zhengzhou 5389 were inoculated and studied in the greenhouse. The

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Table 1 Seedling infection types by 36 known leaf rust resistance genes and two varieties inoculated with 15 Chinese Puccinia triticina pathotypes Infection types to Puccinia triticina pathotypes1) PHJS MH JS FHNQ FGBQ FHBR FHBQ FGBR THJL FHDR FGDQ FHDS THJP TGTT PHGN THJC RL6003 Lr1 4 4 ; ; ; ; ; 4 0 ; 0 4 4 4 4 RL6016 Lr2a ; ; 1+ ; ; 1 1 3 ; ; 2 3 3 ; 4 RL6047 Lr2c 4 1 4 4 4 4 4 4 4 4 4 4 4 4 4 RL6002 Lr3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 RL6010 Lr9 ; ; ; ; 0; 0 0 ; 0 0 ; ; ; 0 ; RL6005 Lr16 4 4 4 4 4 4 3+ 3+ 4 4 4 4 4 3 4 RL6064 Lr24 ;1 ; ; ; ; ; ; ; ; ; ; ; ; ; ; RL6078 Lr26 4 4 4 1 4 4 ; 4 4 1 4 4 2 4 4 RL6007 Lr3ka X X ; ; ; ; 1 1 ; ; ; 1 4 ; X RL6053 Lr11 4 4 1 ; ; 1+ 2 3+ 1 1 2 4 3+ 4 4 RL6008 Lr17 4 3+ 3+ 2 2 2 2+ 4 3+ 4 4 4 4 2+ 4 RL6049 Lr30 3C 1 1 ; ; ; ; 1 ; ; ; ; 4 ; 1 RL6051 LrB 3+ 4 4 4 3+ 4 4 3+ 4 4 4 4 4 4 X RL6004 Lr10 3 3 4 4 4 4 4 2 4 4 4 2+ 4 1 X RL6013 Lr14a 4 4 X X X X X X X 2 4 4 4 3+ X RL6009 Lr18 1 1+ 2 2 4 2 4 1+ 4 2+ 2 4 3+ 3C 3 RL6019 Lr2b 1 0; 4 ; 3 3+ 2 4 3 3+ 3+ 2 4 3C 4 RL6042 Lr3bg 4 4 4 4 4 3+ 4 4 4 4 4 4 4 4 4 RL4031 Lr13 3 4 4 3 3 4 4 3 3 2 3+ 4 4 4 4 RL6006 Lr14b 4 4 4 4 4 4 4 4 4 4 4 X 4 X 4 RL6052 Lr15 1 ; ; ; ; ; ; 4 1 ; ; 4 3+ 4 4 RL6040 Lr19 0 0 ; 0 0 ; 0 0 0 0 ; 0 0 0 ; RL6092 Lr20 4 4 ; ; ; ; 0 ; ; ; ; 4 1 4 ; RL6043 Lr21 4 2 2 ; 2+ 3 2 ; 1 ; 1+ ; 3 1 1 RL6012 Lr23 4 4 4 3+ 3+ 4 3+ 1 4 4 4 4 4 3+ 4 RL6079 Lr28 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 RL6080 Lr29 0 0 0 0 ; 0 0 ; ; 0 3+ 4 ; 0 0 RL6057 Lr33 3 4 3+ 3+ 3+ 4 2+ 3C 3+ 3 4 4 4 3+ 3+ E84018 Lr36 4 2 1+ ; 2 2 1 1 2 2+ 3 2+ 3+ 2+ 3+ KS86 NGRC02 Lr39 ; ;1 ; ; ; ; ; ; ; ; ; ; ; ; ; KS91 WGRC11 Lr42 ; ; ; 0 0 ; ; ; 1 ; ; 0 ; 0 1 RL6147 Lr44 1 ; 4 4 4 4 4 1 4 4 4 ; 1+ ;1 1 RL614 Lr45 4 4 4 4 4 4 4 ; 4 4 4 4 ; ; ; PAVON76 Lr47 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 C78.5 Lr51 ; ; ; ; 1 ; 0 ; ; ; ; 0 ; ; ; -98M71 Lr53 ; 0 0 0 0 0 0 ; 0 0 0 0 0 0 0 Yu 356-9 LrYu 1+ 1+ ; 0 1 1 0 2+ ; 0 ; ; ; ; 2 Zhou 8425B LrZH84 4 3+ 2+ 0 3C 4 0 4 3+ 0 2 4 2 4 4 Zhengzhou 5389 4 4 4 4 3+ 4 4 4 4 4 4 4 4 4 4 Line

1)

Gene

Infection types (IT) are according to the 0–4 Stakman scale as modified by Roelfs et al. (1992). Uredinia somewhat larger than normal for the IT. The same as below.

results of chi-square test, confirmed a 3:1 segregation ratio, suggesting that leaf rust resistance at the seedling stage in Yu 356-9 is controlled by a single dominant gene. The resistance gene in the seedling stage was mapped to chromosome 2BS using SSR molecular markers; the closest molecular marker to LrYu is Xwmc770, with a distance of 5.7 cM.

3.2. Relationship with known genes A total of 71 leaf rust resistance genes have been identified to date. Among these genes, Lr13, Lr16, Lr23, Lr35, Lr48,

Lr50, and Lr58 are located on chromosome 2B. Wheat varieties harboring Lr13 constitute approximately 5.40% of China’s wheat production (Yuan and Chen 2011), and it has been confirmed that Lr13 is an adult resistance gene on wheat chromosome 2BS both in China and abroad. Although few Lr13-avirulent races can induce resistance in the seedling stage (Goyeau and Lannou 2011), resistance is observed in the three-leaf stage (Pretorius et al. 1984). Lr13 also confers high-temperature slow-rusting resistance (Chen and Qin 2002). The position of Lr13 is near that of LrYu. But the resistance of Lr13 is race specific. Lr13 is

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Table 2 Seedling phenotypes of Yu 356-9, Zhengzhou 5389, F1, and F2 lines inoculated with Chinese P. triticina race FHNQ Material

Total

Zhengzhou 5389 Yu 356-9 F1 plants F2 plants

20 20 20 159

1)

Infection types1) R S 0 20 20 0 20 0 110 49

Xwmc25

P cM 22.7 0.09

R, resistant; S, susceptible. LrYu 5.7

susceptible to the new races in recent years. Lr16 and Lr23 are located on 2BS and have largely lost the ability to confer resistance in China. Pathtype FHNQ used to map LrYu is virulent to Lr16 and Lr23 (Table 1). Lr35 and Lr48 are adult plant resistance genes. Lr35 is derived from Aegilops speltoides. Lr48 is a recessive resistance gene (Samsampour et al. 2010). Lr50 and Lr58 are major leaf rust resistance genes located on chromosome 2BL. LrYu on 2BS confers seedling leaf rust resistance. Thus, LrYu is different from these known leaf rust resistance genes. Identifying molecular markers closely linked to this gene will be important in efforts to enrich the disease-resistant gene pool, thereby providing a more abundant source of resistant breeding lines. The genetic analysis of leaf rust resistance in Yu 356-9 using molecular markers indicates that the leaf rust resistance of Yu 356-9 may be controlled by a single dominant gene, temporarily designated LrYu. According to the linkage map constructed by Somers et al. (2004), Xwmc770 is located on chromosome arm 2BS, and the results of linkage analysis show that the genetic distance between Xwmc770 and LrYu is 5.7 cM (Fig. 2). The genetic linkage map of LrYu indicates an association with four other SSR molecular markers (Xgwm410, Xgwm148, Xbarc55,

bp 123

M P1 P2 Br Bs R R

R R

Xwmc770

5.5

Xgwm410

4.5

Xgwm148

12.3 Xbarc55

2BS Fig. 1 Linkage map around the resistance gene LrYu constructed using simple sequence repeat (SSR) markers.

and Xwmc25), all of which are located on chromosome 2BS. Our results showed that LrYu is located on chromosome arm 2BS, and the positions of five SSR markers on the map are nearly identical to those proposed by Somers et al. (2004). Adult leaf rust resistance genes located on 2BS include Lr13, Lr35, and Lr48. As mentioned above, Lr13 confers temperature-sensitive leaf rust resistance. However, in the temperature-sensitivity experiments conducted at the seedling stage, the results obtained with LrYu were

R R R R R R S S S S

S S S S S S M

110 90 Fig. 2 Polyacrylamide gel electrophoresis of PCR products amplified with the SSR marker Xwmc770. The arrows indicate the specific bands of the parents and the F2 lines. P1 corresponds to the resistant parent Yu 356-9, P2 corresponds to the susceptible parent Zhengzhou 5389, Br indicates the resistant bulk, Bs indicates the susceptible bulk, R indicates the 10 resistant lines, S indicates the 10 susceptible lines, and M indicates pBR322/MspI marker.

Table 3 Infection types (FHDQ, THJP and FHDS) of wheat inoculated with three P. triticina pathotypes at three temperatures Wheat variety Yu356-9 Zhengzhou 5389 RL4031 (Lr13)

FHDQ ; 3+ X

18°C THJP 0 4 4

FHDS 0 4 3+

FHDQ ; 4 4

22°C THJP ; 4 4

FHDS ; 4 3+

FHDQ 1+ 3 Dry

25°C THJP ; 3 3

FHDS ; 3 3

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different from those obtained with Lr13; therefore, LrYu is different from Lr13. LrYu also differs from Lr35 and, unlike Lr48, is a dominant gene. Thus, it is inferred that LrYu is different from other known genes and represents a new resistance gene.

4. Conclusion Results from the F2 seedling resistance identification indicated that a single dominant gene conferred resistance to the wheat line Yu 356-9 in response to pathotype FHNQ and has been temporarily designated LrYu. Using molecular markers, LrYu was located on chromosome 2BS and closely linked to Xwmc770, with a genetic distance of 5.7 cM.

5. Materials and methods 5.1. Plant materials and P. triticina isolates The resistant parent Yu 356-9, the susceptible parent Zhengzhou 5389, their derived F1 and F2 plants developed previously were used for genetic analyses. For gene postulations, 36 leaf rust differentials (near isogenic lines) carrying known resistance genes in Thatcher wheat background were obtained from the International Maize and Wheat Improvement Center (CIMMYT), Mexico. The P. triticina pathotypes used were provided by the wheat leaf rust laboratory of Hebei Agricultural University, China. Pathotypes were previously collected from major wheat-growing areas in China, increased, maintained and characterized previously for virulence spectra using the coding system of Long and Kolmer (1989).

5.2. Seedling resistance analysis in the greenhouse Fifteen pathotypes of P. triticina were individually inoculated to host sets including Yu 356-9, Zhengzhou 5389 and leaf rust near-isogenic lines to examine the possible roles of known genes in test genotype Yu 356-9 by comparing its response with the near-isogenic lines. Seeds of F1 and F2 progeny and parents were planted in the greenhouse and after the expansion of the first leaf, inoculation was carried out using a sweep method. Host-pathogen interaction phenotypes were scored after two weeks of inoculation (Kuraparthy et al. 2007). The phenotypes were grouped into 6 classes (0, ;, 1, 2, 3, and 4) based on the presence of necrosis and the size of the pustules. Infection types 0–2 indicated host resistance, and infection types 3–4 indicated host susceptibility. The results were summarized and the resistance phenotypes were postulated using the methods proposed by Dubin et al. (1989).

5.3. Genomic DNA extraction and bulk construction

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Genomic DNA from F2 leaves of wheat seedlings and adult plants was extracted using CTAB dissolved in 1× TE and diluted to a final concentration of 30 ng μL–1. Based on phenotypic rust data from F2 plants, DNA of 10 each of highly resistant and highly susceptible plants were selected and mixed equally to form a resistant and susceptible DNA bulk samples (Gupta et al. 2005).

5.4. SSR analyses and electrophoresis detection A total of 1 082 SSR primers (Röder et al. 1998, http:// wheat.pw.usda.gov/cgi-bin/graingenes/browse.cgi) distributed across 21 wheat chromosomes were screened on the parents and bulk DNAs. The primers were synthesized by Shanghai Sangon Biological Engineering Technology and Services Co., Ltd. (China). PCR was performed in 10 μL volumes containing 1× PCR buffer, 0.2 mmol L–1 dNTP, 0.4 μmol L–1 primers, 3.0 μg mL–1 template DNA, 0.05 U of Taq polymerase (Zexing Biotechnology Co. Ltd., Beijing, China), and 6.7 μL of ddH2O. PCR program included: initial denaturation at 94°C for 5 min; 35 cycles of denaturation at 94°C for 1 min, annealing at 50, 55, or 60°C (according to the primer) for 1 min, and extension at 72°C for 1 min; and a final extension at 72°C for 10 min. The PCR products were then separated using 10% non-denaturing polyacrylamide gel electrophoresis, and the gel was visualized following silver nitrate staining (Bassam et al. 1991).

5.5. Linkage analysis and genetic mapping Genotyping of 159 F2 plants was completed using PCR amplification with markers which were polymorphic between the resistant and susceptible bulks and parents. Phenotypic data were combined with the results of PCR amplification for linkage analysis. MapManager QTXb20 software was used to calculate the genetic distances between the markers and resistance genes and to construct a genetic linkage map.

5.6. Temperature sensitivity experiment Yu 356-9, Zhengzhou 5389, and near-isogenic lines containing the known gene Lr13 were inoculated with three P. triticina pathotypes (FHDQ, THJP, and FHDS) at three temperatures (18, 20, and 25°C).

Acknowledgements This study was supported by the National Natural Science Foundation of China (International/Regional Cooperation and Exchange Program) (31361140367) and the Hebei Provincial Outstanding Youth Project, China (YQ2013024).

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