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Yr18 and Yr36 in Chinese Wheat Cultivars
Available online at www.sciencedirect.com
Journal of Genetics and Genomics 39 (2012) 587e592
JGG ORIGINAL RESEARCH
Distribution, Frequency and Variation of Stripe Rust Resistance Loci Yr10, Lr34/Yr18 and Yr36 in Chinese Wheat Cultivars Cuiling Yuan1, Hui Jiang1, Honggang Wang, Kun Li, Heng Tang, Xianbin Li, Daolin Fu* State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Shandong Agricultural University, 61 Daizong Street, Tai’an, Shandong 271018, China Received 8 February 2012; revised 25 September 2012; accepted 8 October 2012 Available online 15 October 2012
ABSTRACT Wheat stripe rust is a devastating disease in many regions of the world. In wheat, 49 resistance genes for stripe rust have been officially documented, but only three genes are cloned, including the race-specific resistance Yr10 candidate gene (Yr10CG) and slow-rusting genes Lr34/Yr18 (hereafter designated as Yr18) and Yr36. In this study, we developed gene-specific markers for these genes and used them to screen a collection of 659 wheat accessions, including 485 Chinese cultivars. Thirteen percent and eleven percent of the tested Chinese cultivars were positive for the markers for Yr10CG and Yr18RH (the resistant haplotype of Yr18), respectively, but none were positive for the Yr36 marker. Since there is a limited use of the Yr10 gene in Chinese wheat, the relatively high frequency of wheat varieties with the Yr10CG marker suggests that the identity of the Yr10 gene is unknown. With regards to the Yr18 gene, 29% of the tested cultivars that are used in the Middle and Lower Yangtze Valleys’ winter wheat zone were positive for Yr18RH markers. A non-functional allele of Yr18RH was identified in ‘Mingxian 169’, a commonly used susceptible check for studying stripe rust. The data presented here will provide useful information for marker-assisted selection for wheat stripe rust resistance. KEYWORDS: Puccinia striiformis; Stripe rust; Triticum aestivum
1. INTRODUCTION Wheat is one of the most important cereal crops worldwide. In China, wheat is second only to rice in production; the total yield of wheat is ca. 115 million metric tons per year. However, wheat production is threatened by wheat stripe rust (Puccinia striiformis f. sp. tritici, Pst). The causal agent Pst is an obligate biotrophic pathogen; its genome and transcriptome have recently become available (Cantu et al., 2011; Huang et al., 2011). Epidemics of the disease can reduce grain yield as high as 50% (Roelfs et al., 1992). In China, the disease in 1964, 1990 and 2002 caused yield losses of ca. 3.2, 1.8 and 1.3 million metric tons, respectively (Wan et al., 2007). * Corresponding author. Tel: þ86 538 824 9355. E-mail addresses: [email protected], [email protected] (D. Fu). 1 These authors contributed equally to this work.
To develop resistant cultivars is the most economical and environmentally friendly approach to control wheat stripe rust. In wheat, 49 officially named genes (Yr1 to Yr49) have been reported for stripe rust resistance (McIntosh et al., 2011). The Yr10 region on chromosome 1BS contains two nucleotide binding site-leucine rich repeat (NBS-LRR) genomic sequences 4B and 4E; genetic complementation suggested that the 4B sequence is the Yr10 candidate gene (Yr10CG) (Laroche et al., 2002). The Lr34/Yr18 complex on chromosome 7DS is effective against both leaf rust and stripe rust and confers moderate resistance to powdery mildew (Spielmeyer et al., 2005). Recent cloning of Lr34/Yr18 proved that the gene encodes a pleiotropic drug resistance (PDR)-like adenosine triphosphate-binding cassette (ABC) transporter (Krattinger et al., 2009). In the current study, Yr18 is used to designate the Lr34/Yr18 locus. Gene Yr36 encodes a novel kinase-START protein, which is characterized by the presence of the kinase domain and the
1673-8527/$ - see front matter Copyright Ó 2012, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Limited and Science Press. All rights reserved. http://dx.doi.org/10.1016/j.jgg.2012.03.005
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annealing (appropriate temperature, 30 s) and extension (72 C, appropriate time) with a final extension (72 C, 10 min) and a final holding (4 C, indefinite). PCR reactions were performed on a GeneAmpÒ 9700 Thermal Cycler (Applied Biosystems, Foster City, USA). PCR products were separated on either an agarose gel (1%e2%) or a 6% acrylamide gel. Diagnostic bands were scored as positive or negative to represent the presence and absence of the bands.
steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain (Fu et al., 2009). High levels of resistance to leaf and stripe rust in wheat have been achieved by pyramiding multiple resistance genes with small to intermediate additive effects (Singh et al., 2000; Castro et al., 2003). Yr18 and Yr36 provide non race-specific, durable resistance to wheat stripe rust. Pyramiding Yr18 and Yr36 genes in ‘Anza’ significantly enhanced resistance to stripe rust in comparison to Yr18 alone (Uauy et al., 2005). This suggests that pyramiding of partial resistance genes may provide an effective and sustainable control of wheat stripe rust. Up to date, the three known loci Yr10, Yr18 and Yr36 are still effective for stripe rust resistance. However, economic and robust molecular markers are lacking for these loci, and their deployment in Chinese wheat is largely unknown. The genespecific markers developed in this experiment enable us to profile the distribution and frequency of the genes of Yr10, Yr18 and Yr36 in Chinese wheat and to identify novel variations. Knowledge acquired in this study will be used for marker-assisted selection and gene pyramiding to improve stripe rust resistance in Chinese wheat.
Total RNA was extracted using the TRIZOL reagent (Invitrogen, Carlsbad, USA). The first strand cDNA was synthesized using the SuperScriptÔ First-Strand Synthesis System (Invitrogen). The RT-PCR products were cloned into the pMD18-T vector (TaKaRa, Dalian, China). The D allele of Yr18 was identified using the Yr18E11a marker (Table S2). Multiple alignments were performed using the ClustalW2 program maintained by the European Bioinformatics Institute (EMBL-EBI). The SIFT algorithm was used to examine putative effects of nonsynonymous mutation on protein function (Kumar et al., 2009).
2. MATERIALS AND METHODS
3. RESULTS
2.1. Plant materials
3.1. Yr10CG markers were detected in 13% of Chinese wheat cultivars
This study was conducted on 659 common wheat (Triticum aestivum L.), including 7 Chinese landraces, 485 Chinese cultivars, 147 Chinese breeding lines and 20 exotic genotypes. Chinese wheat genotypes were collected from 10 wheat growing zones (Table S1). About 79% (or 88% of the Chinese cultivars) was developed in or adapted to the Northern winter wheat zone (Zone I, 18% of the Chinese cultivars), the Yellow and Huai River Valleys’ winter wheat zone (Zone II, 40%), the Middle and Lower Yangtze Valleys’ winter wheat zone (Zone III, 16%), the Southwestern winter wheat zone (Zone V, 9%), and the Northwestern spring wheat zone (Zone VIII, 6%). 2.2. Gene-specific PCR markers and genotyping Gene sequences of Yr10CG, Yr18 and Yr36 were used to develop gene-specific PCR markers (Table S2). The Yr10CG specific markers were designed based on the sequences of Yr10CG (GenBank Accession No. AF149112), its nonfunctional duplication (AF149113) and other homologues. The Yr18 specific markers were developed using sequences from the resistant and susceptible haplotypes of the Yr18 alleles (FJ436983 and FJ436985). The Yr36 specific marker was designed to detect the WKS1 and WKS2 genes (EU835198). Details for each PCR marker are shown in Table S2. Wheat DNA was extracted using the Sarkosyl method (see Supplementary material). PCR was performed using GoTaqÒ DNA Polymerase (Promega, Madison, USA). In a 25 mL reaction, there were 1 PCR buffer, PCR primers (0.4 mmol/L each), dNTP (200 mmol/L each) and GoTaqÒ DNA Polymerase (1 U). PCR conditions included an initial denaturation (94 C, 5 min), multiple cycles of denaturation (94 C, 30 s),
2.3. cDNA cloning and sequence analysis
Close homologues of the Yr10CG gene exist in common wheat (GenBank Accession No. AF149113) and other Triticeae species (AF446141, AF509533, AF509534, AY613783, AY613785, AY613786 and EU428764). They share 80%e91% sequence identity to the published Yr10CG (AF149112). Therefore, gene specific PCR primers were designed based on the last 500 bp of Yr10CG coding region, which excludes the highly conserved region of the NBS-LRR gene family. Two dominant PCR makers, Yr10CGE2a and Yr10CGE2b, were developed to detect the Yr10CG gene in wheat germplasm (Fig. 1 and S1, Table S2). The PCR specificity was tested on isogenic lines of ‘Avocet S’, ‘Avocet R’ and ‘Avocet SþYr10’. Both Yr10CGE2a and Yr10CGE2b worked in Avocet SþYr10, but not in Avocet S and Avocet R (Fig. S2). The specificity of Yr10CGE2a and Yr10CGE2b was further confirmed by sequencing the PCR products from two Yr10 carriers including Avocet SþYr10 and Moro. In 659 wheat lines screened, 14% were positive for the Yr10CG markers; 13% of the advanced cultivars had the Yr10CG gene (Tables 1 and S3). Wheat cultivars positive for the Yr10CG markers were distributed over 10 wheat growing zones (Table 1). Within wheat growing zones I, II, III, V and VIII, the frequency of cultivars positive for the Yr10CG markers were 5%, 4%, 26%, 33% and 11%, respectively. Among cultivars positive for the Yr10CG marker, the fraction of growing zones I, II, III, V and VIII were 7%, 11%, 32%, 23% and 5%, respectively. Consequently, cultivars from wheat zones III and V have a higher frequency of the Yr10CG markers. A pedigree comparison suggested that the Yr10CG gene in more than 55% of Chinese wheat cultivars was derived from ‘Nanda 2419’, a selection of Italian wheat ‘Mentana’.
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1 was also present in other Yr10CG homologues (AF149112, AF149113, AF446141, AF509533, AF509534, AY613783, AY613785, AY613786 and EU428764), the G-to-T mutation (Val558Leu) in exon 2 was unique to Jiangdongmen. A diagnostic marker Yr10JDM was developed to detect the G-toT mutation, which was only detected in Jiangdongmen, ‘Baiquan 565’ and ‘Fufan 916’ (Fig. 2A and Table S2). According to the SIFT analysis, the Lys30Arg and Val558Leu substitutions would be tolerated, suggesting that the Yr10CG gene from Jiangdongmen might be functional. Semiquantitative RT-PCR revealed that the level of Yr10CG mRNA was comparable in Jiangdongmen and Moro, but seemed lower in Nanda 2419 than in the others (Fig. 3). 3.2. Yr18RH is present in 11% of Chinese wheat genotypes
Fig. 1. Detection of Yr10CG, Yr18, and Yr36 genes in seven wheat lines using the developed PCR markers. PCR products of Yr10CG and Yr36 genes were separated on 1%e2% agarose gels, while the separation of Yr18 products was made on the 6% acrylamide gel. Seven representative wheat lines are 98M71, Chinese Spring (CS), Nanda 2419 (ND), Fan 6, Moro, UC1041, and UC1041þYr36.
Some native wheat such as ‘Jiangdongmen’ also was positive for the Yr10CG markers. Genomic and cDNA sequences of Yr10CG were obtained from Jiangdongmen (HM231238 and HM231239), Nanda 2419 (HM461975 and HM461976), and Moro. The Yr10CG sequence (start to stop codons) from Nanda 2419 was identical to that of the Moro. In comparison, the Yr10CG cDNA from Jiangdongmen had two missense point mutations. Although the A-to-G mutation (Lys30Arg) in exon
Resistant and susceptible haplotypes of the Yr18 gene have been identified (Krattinger et al., 2009). The resistant haplotype of Yr18 (Yr18RH) is characterized by a 3-bp deletion (TTC) in exon 11 and a T-to-C point mutation (Tyr634His) in exon 12. The dominant marker Yr18E11a was designed to detect the 3-bp deletion (Fig. 1 and Table S2). To confirm that the 3-bp deletion involves the expected TTC sequence, lines with the Yr18E11a marker were also tested by the Yr18E11b that produced diagnostic band only in wheat lines without the TTC deletion (Fig. 1 and Table S2). Yr18E12a is a dominant marker for the resistant haplotype and detects the T-to-C point mutation (Fig. 1 and Table S2). Analysis of Yr18E11a and Yr18E12a markers in nulli-tetrasomic lines of ‘Chinese Spring’ demonstrated that the diagnostic bands are 7D specific (Fig. S2). Fifteen percent of all 660 lines or 11% of the 486 advanced cultivars had the Yr18RH allele (Tables 1 and S3). Wheat cultivars positive for the Yr18RH markers were distributed over all 10 wheat growing zones. Within wheat growing zones I, II, III, V and VIII, the frequencies of cultivars positive for the Yr18RH markers were 5%, 6%, 29%, 12% and 11%, respectively. Among cultivars positive for the Yr18RH marker, the
Table 1 Wheat cultivars positive for Yr10CG or Yr18RH markers Yr10CGa
Yr18RHb
No.
Wheat germplasmc
Positive
Negative
44
Aiganzao, Baiquan 565d, Chuan 533, Chuanmai 4, Chuanmai 28, Chuanyu 20, e 1161, Fan 6, Fengmai 2, Fengmai 13, Fufan 17, Fufan 24, Gahai 1, Ji’nan 13, Jiangdongmen, Jianyang 73, Jingchun 70-5321, Jinghong 9, Jurong 03, Kehan 4, Linmai 4, Longchun 11, Lumai 20, Mianyang 11, Mianyang 12, Mianyang 15, Mianyang 62-31, Nanyuan 1, Nongda 311, Pulin 5, Qingxuan 15, Rikaze 7, Sufu, Sumai 1, Wanpin 8029, Wuyimai, Xi’anshixinmai, Xichang 177, Xinyang 1, Zhongshan 2, Jubileina Ie, Mexipak 66e, Rieti 75e, Virgilioe
Negative
Positive
39
Bosu 16, Fufan 76, Gan 174, Huamai 7, Ji’nan 9, Jingzhou 1, Jinmai 7, Kechun 5, Kehan 9, Kejin 6, Longjian 9811, Miannong 4, Pingliang 32, Shan 229, Shuwan 8, Simai 2, Tianxuan 44, Wanmai 32, Wanyou (1), Weidong 1, Xiangyang 4, Yanda 24, Youaola, Youjing’ai, Youyimai, Yunshi 1, Zhen 7495, Zheng 6 fu, Zhengzhou 6, Zhengzhou 18, Zhengzhou 742, Zhengzhou 743, Zhengzhou 9285, Chengduguangtouf, Chinese Springf, Mazhamaif, Mingxian 169f, Pingyaoxiaobaimaif, Youzimaif
Positive
Positive
22
Bosu 1, e’mai 6, Fengkang 11, Fengqiang 7, Gan 162, Ganmai 7, Gui 83-7, Huazhong 6, Jingzhou 47, Nanda 2419, Nannongdaheimang, Neimai 14, Neixiang 19, Ningmai 1, Rikaze 8, Sumai 2, Sumai 3, Wangmai 17, Wangmai 19, Wannian 2, Xiangnong 3, Youxiang (2)
a d
Markers of Yr10CGE2a and Yr10CGE2b. b Markers of Yr18E11a and Yr18E12a. c Wheat germplasm includes cultivars, landraces, and exotic materials. Cultivars highlighted by underline have ‘Mentana/Ardito’ in their pedigree. e Exotic materials. f Landraces from China.
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Fig. 2. Genotyping unique alleles of Yr10CG and Yr18 genes. A: the 136 bp PCR marker Yr10JDM was developed to detect the point mutation in the Yr10CG coding region in Chinese wheat ‘Jiangdongmen’ (JDM). The 118 bp band indicates absence of the point mutation identified in JDM. B: PCR markers were developed to detect informative SNPs in exon 17 of Yr18RH in Chinese wheat ‘Mingxian 169’ (MX). Representative wheat genotypes included Jiangdongmen (JDM), Nanda 2419 (ND), Mingxian 169 (MX), and Chinese Spring (CS).
fraction of growing zones I, II, III, V and VIII were 7%, 22%, 40%, 9% and 6%, respectively. Clearly, cultivars from wheat zone III had the highest frequency of Yr18RH. Pedigree analysis suggested that Yr18RH in more than 38% of Chinese cultivars can be traced back to Nanda 2419. On the other hand, several native landraces carrying Yr18RH, such as ‘Chengduguangtou’, ‘Mazhamai’ and ‘Youzimai’, have been used as core germplasm in wheat breeding. ‘Mingxian 169’ has been widely used as a susceptible parent in genetic studies and as a susceptible check to test stripe rust resistance. However, it scored positively for Yr18RH markers. To identify the putative cause for lack of function, the Yr18 cDNA was isolated from Mingxian 169. In total, we sequenced five full-length cDNA clones representing the D genome. In comparison to Chinese Spring (FJ436983), the D allele in Mingxian 169 was found to have an alternative splicing (Fig. S3). For example, either intron 6 or 9 was present in cDNA clones (GU929206 and GU929207). Alternative splicing also occurred in exons; either the first 92 bp or 44 bp in exon 10 or 12, respectively, was absent in cDNA clones
(GU929206 and GU929207). However, cDNA clone A13 had a complete exon 12. Taken together, the coding sequence of Mingxian 169 has a 99% identity with the Yr18RH gene from Chinese Spring, but Mingxian 169 has a C-to-T point mutation (Leu913Phe) in exon 17. According to the SIFT analysis, the Leu913Phe mutation likely has negated the function of the Yr18RH allele in Mingxian 169. We also developed three PCR markers, Yr18E17a, Yr18E17b and Yr18E17c, to detect the frequency of the C-to-T mutation in exon 17 (Fig. 2B and Table S2). This mutation is unique to Mingxian 169 and is absent in other Yr18RH alleles in the current collection. Transcriptional analysis indicated that alternative splicing in Yr18 is common (Fig. S4). Chinese spring and other Triticeae lines (Aegilops tauschii, Hordeum vulgare, and T. turgidum ssp. Durum) had the expected splicing for Yr18. However, additional splicing patterns appeared to be present (Fig. S4). 3.3. Yr36 gene is not present in current Chinese wheat In the Yr36 region, there are two duplicated genes, Wheat Kinase-START 1 and 2 (WKS1 and WKS2), and WKS1 is the Yr36 gene (Fu et al., 2009). WKS1 and WKS2 encode 86% identical proteins. To investigate the presence of the Yr36 gene in Chinese wheat, we developed the PCR marker Yr36E1a (Fig. 1 and Table S2). YrR36E1a was designed based on the common sequence flanking the kinase domain in both WKS1 and WKS2 genes. Screening of the current collection suggested that the WKS genes were absent in Chinese common wheat. 4. DISCUSSION 4.1. Gene-specific markers are developed for known resistance loci to wheat stripe rust The preferred method for controlling wheat stripe rust is utilizing the host resistance. So far, two non race-specific resistance genes, Yr18 and Yr36, and one race-specific resistance candidate gene Yr10CG have been cloned. Gene-specific markers have been reported for each of them (Temel et al., 2008; Fu et al., 2009; Lagudah et al., 2009; Singh et al., 2009; Dakouri et al., 2010). However, there are certain disadvantages associated with the existing markers. For example, the Yr10CG markers appear to be less specific when compared to other Yr10CG homologues (Fig. S1). Markers of the Yr18 gene either requires specialized instrument (Dakouri et al., 2010) or relies on expensive restriction enzyme (Lagudah et al., 2009). Although four sets of PCR markers are available for the Yr36 gene (Fu et al., 2009), a single robust PCR marker is necessary for marker-assisted selection. Here, we developed new diagnostic PCR markers for all three genes and used them to genotyping the current collection on Chinese wheat. 4.2. Molecular function of the Yr10CG gene is poorly understood
Fig. 3. Transcriptional analysis of Yr10CG using two markers. Semi-quantitative RT-PCR was used to assess the Yr10CG mRNA in three representative wheat genotypes with Yr10CG, Jiangdongmen (JDM), Nanda 2419 (ND), and Moro.
The Yr10 locus was first identified in a Turkish wheat line, PI 178383, which was further used as the Yr10 donor to developing wheat cultivars such as Moro, ‘Crest’ and ‘Jacman’ (Line and
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Qayoum, 1991). Unfortunately, the Yr10 gene in Moro remained effective for only 3 years in the U.S. Pacific Northwest (Sharp and Volin, 1970). However, the Yr10 gene is still considered to be effective in many wheat growing regions in the world. The Yr10CG gene encodes a classic NBS-LRR protein. Singh et al. (2009) reported that markers derived from the Yr10CG sequence (GenBank accession No. AF149112) are completely linked to resistance to the rust pathotype 46S119. In the present study, Yr10CGE2a and Yr10CGE2b were designed to amplify the last 500 bp, the most variable region in the gene. By choosing unique primer ends, Yr10CGE2a and Yr10CGE2b markers are more specific for the Yr10CG gene than previously published Yr10 markers (Fig. S1). When the Yr10CGE2a sequence was used to blast the Chinese Spring genomic sequence (version 2011.06, http://www.cerealsdb.uk. net), the closest homologue was F676MBK02FZL6K which is a NBS-LRR-like gene from Ae. tauschii (AY613785). Yr10CG from Moro and the GenBank entry AY613785 share 91% identity in the predicted coding region. In the current collection, Yr10CG markers were detected in 62 (13%) Chinese cultivars including Nanda 2419 and ‘Fan 6’. Surprisingly, 34 of them can be traced back to Nanda 2419 or ‘Mentana/Ardito’ lineage. ‘Ardito’ and ‘Mentana’ are founder cultivars for Yr18 germplasm in North/South America wheat and European winter wheat, respectively (Kolmer et al., 2008). Nanda 2419 is a core germplasm in wheat breeding in China; more than one hundred wheat cultivars have been developed from Nanda 2419. Moro and Nanda 2419 are identical on the Yr10CG gene; however, the mRNA level of the Yr10CG gene is lower in Nanda 2419 than in Moro, and the Yr10 carriers Moro and PI 178383 have a different resistance spectrum than Nanda 2419 (Qi et al., 2002). Whether the expression of Yr10CG gene affects the resistance level remains to be investigated. In this study, Pst races CYR29, CYR32, and Su-14 were inoculated on the seedling plants of selected cultivars. Cultivars with the Yr10CG gene displayed varying infection types from susceptible to fully resistant (Fig. S5). Because a greater percentage of Chinese cultivars have the Yr10CG but not the apparent resistance of Yr10, it remains inconclusive that the Yr10CG is Yr10. 4.3. Yr18 gene has been a long-standing resistance resource widely-deployed in Chinese wheat The Yr18 gene encodes a PDR-like ABC transporter. The slow rusting resistance conferred by the YrI8 gene reduces grain yield loss by 36%e58% under high disease pressure (Ma and Singh, 1996). PCR markers are developed to identify the resistant and susceptible alleles of Yr18 gene (Lagudah et al., 2009; Dakouri et al., 2010). New PCR markers, Yr18E11a and Yr18E12a, amplify Yr18 genes from D and other genomes, but the diagnostic bands are D genome specific (Fig. S2). The Yr18E11a marker can be easily scored by running a 6% acrylamide gel, and the Yr18E12a marker uses a less expensive restriction enzyme (Nla III). Dakouri et al. (2010) studied the distribution of the resistant haplotype on the Yr18 gene in 700 wheat lines, including 337 lines from various geographical areas of the world and 363 lines
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mostly from North America. In average, about 26% of the screened germplasm are positive for the Yr18RH gene. In the current collection, 11% of the cultivars have the resistant haplotype on the Yr18 gene. Seedling tests suggested that the Yr18RH gene alone might not provide adequate resistance to stripe rust (Fig. S5). However, cultivars negative for Yr10CG and Yr18RH markers also showed different infection types (Fig. S5). In addition, a non-functional Yr18RH allele was found in Mingxian 169. The Leu913Phe substitution might account for the loss-of-function of the Yr18RH allele in Mingxian 169. In other studies, allelic diversities also occurred in exon 10 or exon 22. The cultivar ‘Invader’ has an additional A in exon 10, which causes a frameshift in mRNA (Dakouri et al., 2010); the cultivar ‘Jagger’ has a G-to-T point mutation in exon 22, which results in a premature stop codon (Lagudah et al., 2009). Chinese wheat is amongst the earliest documented sources of Lr34/Yr18 (Dyck, 1977). In particular, the Yr18RH gene carrier Chinese Spring was developed in the early last century. In China, several Yr18RH carrier landraces, such as ‘Chengduguangtou’, ‘Mazhamai’ and ‘Youzimai’, have been used as core germplasm in wheat breeding, generating over 100 cultivars. On the other hand, Nanda 2419 is positive for the Yr10CG and Yr18RH markers. Of the 22 Chinese wheat cultivars positive for both markers, 15 have Nanda 2419 in their pedigrees. Therefore, Yr18RH in many Chinese wheat cultivars originated from the ‘Mentana/Ardito’ lineage. Yr18 has higher expression in adult plants than in seedlings (Krattinger et al., 2009). In adult leaves, the correctly spliced Yr18 product accumulates in both leaf bases and leaf tips, however the unspliced transcripts appears to be predominant in leaf tips (Krattinger et al., 2009). Our study proved that the Yr18 gene has alternative splicing sites and there is enrichment of certain transcriptional variants during plant development. 4.4. Yr36 gene represents a novel source for stripe rust resistance in China Wild emmer wheat (T. dicoccoides) is the progenitor of modern tetraploid and hexaploid cultivated wheat. So far, four stripe rust resistance genes, including YrH52, Yr15, Yr35 and Yr36, have been identified in this species. Yr36 confers resistance to all tested Pst races in the U.S., including PST-43, 45, 78, 98, 100, 101, 111, 112, 113 and 116 under high temperatures (e.g. 25 C) (Fu et al., 2009). Excepting PST-43 and PST-45, the other races were found after 2000 representing the most predominant races from 2000 to 2005 in the U.S. (Chen, 2007). In China, Yr36 is effective against current prevalent races in the northwestern region of Sichuan Province. In a previous study, PCR markers were developed to detect the kinase domain, the START domain, and the internal region between the kinase and the START domains (Fu et al., 2009). In all their screened materials, the primers from each target region gave identical results. Because it appeared that a single PCR marker could be used to detect the presence of WKS genes, we designed PCR primers for marker Yr36E1a to the kinase domain region. The Yr36E1a marker was based on the common sequence in the WKS1 and WKS2 genes; the PCR primers were
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targeted to the less conserved sequence flanking the kinase domain, increasing the specificity and reliability of PCR. Both primers reside on one single exon, reducing the chance of PCR failure due to large insertions or deletions in introns. In a previous study, Yr36 was found only in the wild species T. turgidum spp. dicoccoides, but not in the accessions of three related populations: domesticated emmer, cultivated durum and common wheat (Fu et al., 2009). Genomic search revealed that the Yr36 gene/ortholog does not exist in Chinese Spring, and unlikely to exist in all other common wheat. In the present study, we further confirmed the lack of Yr36 in all tested Chinese common wheat. Because Yr36 was lost during wheat domestication, the gene has not been widely used in wheat breeding. Only a few wheat genotypes that were selected for the high grain protein content gene have the Yr36, such as ‘Glupro’, ‘Lassik’, ‘Farnum’ and ‘PI 638740’ (Uauy et al., 2005; Fu et al., 2009). In China, the Yr36 gene has been used in wheat breeding since 2008, mainly in the Sichuan and Shandong Provinces. At present, the major donor for Yr36 is breeding lines ‘UC1041þYr36’ and ‘Yecora RojoþYr36’. ACKNOWLEDGEMENTS We thank Dr. Lynn Epstein (University of California, USA) and Dr. Xianming Chen (Washington State University, USA) for their critical reading of the manuscript. This work was supported by the China Research and Development Initiative on Genetically Modified Plants (Grant No. 2009ZX08009053B), the National Basic Research Program of China (973 Program, Grant No. 2011CB100700), and the National Natural Science Foundation of China (Grant No. 30871323). SUPPLEMENTARY DATA Table S1. Wheat growing zones of China. Table S2. PCR markers used for germplasm screening. Table S3. Wheat germplasm negative for Yr10CG, Yr18RH and Yr36 markers. Fig. S1. Primer specificity of Yr10CG markers. Fig. S2. Specificity of Yr10CG and Yr18RH markers. Fig. S3. Sequence alignment of transcribed/predicted cDNAs of the Yr18 gene. Fig. S4. Alternative splicing of the Yr18 gene. Fig. S5. Stripe rust resistance in eight representative wheat lines. Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.jgg.2012.03.005. REFERENCES Cantu, D., Govindarajulu, M., Kozik, A., Wang, M., Chen, X., Kojima, K.K., Jurka, J., Michelmore, R.W., Dubcovsky, J., 2011. Next generation sequencing provides rapid access to the genome of Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust. PLoS ONE 6, e24230. Castro, A.J., Chen, X.M., Hayes, P.M., Johnston, M., 2003. Pyramiding quantitative trait locus (QTL) alleles determining resistance to barley stripe rust: effects on resistance at the seedling stage. Crop Sci. 43, 651e659. Chen, X.M., 2007. Challenges and solutions for stripe rust control in the United States. Aust. J. Agric. Res. 58, 648e655.
Dakouri, A., McCallum, B.D., Walichnowski, A.Z., Cloutier, S., 2010. Finemapping of the leaf rust Lr34 locus in Triticum aestivum (L.) and characterization of large germplasm collections support the ABC transporter as essential for gene function. Theor. Appl. Genet. 121, 373e384. Dyck, P.L., 1977. Genetics of leaf rust reaction in three introductions of common wheat. Can. J. Genet. Cytol. 19, 711e716. Fu, D., Uauy, C., Distelfeld, A., Blechl, A., Epstein, L., Chen, X.M., Sela, H., Fahima, T., Dubcovsky, J., 2009. A kinase-start gene confers temperaturedependent resistance to wheat stripe rust. Science 323, 1357e1360. Huang, X., Chen, X., Coram, T., Wang, M., Kang, Z., 2011. Gene expression profiling of Puccinia striiformis f. sp. tritici during development reveals a highly dynamic transcriptome. J. Genet. Genomics 38, 357e371. Kolmer, J.A., Singh, R.P., Garvin, D.F., Viccars, L., William, H.M., HuertaEspino, J., Ogbonnaya, F.C., Raman, H., Orford, S., Bariana, H.S., Lagudah, E.S., 2008. Analysis of the Lr34/Yr18 rust resistance region in wheat germplasm. Crop Sci. 48, 1841e1852. Krattinger, S.G., Lagudah, E.S., Spielmeyer, W., Singh, R.P., HuertaEspino, J., McFadden, H., Bossolini, E., Selter, L.L., Keller, B., 2009. A putative ABC transporter confers durable resistance to multiple fungal pathogens in wheat. Science 323, 1360e1363. Kumar, P., Henikoff, S., Ng, P.C., 2009. Predicting the effects of coding nonsynonymous variants on protein function using the SIFT algorithm. Nat. Protoc. 4, 1073e1082. Lagudah, E.S., Krattinger, S.G., Herrera-Foessel, S., Singh, R.P., HuertaEspino, J., Spielmeyer, W., Brown-Guedira, G., Selter, L.L., Keller, B., 2009. Gene-specific markers for the wheat gene Lr34/Yr18/Pm38 which confers resistance to multiple fungal pathogens. Theor. Appl. Genet. 119, 889e898. Laroche, A., Eudes, F., Frick, M.M., Huel, R., Nykiforuk, C.L., Conner, R.L., Kuzyk, A., Acharya, S., Jordan, M., 2002. A wheat resistance gene against stripe rust. Can. J. Plant Pathol. 24, 506. Line, R.F., Qayoum, A., 1991. Virulence, aggressiveness, evolution and distribution of races of Puccinia striiformis (the cause of stripe rust of wheat) in North America, 1968e1987. In United States Department of Agriculture e Technical Bulletin No. 1788, Washington, USA. Ma, H., Singh, R.P., 1996. Contribution of adult plant resistance gene Yrl8 in protecting wheat from yellow rust. Plant Dis. 80, 66e69. McIntosh, R.A., Dubcovsky, J., Rogersm, W.J., Morris, C.F., Appels, R., Xia, X.C., 2011. Catalogue of Gene Symbols for Wheat: 2011 Supplement. Annual Wheat Newsletter 57, 303e321. Qi, Q., Niu, Y., Wan, A., Wu, L., 2002. Cluster analysis of seventy-eight important wheat cultivars for stripe rust resistance in seedling stage. Acta Phytophylacica Sinica 29, 210e216. Roelfs, A.P., Singh, R.P., Saari, E.E., 1992. Rust Diseases of Wheat: Concept and Methods of Disease Management. CIMMYT, Mexico. Sharp, E.L., Volin, R.B., 1970. Additive genes in wheat conditioning resistance to stripe rust. Phytopathology 60, 1146e1147. Singh, R., Datta, D., Priyamvada, Singh, S., Tiwari, R., 2009. A diagnostic PCR based assay for stripe rust resistance gene Yr10 in wheat. Acta Phytopathol. Entomol. Hung. 44, 11e18. Singh, R.P., Nelson, J.C., Sorrells, M.E., 2000. Mapping Yr28 and other genes for resistance to stripe rust in wheat. Crop Sci. 40, 1148e1155. Spielmeyer, W., McIntosh, R.A., Kolmer, J., Lagudah, E.S., 2005. Powdery mildew resistance and Lr34/Yr18 genes for durable resistance to leaf and stripe rust cosegregate at a locus on the short arm of chromosome 7D of wheat. Theor. Appl. Genet. 111, 731e735. Temel, A., S‚entu¨rk-Akfirat, F., Ertugrul, F., Yumurtaci, A., Aydin, Y., Talas¨ ., Belen, S., Ogras‚, T., Go¨zu¨kirmizi, N., Bolat, N., Yorgancilar, O ¨ zdemir, E., C Yildirim, M., C ¸ akmak, M., O ¸ etin, L., Mert, Z., Sipahi, H., Albustan, S., Akan, K., Du¨s‚u¨nceli, F., Uncuoglu, A.A., 2008. Yr10 gene polymorphism in bread wheat varieties. Afr. J. Biotechnol. 7, 2328e2332. Uauy, C., Brevis, J.C., Chen, X.M., Khan, I.A., Jackson, L., Chicaiza, O., Distelfeld, A., Fahima, T., Dubcovsky, J., 2005. High-temperature adult plant (HTAP) stripe rust resistance gene Yr36 from Triticum turgidum ssp. dicoccoides is closely linked to the grain protein content locus Gpc-B1. Theor. Appl. Genet. 112, 97e105. Wan, A.M., Chen, X.M., He, Z.H., 2007. Wheat stripe rust in China. Aust. J. Agric. Res. 58, 605e619.
Journal of Genetics and Genomics 39 (2012) 587e592
JGG ORIGINAL RESEARCH
Distribution, Frequency and Variation of Stripe Rust Resistance Loci Yr10, Lr34/Yr18 and Yr36 in Chinese Wheat Cultivars Cuiling Yuan1, Hui Jiang1, Honggang Wang, Kun Li, Heng Tang, Xianbin Li, Daolin Fu* State Key Laboratory of Crop Biology, Shandong Key Laboratory of Crop Biology, Shandong Agricultural University, 61 Daizong Street, Tai’an, Shandong 271018, China Received 8 February 2012; revised 25 September 2012; accepted 8 October 2012 Available online 15 October 2012
ABSTRACT Wheat stripe rust is a devastating disease in many regions of the world. In wheat, 49 resistance genes for stripe rust have been officially documented, but only three genes are cloned, including the race-specific resistance Yr10 candidate gene (Yr10CG) and slow-rusting genes Lr34/Yr18 (hereafter designated as Yr18) and Yr36. In this study, we developed gene-specific markers for these genes and used them to screen a collection of 659 wheat accessions, including 485 Chinese cultivars. Thirteen percent and eleven percent of the tested Chinese cultivars were positive for the markers for Yr10CG and Yr18RH (the resistant haplotype of Yr18), respectively, but none were positive for the Yr36 marker. Since there is a limited use of the Yr10 gene in Chinese wheat, the relatively high frequency of wheat varieties with the Yr10CG marker suggests that the identity of the Yr10 gene is unknown. With regards to the Yr18 gene, 29% of the tested cultivars that are used in the Middle and Lower Yangtze Valleys’ winter wheat zone were positive for Yr18RH markers. A non-functional allele of Yr18RH was identified in ‘Mingxian 169’, a commonly used susceptible check for studying stripe rust. The data presented here will provide useful information for marker-assisted selection for wheat stripe rust resistance. KEYWORDS: Puccinia striiformis; Stripe rust; Triticum aestivum
1. INTRODUCTION Wheat is one of the most important cereal crops worldwide. In China, wheat is second only to rice in production; the total yield of wheat is ca. 115 million metric tons per year. However, wheat production is threatened by wheat stripe rust (Puccinia striiformis f. sp. tritici, Pst). The causal agent Pst is an obligate biotrophic pathogen; its genome and transcriptome have recently become available (Cantu et al., 2011; Huang et al., 2011). Epidemics of the disease can reduce grain yield as high as 50% (Roelfs et al., 1992). In China, the disease in 1964, 1990 and 2002 caused yield losses of ca. 3.2, 1.8 and 1.3 million metric tons, respectively (Wan et al., 2007). * Corresponding author. Tel: þ86 538 824 9355. E-mail addresses: [email protected], [email protected] (D. Fu). 1 These authors contributed equally to this work.
To develop resistant cultivars is the most economical and environmentally friendly approach to control wheat stripe rust. In wheat, 49 officially named genes (Yr1 to Yr49) have been reported for stripe rust resistance (McIntosh et al., 2011). The Yr10 region on chromosome 1BS contains two nucleotide binding site-leucine rich repeat (NBS-LRR) genomic sequences 4B and 4E; genetic complementation suggested that the 4B sequence is the Yr10 candidate gene (Yr10CG) (Laroche et al., 2002). The Lr34/Yr18 complex on chromosome 7DS is effective against both leaf rust and stripe rust and confers moderate resistance to powdery mildew (Spielmeyer et al., 2005). Recent cloning of Lr34/Yr18 proved that the gene encodes a pleiotropic drug resistance (PDR)-like adenosine triphosphate-binding cassette (ABC) transporter (Krattinger et al., 2009). In the current study, Yr18 is used to designate the Lr34/Yr18 locus. Gene Yr36 encodes a novel kinase-START protein, which is characterized by the presence of the kinase domain and the
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annealing (appropriate temperature, 30 s) and extension (72 C, appropriate time) with a final extension (72 C, 10 min) and a final holding (4 C, indefinite). PCR reactions were performed on a GeneAmpÒ 9700 Thermal Cycler (Applied Biosystems, Foster City, USA). PCR products were separated on either an agarose gel (1%e2%) or a 6% acrylamide gel. Diagnostic bands were scored as positive or negative to represent the presence and absence of the bands.
steroidogenic acute regulatory protein (StAR)-related lipid transfer (START) domain (Fu et al., 2009). High levels of resistance to leaf and stripe rust in wheat have been achieved by pyramiding multiple resistance genes with small to intermediate additive effects (Singh et al., 2000; Castro et al., 2003). Yr18 and Yr36 provide non race-specific, durable resistance to wheat stripe rust. Pyramiding Yr18 and Yr36 genes in ‘Anza’ significantly enhanced resistance to stripe rust in comparison to Yr18 alone (Uauy et al., 2005). This suggests that pyramiding of partial resistance genes may provide an effective and sustainable control of wheat stripe rust. Up to date, the three known loci Yr10, Yr18 and Yr36 are still effective for stripe rust resistance. However, economic and robust molecular markers are lacking for these loci, and their deployment in Chinese wheat is largely unknown. The genespecific markers developed in this experiment enable us to profile the distribution and frequency of the genes of Yr10, Yr18 and Yr36 in Chinese wheat and to identify novel variations. Knowledge acquired in this study will be used for marker-assisted selection and gene pyramiding to improve stripe rust resistance in Chinese wheat.
Total RNA was extracted using the TRIZOL reagent (Invitrogen, Carlsbad, USA). The first strand cDNA was synthesized using the SuperScriptÔ First-Strand Synthesis System (Invitrogen). The RT-PCR products were cloned into the pMD18-T vector (TaKaRa, Dalian, China). The D allele of Yr18 was identified using the Yr18E11a marker (Table S2). Multiple alignments were performed using the ClustalW2 program maintained by the European Bioinformatics Institute (EMBL-EBI). The SIFT algorithm was used to examine putative effects of nonsynonymous mutation on protein function (Kumar et al., 2009).
2. MATERIALS AND METHODS
3. RESULTS
2.1. Plant materials
3.1. Yr10CG markers were detected in 13% of Chinese wheat cultivars
This study was conducted on 659 common wheat (Triticum aestivum L.), including 7 Chinese landraces, 485 Chinese cultivars, 147 Chinese breeding lines and 20 exotic genotypes. Chinese wheat genotypes were collected from 10 wheat growing zones (Table S1). About 79% (or 88% of the Chinese cultivars) was developed in or adapted to the Northern winter wheat zone (Zone I, 18% of the Chinese cultivars), the Yellow and Huai River Valleys’ winter wheat zone (Zone II, 40%), the Middle and Lower Yangtze Valleys’ winter wheat zone (Zone III, 16%), the Southwestern winter wheat zone (Zone V, 9%), and the Northwestern spring wheat zone (Zone VIII, 6%). 2.2. Gene-specific PCR markers and genotyping Gene sequences of Yr10CG, Yr18 and Yr36 were used to develop gene-specific PCR markers (Table S2). The Yr10CG specific markers were designed based on the sequences of Yr10CG (GenBank Accession No. AF149112), its nonfunctional duplication (AF149113) and other homologues. The Yr18 specific markers were developed using sequences from the resistant and susceptible haplotypes of the Yr18 alleles (FJ436983 and FJ436985). The Yr36 specific marker was designed to detect the WKS1 and WKS2 genes (EU835198). Details for each PCR marker are shown in Table S2. Wheat DNA was extracted using the Sarkosyl method (see Supplementary material). PCR was performed using GoTaqÒ DNA Polymerase (Promega, Madison, USA). In a 25 mL reaction, there were 1 PCR buffer, PCR primers (0.4 mmol/L each), dNTP (200 mmol/L each) and GoTaqÒ DNA Polymerase (1 U). PCR conditions included an initial denaturation (94 C, 5 min), multiple cycles of denaturation (94 C, 30 s),
2.3. cDNA cloning and sequence analysis
Close homologues of the Yr10CG gene exist in common wheat (GenBank Accession No. AF149113) and other Triticeae species (AF446141, AF509533, AF509534, AY613783, AY613785, AY613786 and EU428764). They share 80%e91% sequence identity to the published Yr10CG (AF149112). Therefore, gene specific PCR primers were designed based on the last 500 bp of Yr10CG coding region, which excludes the highly conserved region of the NBS-LRR gene family. Two dominant PCR makers, Yr10CGE2a and Yr10CGE2b, were developed to detect the Yr10CG gene in wheat germplasm (Fig. 1 and S1, Table S2). The PCR specificity was tested on isogenic lines of ‘Avocet S’, ‘Avocet R’ and ‘Avocet SþYr10’. Both Yr10CGE2a and Yr10CGE2b worked in Avocet SþYr10, but not in Avocet S and Avocet R (Fig. S2). The specificity of Yr10CGE2a and Yr10CGE2b was further confirmed by sequencing the PCR products from two Yr10 carriers including Avocet SþYr10 and Moro. In 659 wheat lines screened, 14% were positive for the Yr10CG markers; 13% of the advanced cultivars had the Yr10CG gene (Tables 1 and S3). Wheat cultivars positive for the Yr10CG markers were distributed over 10 wheat growing zones (Table 1). Within wheat growing zones I, II, III, V and VIII, the frequency of cultivars positive for the Yr10CG markers were 5%, 4%, 26%, 33% and 11%, respectively. Among cultivars positive for the Yr10CG marker, the fraction of growing zones I, II, III, V and VIII were 7%, 11%, 32%, 23% and 5%, respectively. Consequently, cultivars from wheat zones III and V have a higher frequency of the Yr10CG markers. A pedigree comparison suggested that the Yr10CG gene in more than 55% of Chinese wheat cultivars was derived from ‘Nanda 2419’, a selection of Italian wheat ‘Mentana’.
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1 was also present in other Yr10CG homologues (AF149112, AF149113, AF446141, AF509533, AF509534, AY613783, AY613785, AY613786 and EU428764), the G-to-T mutation (Val558Leu) in exon 2 was unique to Jiangdongmen. A diagnostic marker Yr10JDM was developed to detect the G-toT mutation, which was only detected in Jiangdongmen, ‘Baiquan 565’ and ‘Fufan 916’ (Fig. 2A and Table S2). According to the SIFT analysis, the Lys30Arg and Val558Leu substitutions would be tolerated, suggesting that the Yr10CG gene from Jiangdongmen might be functional. Semiquantitative RT-PCR revealed that the level of Yr10CG mRNA was comparable in Jiangdongmen and Moro, but seemed lower in Nanda 2419 than in the others (Fig. 3). 3.2. Yr18RH is present in 11% of Chinese wheat genotypes
Fig. 1. Detection of Yr10CG, Yr18, and Yr36 genes in seven wheat lines using the developed PCR markers. PCR products of Yr10CG and Yr36 genes were separated on 1%e2% agarose gels, while the separation of Yr18 products was made on the 6% acrylamide gel. Seven representative wheat lines are 98M71, Chinese Spring (CS), Nanda 2419 (ND), Fan 6, Moro, UC1041, and UC1041þYr36.
Some native wheat such as ‘Jiangdongmen’ also was positive for the Yr10CG markers. Genomic and cDNA sequences of Yr10CG were obtained from Jiangdongmen (HM231238 and HM231239), Nanda 2419 (HM461975 and HM461976), and Moro. The Yr10CG sequence (start to stop codons) from Nanda 2419 was identical to that of the Moro. In comparison, the Yr10CG cDNA from Jiangdongmen had two missense point mutations. Although the A-to-G mutation (Lys30Arg) in exon
Resistant and susceptible haplotypes of the Yr18 gene have been identified (Krattinger et al., 2009). The resistant haplotype of Yr18 (Yr18RH) is characterized by a 3-bp deletion (TTC) in exon 11 and a T-to-C point mutation (Tyr634His) in exon 12. The dominant marker Yr18E11a was designed to detect the 3-bp deletion (Fig. 1 and Table S2). To confirm that the 3-bp deletion involves the expected TTC sequence, lines with the Yr18E11a marker were also tested by the Yr18E11b that produced diagnostic band only in wheat lines without the TTC deletion (Fig. 1 and Table S2). Yr18E12a is a dominant marker for the resistant haplotype and detects the T-to-C point mutation (Fig. 1 and Table S2). Analysis of Yr18E11a and Yr18E12a markers in nulli-tetrasomic lines of ‘Chinese Spring’ demonstrated that the diagnostic bands are 7D specific (Fig. S2). Fifteen percent of all 660 lines or 11% of the 486 advanced cultivars had the Yr18RH allele (Tables 1 and S3). Wheat cultivars positive for the Yr18RH markers were distributed over all 10 wheat growing zones. Within wheat growing zones I, II, III, V and VIII, the frequencies of cultivars positive for the Yr18RH markers were 5%, 6%, 29%, 12% and 11%, respectively. Among cultivars positive for the Yr18RH marker, the
Table 1 Wheat cultivars positive for Yr10CG or Yr18RH markers Yr10CGa
Yr18RHb
No.
Wheat germplasmc
Positive
Negative
44
Aiganzao, Baiquan 565d, Chuan 533, Chuanmai 4, Chuanmai 28, Chuanyu 20, e 1161, Fan 6, Fengmai 2, Fengmai 13, Fufan 17, Fufan 24, Gahai 1, Ji’nan 13, Jiangdongmen, Jianyang 73, Jingchun 70-5321, Jinghong 9, Jurong 03, Kehan 4, Linmai 4, Longchun 11, Lumai 20, Mianyang 11, Mianyang 12, Mianyang 15, Mianyang 62-31, Nanyuan 1, Nongda 311, Pulin 5, Qingxuan 15, Rikaze 7, Sufu, Sumai 1, Wanpin 8029, Wuyimai, Xi’anshixinmai, Xichang 177, Xinyang 1, Zhongshan 2, Jubileina Ie, Mexipak 66e, Rieti 75e, Virgilioe
Negative
Positive
39
Bosu 16, Fufan 76, Gan 174, Huamai 7, Ji’nan 9, Jingzhou 1, Jinmai 7, Kechun 5, Kehan 9, Kejin 6, Longjian 9811, Miannong 4, Pingliang 32, Shan 229, Shuwan 8, Simai 2, Tianxuan 44, Wanmai 32, Wanyou (1), Weidong 1, Xiangyang 4, Yanda 24, Youaola, Youjing’ai, Youyimai, Yunshi 1, Zhen 7495, Zheng 6 fu, Zhengzhou 6, Zhengzhou 18, Zhengzhou 742, Zhengzhou 743, Zhengzhou 9285, Chengduguangtouf, Chinese Springf, Mazhamaif, Mingxian 169f, Pingyaoxiaobaimaif, Youzimaif
Positive
Positive
22
Bosu 1, e’mai 6, Fengkang 11, Fengqiang 7, Gan 162, Ganmai 7, Gui 83-7, Huazhong 6, Jingzhou 47, Nanda 2419, Nannongdaheimang, Neimai 14, Neixiang 19, Ningmai 1, Rikaze 8, Sumai 2, Sumai 3, Wangmai 17, Wangmai 19, Wannian 2, Xiangnong 3, Youxiang (2)
a d
Markers of Yr10CGE2a and Yr10CGE2b. b Markers of Yr18E11a and Yr18E12a. c Wheat germplasm includes cultivars, landraces, and exotic materials. Cultivars highlighted by underline have ‘Mentana/Ardito’ in their pedigree. e Exotic materials. f Landraces from China.
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Fig. 2. Genotyping unique alleles of Yr10CG and Yr18 genes. A: the 136 bp PCR marker Yr10JDM was developed to detect the point mutation in the Yr10CG coding region in Chinese wheat ‘Jiangdongmen’ (JDM). The 118 bp band indicates absence of the point mutation identified in JDM. B: PCR markers were developed to detect informative SNPs in exon 17 of Yr18RH in Chinese wheat ‘Mingxian 169’ (MX). Representative wheat genotypes included Jiangdongmen (JDM), Nanda 2419 (ND), Mingxian 169 (MX), and Chinese Spring (CS).
fraction of growing zones I, II, III, V and VIII were 7%, 22%, 40%, 9% and 6%, respectively. Clearly, cultivars from wheat zone III had the highest frequency of Yr18RH. Pedigree analysis suggested that Yr18RH in more than 38% of Chinese cultivars can be traced back to Nanda 2419. On the other hand, several native landraces carrying Yr18RH, such as ‘Chengduguangtou’, ‘Mazhamai’ and ‘Youzimai’, have been used as core germplasm in wheat breeding. ‘Mingxian 169’ has been widely used as a susceptible parent in genetic studies and as a susceptible check to test stripe rust resistance. However, it scored positively for Yr18RH markers. To identify the putative cause for lack of function, the Yr18 cDNA was isolated from Mingxian 169. In total, we sequenced five full-length cDNA clones representing the D genome. In comparison to Chinese Spring (FJ436983), the D allele in Mingxian 169 was found to have an alternative splicing (Fig. S3). For example, either intron 6 or 9 was present in cDNA clones (GU929206 and GU929207). Alternative splicing also occurred in exons; either the first 92 bp or 44 bp in exon 10 or 12, respectively, was absent in cDNA clones
(GU929206 and GU929207). However, cDNA clone A13 had a complete exon 12. Taken together, the coding sequence of Mingxian 169 has a 99% identity with the Yr18RH gene from Chinese Spring, but Mingxian 169 has a C-to-T point mutation (Leu913Phe) in exon 17. According to the SIFT analysis, the Leu913Phe mutation likely has negated the function of the Yr18RH allele in Mingxian 169. We also developed three PCR markers, Yr18E17a, Yr18E17b and Yr18E17c, to detect the frequency of the C-to-T mutation in exon 17 (Fig. 2B and Table S2). This mutation is unique to Mingxian 169 and is absent in other Yr18RH alleles in the current collection. Transcriptional analysis indicated that alternative splicing in Yr18 is common (Fig. S4). Chinese spring and other Triticeae lines (Aegilops tauschii, Hordeum vulgare, and T. turgidum ssp. Durum) had the expected splicing for Yr18. However, additional splicing patterns appeared to be present (Fig. S4). 3.3. Yr36 gene is not present in current Chinese wheat In the Yr36 region, there are two duplicated genes, Wheat Kinase-START 1 and 2 (WKS1 and WKS2), and WKS1 is the Yr36 gene (Fu et al., 2009). WKS1 and WKS2 encode 86% identical proteins. To investigate the presence of the Yr36 gene in Chinese wheat, we developed the PCR marker Yr36E1a (Fig. 1 and Table S2). YrR36E1a was designed based on the common sequence flanking the kinase domain in both WKS1 and WKS2 genes. Screening of the current collection suggested that the WKS genes were absent in Chinese common wheat. 4. DISCUSSION 4.1. Gene-specific markers are developed for known resistance loci to wheat stripe rust The preferred method for controlling wheat stripe rust is utilizing the host resistance. So far, two non race-specific resistance genes, Yr18 and Yr36, and one race-specific resistance candidate gene Yr10CG have been cloned. Gene-specific markers have been reported for each of them (Temel et al., 2008; Fu et al., 2009; Lagudah et al., 2009; Singh et al., 2009; Dakouri et al., 2010). However, there are certain disadvantages associated with the existing markers. For example, the Yr10CG markers appear to be less specific when compared to other Yr10CG homologues (Fig. S1). Markers of the Yr18 gene either requires specialized instrument (Dakouri et al., 2010) or relies on expensive restriction enzyme (Lagudah et al., 2009). Although four sets of PCR markers are available for the Yr36 gene (Fu et al., 2009), a single robust PCR marker is necessary for marker-assisted selection. Here, we developed new diagnostic PCR markers for all three genes and used them to genotyping the current collection on Chinese wheat. 4.2. Molecular function of the Yr10CG gene is poorly understood
Fig. 3. Transcriptional analysis of Yr10CG using two markers. Semi-quantitative RT-PCR was used to assess the Yr10CG mRNA in three representative wheat genotypes with Yr10CG, Jiangdongmen (JDM), Nanda 2419 (ND), and Moro.
The Yr10 locus was first identified in a Turkish wheat line, PI 178383, which was further used as the Yr10 donor to developing wheat cultivars such as Moro, ‘Crest’ and ‘Jacman’ (Line and
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Qayoum, 1991). Unfortunately, the Yr10 gene in Moro remained effective for only 3 years in the U.S. Pacific Northwest (Sharp and Volin, 1970). However, the Yr10 gene is still considered to be effective in many wheat growing regions in the world. The Yr10CG gene encodes a classic NBS-LRR protein. Singh et al. (2009) reported that markers derived from the Yr10CG sequence (GenBank accession No. AF149112) are completely linked to resistance to the rust pathotype 46S119. In the present study, Yr10CGE2a and Yr10CGE2b were designed to amplify the last 500 bp, the most variable region in the gene. By choosing unique primer ends, Yr10CGE2a and Yr10CGE2b markers are more specific for the Yr10CG gene than previously published Yr10 markers (Fig. S1). When the Yr10CGE2a sequence was used to blast the Chinese Spring genomic sequence (version 2011.06, http://www.cerealsdb.uk. net), the closest homologue was F676MBK02FZL6K which is a NBS-LRR-like gene from Ae. tauschii (AY613785). Yr10CG from Moro and the GenBank entry AY613785 share 91% identity in the predicted coding region. In the current collection, Yr10CG markers were detected in 62 (13%) Chinese cultivars including Nanda 2419 and ‘Fan 6’. Surprisingly, 34 of them can be traced back to Nanda 2419 or ‘Mentana/Ardito’ lineage. ‘Ardito’ and ‘Mentana’ are founder cultivars for Yr18 germplasm in North/South America wheat and European winter wheat, respectively (Kolmer et al., 2008). Nanda 2419 is a core germplasm in wheat breeding in China; more than one hundred wheat cultivars have been developed from Nanda 2419. Moro and Nanda 2419 are identical on the Yr10CG gene; however, the mRNA level of the Yr10CG gene is lower in Nanda 2419 than in Moro, and the Yr10 carriers Moro and PI 178383 have a different resistance spectrum than Nanda 2419 (Qi et al., 2002). Whether the expression of Yr10CG gene affects the resistance level remains to be investigated. In this study, Pst races CYR29, CYR32, and Su-14 were inoculated on the seedling plants of selected cultivars. Cultivars with the Yr10CG gene displayed varying infection types from susceptible to fully resistant (Fig. S5). Because a greater percentage of Chinese cultivars have the Yr10CG but not the apparent resistance of Yr10, it remains inconclusive that the Yr10CG is Yr10. 4.3. Yr18 gene has been a long-standing resistance resource widely-deployed in Chinese wheat The Yr18 gene encodes a PDR-like ABC transporter. The slow rusting resistance conferred by the YrI8 gene reduces grain yield loss by 36%e58% under high disease pressure (Ma and Singh, 1996). PCR markers are developed to identify the resistant and susceptible alleles of Yr18 gene (Lagudah et al., 2009; Dakouri et al., 2010). New PCR markers, Yr18E11a and Yr18E12a, amplify Yr18 genes from D and other genomes, but the diagnostic bands are D genome specific (Fig. S2). The Yr18E11a marker can be easily scored by running a 6% acrylamide gel, and the Yr18E12a marker uses a less expensive restriction enzyme (Nla III). Dakouri et al. (2010) studied the distribution of the resistant haplotype on the Yr18 gene in 700 wheat lines, including 337 lines from various geographical areas of the world and 363 lines
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mostly from North America. In average, about 26% of the screened germplasm are positive for the Yr18RH gene. In the current collection, 11% of the cultivars have the resistant haplotype on the Yr18 gene. Seedling tests suggested that the Yr18RH gene alone might not provide adequate resistance to stripe rust (Fig. S5). However, cultivars negative for Yr10CG and Yr18RH markers also showed different infection types (Fig. S5). In addition, a non-functional Yr18RH allele was found in Mingxian 169. The Leu913Phe substitution might account for the loss-of-function of the Yr18RH allele in Mingxian 169. In other studies, allelic diversities also occurred in exon 10 or exon 22. The cultivar ‘Invader’ has an additional A in exon 10, which causes a frameshift in mRNA (Dakouri et al., 2010); the cultivar ‘Jagger’ has a G-to-T point mutation in exon 22, which results in a premature stop codon (Lagudah et al., 2009). Chinese wheat is amongst the earliest documented sources of Lr34/Yr18 (Dyck, 1977). In particular, the Yr18RH gene carrier Chinese Spring was developed in the early last century. In China, several Yr18RH carrier landraces, such as ‘Chengduguangtou’, ‘Mazhamai’ and ‘Youzimai’, have been used as core germplasm in wheat breeding, generating over 100 cultivars. On the other hand, Nanda 2419 is positive for the Yr10CG and Yr18RH markers. Of the 22 Chinese wheat cultivars positive for both markers, 15 have Nanda 2419 in their pedigrees. Therefore, Yr18RH in many Chinese wheat cultivars originated from the ‘Mentana/Ardito’ lineage. Yr18 has higher expression in adult plants than in seedlings (Krattinger et al., 2009). In adult leaves, the correctly spliced Yr18 product accumulates in both leaf bases and leaf tips, however the unspliced transcripts appears to be predominant in leaf tips (Krattinger et al., 2009). Our study proved that the Yr18 gene has alternative splicing sites and there is enrichment of certain transcriptional variants during plant development. 4.4. Yr36 gene represents a novel source for stripe rust resistance in China Wild emmer wheat (T. dicoccoides) is the progenitor of modern tetraploid and hexaploid cultivated wheat. So far, four stripe rust resistance genes, including YrH52, Yr15, Yr35 and Yr36, have been identified in this species. Yr36 confers resistance to all tested Pst races in the U.S., including PST-43, 45, 78, 98, 100, 101, 111, 112, 113 and 116 under high temperatures (e.g. 25 C) (Fu et al., 2009). Excepting PST-43 and PST-45, the other races were found after 2000 representing the most predominant races from 2000 to 2005 in the U.S. (Chen, 2007). In China, Yr36 is effective against current prevalent races in the northwestern region of Sichuan Province. In a previous study, PCR markers were developed to detect the kinase domain, the START domain, and the internal region between the kinase and the START domains (Fu et al., 2009). In all their screened materials, the primers from each target region gave identical results. Because it appeared that a single PCR marker could be used to detect the presence of WKS genes, we designed PCR primers for marker Yr36E1a to the kinase domain region. The Yr36E1a marker was based on the common sequence in the WKS1 and WKS2 genes; the PCR primers were
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targeted to the less conserved sequence flanking the kinase domain, increasing the specificity and reliability of PCR. Both primers reside on one single exon, reducing the chance of PCR failure due to large insertions or deletions in introns. In a previous study, Yr36 was found only in the wild species T. turgidum spp. dicoccoides, but not in the accessions of three related populations: domesticated emmer, cultivated durum and common wheat (Fu et al., 2009). Genomic search revealed that the Yr36 gene/ortholog does not exist in Chinese Spring, and unlikely to exist in all other common wheat. In the present study, we further confirmed the lack of Yr36 in all tested Chinese common wheat. Because Yr36 was lost during wheat domestication, the gene has not been widely used in wheat breeding. Only a few wheat genotypes that were selected for the high grain protein content gene have the Yr36, such as ‘Glupro’, ‘Lassik’, ‘Farnum’ and ‘PI 638740’ (Uauy et al., 2005; Fu et al., 2009). In China, the Yr36 gene has been used in wheat breeding since 2008, mainly in the Sichuan and Shandong Provinces. At present, the major donor for Yr36 is breeding lines ‘UC1041þYr36’ and ‘Yecora RojoþYr36’. ACKNOWLEDGEMENTS We thank Dr. Lynn Epstein (University of California, USA) and Dr. Xianming Chen (Washington State University, USA) for their critical reading of the manuscript. This work was supported by the China Research and Development Initiative on Genetically Modified Plants (Grant No. 2009ZX08009053B), the National Basic Research Program of China (973 Program, Grant No. 2011CB100700), and the National Natural Science Foundation of China (Grant No. 30871323). SUPPLEMENTARY DATA Table S1. Wheat growing zones of China. Table S2. PCR markers used for germplasm screening. Table S3. Wheat germplasm negative for Yr10CG, Yr18RH and Yr36 markers. Fig. S1. Primer specificity of Yr10CG markers. Fig. S2. Specificity of Yr10CG and Yr18RH markers. Fig. S3. Sequence alignment of transcribed/predicted cDNAs of the Yr18 gene. Fig. S4. Alternative splicing of the Yr18 gene. Fig. S5. Stripe rust resistance in eight representative wheat lines. Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.jgg.2012.03.005. REFERENCES Cantu, D., Govindarajulu, M., Kozik, A., Wang, M., Chen, X., Kojima, K.K., Jurka, J., Michelmore, R.W., Dubcovsky, J., 2011. Next generation sequencing provides rapid access to the genome of Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe rust. PLoS ONE 6, e24230. Castro, A.J., Chen, X.M., Hayes, P.M., Johnston, M., 2003. Pyramiding quantitative trait locus (QTL) alleles determining resistance to barley stripe rust: effects on resistance at the seedling stage. Crop Sci. 43, 651e659. Chen, X.M., 2007. Challenges and solutions for stripe rust control in the United States. Aust. J. Agric. Res. 58, 648e655.
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