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Published July 7, 2016 o r i g i n a l r es e a r c h Fine Mapping of Two Wheat Powdery Mildew Resistance Genes Located at the Pm1 Cluster Junchao Liang, Bisheng Fu, Wenbin Tang, Nasr U. Khan, Na Li, and Zhengqiang Ma* Abstract Core Ideas Powdery mildew caused by Blumeria graminis (DC.) f. sp. tritici (Bgt) is a globally devastating foliar disease of wheat (Triticum aestivum L.). More than a dozen genes against this disease, identified from wheat germplasms of different ploidy levels, have been mapped to the region surrounding the Pm1 locus on the long arm of chromosome 7A, which forms a resistance (R)-gene cluster. Mlm2033 and Mlm80 from einkorn wheat (T. monococcum L.) were two of the R genes belonging to this cluster. This study was initiated to fine map these two genes toward map-based cloning. Comparative genomics study showed that macrocolinearity exists between Brachypodium distachyon L. chromosome 1 (Bd1) and the Mlm2033–Mlm80–Pm1a region, which allowed us to develop markers based on the wheat sequences orthologous to genes contained in the Bd1 region. With these and other newly developed and published markers, high-resolution maps were constructed for both Mlm2033 and Mlm80 using large F2 populations. Moreover, a physical map of Mlm2033 was constructed through chromosome walking with bacterial artificial chromosome (BAC) clones and comparative mapping. Eventually, Mlm2033 and Mlm80 were restricted to a 0.12- and 0.86-cM interval, respectively. Based on the closely linked common markers, Mlm2033, Mlm80, and MlIW172 (another powdery mildew resistance gene in the Pm1 cluster) were not allelic to one another. Severe recombination suppression and disruption of synteny were noted in the region encompassing Mlm2033. These results provided useful information for mapbased cloning of the R genes in the Pm1 cluster and interpretation of their evolution. Published in Plant Genome Volume 9. doi: 10.3835/plantgenome2015.09.0084 © Crop Science Society of America 5585 Guilford Rd., Madison, WI 53711 USA This is an open access article distributed under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). the pl ant genome ju ly 2016 vol . 9, no . 2 • High-resolution maps were constructed for both Mlm2033 and Mlm80. Mlm2033, Mlm80, and MlIW172 are different powdery mildew resistance genes in the Pm1 cluster. New markers tightly linked to Mlm2033 were developed. • • P is an important foliar wheat disease caused by biotrophic fungus Bgt. Severe epidemic of this disease could cause significant yield reduction especially in the areas with cool or maritime climates (Everts and Leath 1992). Deployment of host R genes is the most effective and environmentally safe approach to control this disease in agricultural production. However, R genes with large effect are often defeated, when widely employed over a large geographical area, by rapidly evolved pathogen populations within a short period (McDonald and Linde 2002). Thus, it is necessary to constantly discover and use new powdery mildew R genes in the breeding program. To overcome powdery mildew in wheat, over 70 R genes, mapping to 50 distinct loci of the genome, have owdery mildew J.C. Liang, B. Fu, W. Tang, N.U. Khan, N. Li, and Z. Ma, The Applied Plant Genomics Laboratory of Crop Genomics and Bioinformatics Centre, Nanjing Agricultural University, Nanjing 210095, Jiangsu, China; B.S. Fu, current address, Crop Research Institute, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, Jiangsu, China. Received 11 Sept. 2015. Accepted 29 Nov. 2015. *Corresponding author (zqm2@ njau.edu.cn). Abbreviations: BAC, bacterial artificial chromosome; Bd1, Brachypodium distachyon L. chromosome 1; Bgt, Blumeria graminis (DC.) f. sp. tritici; CAPS, cleaved amplified polymorphic sequence; CC-NBS-LRR, coiled-coil–nucleotide-binding-site–leucine-rich repeat; CDS, coding DNA sequences; EST, expressed sequence tag; PCR, polymerase chain reaction; R, resistance; RGA, resistance gene analog; SSR, simple-sequence repeat; STS, sequence-tagged site. 1 of 9 been identified in common wheat and its relatives (McIntosh et al., 2013, 2014; Hao et al., 2015; Wang et al., 2015; Ma et al., 2015). Most of these genes confer race-specific resistance to wheat powdery mildew. Yahiaoui et al. (2004) isolated Pm3b on chromosome 1A of common wheat through positional cloning, which is an allele of the R locus encoding coiled-coil–nucleotide-binding-site– leucine-rich repeat (CC-NBS-LRR) proteins. The previously popular Pm8, originated from rye (Secale cereale L.), is the ortholog of Pm3 (Hurni et al., 2013). An RGA gene isolated from chromosome 6V of Haynaldia villosa (L.) Schur, the carrier of Pm21, encodes a serine–threonine kinase and provided powdery mildew resistance (Cao et al., 2011). However, in consideration of the vast amount of wheat R genes, lack of knowledge about their evolution, products, and functions has limited our understanding of the strategies employed by wheat to combat diseases. Resistance-gene clusters widely exist in plants. In Arabidopsis thaliana L., two-thirds of the NBS-LRR R genes were in clusters (Meyers et al., 2003); 74.3% R genes in rice (Oryza sativa L.) were in clusters (Monosi et al., 2004; Luo et al., 2012). Barley (Hordeum vulgare L.) Mla (Wei et al., 1999), maize (Zea mays L.) Rp1 (Ramakrishna et al., 2002; Smith et al., 2004), and lettuce (Lactuca sativa L.) RGC2 (Meyers et al., 1998) are all known R-gene clusters. The Rp1 encompassed up to >50 copies of R genes was probably the largest R-gene cluster known so far in plants. Of the 50 loci identified for wheat powdery mildew resistance, six, including Pm1, Pm2, Pm3, Pm4, Pm5, and Pm24, were known to have multiple alleles according to allelic tests or mapping results. For instance, at Pm3, 17 functional alleles have been identified at the sequence level (Yahiaoui et al., 2004, 2006, 2009; Srichumpa et al., 2005; Bhullar et al., 2009, 2010), which form a true allelic series. However, some of the socalled allelic relationships might result from tight linkage of different R genes. At Pm1, apart from Pm1a to Pm1e, PmU, Mlm2033, Mlm80, MlIM72, MIAG12, PmG16, MlIM172, HSM1 and PmTb7A.2, were all mapped to the same chromosomal interval (Hsam et al., 1998; Singrün et al., 2003; Qiu et al., 2005; Yao et al., 2006; Ji et al., 2008; Maxwell et al., 2009; Ben-David et al., 2010; Ouyang et al., 2014; Li et al., 2014; Chhuneja et al., 2012, 2015), which was subjected to recombination suppression (Neu et al., 2002). Therefore, Pm1 might represent a significant R-gene cluster with genes identified in wheat accessions of different ploidy levels. Mlm2033 and Mlm80 were two powdery mildew R genes identified in einkorn accessions and were both mapped to the Pm1 cluster by our group (Yao et al., 2006). This study was initiated to fine map these two genes and determine their physical positions. Materials and Methods Plant Materials Triticum monococcum accession TA2033, possessing powdery mildew resistance gene Mlm2033; T. boeoticum accession M80, possessing powdery mildew resistance gene Mlm80; and susceptible T. monococcum accessions 2 of 9 M389 and TA2725 were used in this study. Three thousand three hundred ninety-one F2 plants derived from the cross of M389 ´ TA2033 and 1411 F2 plants derived from TA2725 ´ M80 were used for fine mapping. Resistance Evaluation Powdery mildew resistance evaluation was conducted following the same procedures as previously described by Yao et al. (2006). The seedlings were grown in rectangular trays under controlled condition with a 22 and 18°C day vs. night cycle. All plants were inoculated at the one-leaf stage with isolate Bgt19 and scored 7 d after inoculation for the resistance level, using a scale of 0 to 5, which represents no visible symptom (0), necrosis without sporulation (1), sparse sporulation (2), moderate sporulation (3), abundant sporulation (4), and abundant sporulation (5) with more than 80% of the leaf area covered with mycelia, respectively. Polymerase Chain Reaction Amplification DNA was extracted from young seedling tissues following the procedures described by Ma et al. (1994). Resistant and susceptible bulks were made by combination of equal amount of DNA from six F2 plants showing resistant and susceptible phenotypes, respectively. Polymerase chain reaction of markers was performed in a PE9600 thermal cycler (PerkinElmer) in a volume of 10 mL containing 10 to 20 ng of template, 2 pmol of each of the primers, 2 nmol of each of the deoxynucleoside triphosphates, 15 nmol of MgCl2, 0.1 U Taq DNA polymerase, and 1´ PCR buffer. The PCR profile was as follows: one cycle of 94°C for 3 min, followed by 35 cycles of 94°C for 30 s, 50 to 60°C (depending on the specific primers) for 40 s, and 72°C for 50 s, and a final extension at 72°C for 5 min. The PCR products were separated in 8% nondenaturing polyacrylamide gels with a 19:1 or 25:1 or 39:1 acrylamide/bis-acrylamide ratio and then silver-stained as described by Santos et al. (1993). Marker Development and Recombinant Screening Wheat expressed sequence tags (ESTs) deposited in the NCBI (http://www.ncbi.nlm.nih.gov/nucest), T. urartu genome scaffolds (Ling et al., 2013) and survey sequence contigs of the wheat cultivar Chinese Spring (International Wheat Genome Sequencing Consortium, www.wheatgenome.org) that are homologous to the predicted coding DNA sequences (CDS) in the B. distachyon chromosomal region collinear to the Mlm2033–Mlm80 interval were used for development of simple-sequence repeat (SSR) or sequence-tagged site (STS) markers. Additional markers were designed based on the tetraploid wheat BAC sequence LDN133-865 (GI: 636536483) (Ouyang et al., 2014), courtesy of Dr. Zhiyong Liu of China Agricultural University. Triticum urartu sequence scaffold9207 (GI: 473888380) (Ling et al., 2013), which contains one CDS homologous to Bradi1 g29693, was also used for marker design. Primers of markers were designed using MacVector v12.0 software (MacVector Inc., 2011). Some monomorphic STS markers the pl ant genome ju ly 2016 vol . 9, no . 2 Table 1. Markers developed in this study that detected polymorphism between the parents Marker name MAG5240† MAG5825† MAG5827† MAG6385 MAG6546 MAG7310 MAG7914 MAG8220 MAG8223 MAG8415 MAG8626 MAG9060 WGRC253 WGRC271 Sources Restriction enzyme needed Forward primer (5¢-3¢) Reverse primer (5¢-3¢) BQ240837 CJ664083 CJ579028 H194P1 B89H15 H194P1 B460F16 B588P8 B270E13 B588P8 B270E13 LDN133-865 Scaffold9207 Scaffold9207 EcoRI RsaI – – – – – – TaqI MboI – – – MnlI GGAACATACGACCCAGATTTACGAG GATGATAATGACTCCCTATG CAGGGATGTCGGAGAAGA CGCCTATGTCTCTAAGTCGTGCC CGAAACAACGAGGATGATGACTTG ATTCCTGTTCATCTGCCTCC ATAAGCACTTGACGGTAGGGGC CACACGTCTAGCCAATACTTCAC TCGTCTTTGCTGTTCTCGGC CCCCCGTGATTATGAGAAACG AAGAGCACCAGAGCGACGAGAATG GGGCTGAAAAAGGAACTGAAACTC CGCACTCCACGACTCACG AACTTGAATACGCCCTTCCTC TGATGCCGACTGTCACTGCTTAC AGAATCCCTTCTGCTGACC GGTGACGACTCCTTTGAA CTGGGATACACTAAAAGTAACTGCCG GCACCAACGAGGAACAACCTATG GATACATTCCCTCTAACCCTGAC ATAGGGAAGGCATCAGAAGGGGAC CTGTACCAACGAAAGAGAAAGG GCTCCTTACGCTTCAGGGATAAATC AAGACGACGAACAGCCAAAGTG TGAGGCGAGCGTGTGATAGATAC CGCGAATGTACACACAACACTG CCACGAAAGGCAAAGGCAG TCCAGACGCACGACGATG † Markers corresponding to B. distachyon genes. were converted to cleaved amplified polymorphic sequence (CAPS) markers by digesting the amplified PCR products with restriction enzymes following the standard protocols. Recombinants in the Mlm2033 population were screened with flanking markers Xmag5825 and Xmag5827, and those in the Mlm80 population were screened with flanking markers Xmag1757 and Xmag2185. Bacterial Artificial Chromosome Library Screening, Sequencing, and Sequence Analysis A BAC library of about sixfold genome coverage, constructed with the TA2033 genomic DNA in the authors’ lab, was used for BAC screening. End sequences of positive BAC clones were obtained by plasmid rescue (Woo et al., 1994); markers were subsequently designed for chromosome walking. Bacterial artificial chromosome DNA was prepared following the protocol described in Sambrook et al. (1989). Bacterial artificial chromosome contigs were assembled according to the fingerprints after single or double digestions with restriction enzymes NotI, HindIII, BamHI, PstI, and EcoRI and band patterns from PCR amplification of BAC clones with polymorphic markers. Insert sizes of BACs were estimated by pulse-field gel electrophoresis of the NotI digests of BAC DNA along with high molecular weight markers (#N0350S, New England Biolabs). Sequencing of BAC clones was performed in BGI-Shenzhen, Shenzhen, China. Gene prediction was performed using the gene predictor program FGENESH (http://www.softberry. com) complemented with BLASTn search against dbEST, BLASTx, and BLASTp search against the nonredundant protein database at GenBank. A homologous search of the predicted genes was also performed with the BLASTn algorithms in the B. distachyon CDS (http://www.plantgdb.org/BdGDB/cgi-bin/blastGDB.pl) and the T. urartu genome scaffolds (Ling et al., 2013). The Triticum survey sequence databases publicly available in Unité de Recherche Génomique Info (https://urgi.versailles.inra.fr/blast/ blast.php) were similarly surveyed. liang et al .: fine m apping of the pm1 cluster Linkage Analysis Recombinant frequencies (r) were calculated using the susceptible plants of segregating F2 populations with formula: r = (2f1+f2)/2N (Liu 1998), where N is the number of total susceptible plants of each population, and f1 and f2 are the numbers of homozygous and heterozygous recombinants of each marker, respectively. Linkage distances (d) were determined using formula: d = ln[(1+2r)/ (1–2r)]/4. Linkage maps were drawn with MapDraw Version 2.1 (Liu and Meng 2003). Results Macrocolinearity of the Mlm2033-Mlm80 Region with Brachypodium distachyon Genome The B. distachyon genome has substantial colinearity to cereal genomes (The International Brachypodium Initiative, 2010) and could be taken advantage of to identify wheat sequences suitable for marker development. As the first step, the macrocolinearity of the Mlm2033–Mlm80 region with the B. distachyon genome was determined. Thus, the EST sequences corresponding to STS markers MAG1810, MAG1757, MAG1759, MAG2185, MAG2166, MAG1986, and MAG1705 that cosegregated or showed close linkage with Mlm2033 or Mlm80 (Yao et al., 2006; Fig. 1) were used as queries to search homologous B. distachyon sequences in the B. distachyon genome sequence database (http://www. brachypodium.org/) via the BLASTn algorithm. Sequences orthologous to all these queries but MAG1705 were identified on B. distachyon chromosome 1 with an alignment similarly to the wheat genes (Fig. 1). In addition to CDO347 (MAG2166) and PSR680 (MAG2185), restriction fragmentlength polymorphism markers C607 and PSR148 that cosegregated with Pm1a (Neu et al., 2002) also have orthologous sequences in the Bradi1 g30190-Bradi1 g29320 interval. These results indicated perfect macrocolinearity between the Mlm2033–Mlm80–Pm1a region and the 750-kb B. 3 of 9 Fig. 1. Colinearity of the Mlm2033-Mlm80 region to Brachypodium distachyon chromosome 1 (Bd1) based on homology of restriction fragment-length polymorphism (RFLP) probes and expressed sequence tags–based markers. (a) and (c) Mlm2033 and MLm80 genetic maps reported by Yao et al. (2006). (b) Gene map of Bd1 in the region collinear to the Mlm2033-Mlm80 region in which the Pm1alinked RFLP probes homologous to the Bd1 genes are shown in brackets. The vertical arrows indicate the direction toward telomere. distachyon region flanked by Bradi1 g30190 and Bradi1 g29320 (unpublished data, 2012). Marker Development and Comparative Mapping of Mlm2033 and Mlm80 To obtain more markers for Mlm2033 and Mlm80, wheat ESTs homologous to the predicted B. distachyon CDS of the Bradi1 g30190-Bradi1 g29320 interval were used for identification of additional markers. Three markers, including MAG5827, MAG5240, and MAG5825, corresponding to Bradi1 g29590, Bradi1 g29658, and Bradi1 g29690, respectively, were obtained and revealed polymorphism between the resistant and susceptible bulks for Mlm2033 without or with enzyme digestion (Table 1). LDN133-865 is a BAC sequence mapping to chromosome 7A of tetraploid wheat and has 88% sequence identity to the RGA-like PSR680 (MAG2185). Annotation of this BAC sequence revealed another four RGA fragments and several single-copy regions. Markers were designed for these sequence fragments, but none of the RGA-derived markers could amplify products or detect polymorphism in the parents, and the marker MAG9060 based on one of the single-copy regions detected polymorphism between the Mlm2033 bulks. In addition, SSR marker WGRC253 and CAPS marker WGRC271 were developed based on T. urartu scaffold scaffold9207 that contains the Bradi1 g29693 homolog (Table 1); the former detected polymorphism between the resistant and susceptible bulks of both the Mlm2033 and Mlm80, and the latter revealed polymorphism between the resistant 4 of 9 and susceptible bulks of Mlm80 after MnlI digestion of the PCR products. Since powdery mildew R gene MlIW172 was mapped to the same chromosomal regions as Mlm2033–Mlm80 (Ouyang et al., 2014), markers linked to MlIW172 were also surveyed for polymorphism. WGGC4659 and WGGC4655 with HaeIII digestion detected polymorphism between the resistant and susceptible bulks of the Mlm2033 and Mlm80. Other markers, including WGGC4653 (with NlaIII digestion), WGGC4654 (with AluI digestion), and WGGC4665, detected polymorphism between the resistant and susceptible bulks of Mlm80. Mapping using the F2 populations developed by Yao et al. (2006) indicated that all these polymorphic markers were either cosegregated or tightly linked to the respective R genes and were mapped between Xmag5825 and Xmag5827 in the Mlm2033 map and between Xmag1757 and Xmag2185 in the Mlm80 map. Fine Mapping of Mlm2033 and Mlm80 To construct a high-resolution genetic map of Mlm2033, an F2 population of 3391 plants from the cross of M389 ´ TA2033 were evaluated for powdery mildew resistance. Of these plants, 832 had a score of 4 or 5 and all others had scores from 0 to 1, indicating, as expected, a single dominant gene controlled the resistance (c23:1 = 0.39). Similarly, to fine map Mlm80, 1411 F2 plants derived from M80 ´ TA2725 was surveyed. In this population, 350 F2 plants had a score from of 4 or 5 and the others had scores from the pl ant genome ju ly 2016 vol . 9, no . 2 Fig. 3. The physical maps of Mlm2033. The marker loci cosegregating with Mlm2033 were put proportionally to their physical positions. The arrow indicates the direction toward telomere. Fig. 2. High-resolution maps of (a) Mlm2033 and (b) Mlm80 constructed using 832 susceptible F2 plants from TA2033 ´ M389 and 350 susceptible F2 plants derived from M80 ´ TA2725, respectively. The vertical arrows indicate the direction toward telomere. Common markers are connected with dotted lines. Markers in boxes were developed based on bacterial artificial chromosome sequences. 0 to 1. The resistance segregation again fit the expected 3:1 ratio (c 23:1 = 0.02). The susceptible plants in both populations were used for recombinant identification between the flanking markers to reduce workload. Thirty-eight recombinants were obtained between Xmag5825 and Xmag5827 in the first population, and 20 were obtained between Xmag1757 and Xmag2185 in the second one. To avoid any phenotyping and genotyping errors, these recombinants and their progenies were re-evaluated phenotypically and double checked with markers mapped between the flanking markers. Finally, high-resolution maps were constructed for Mlm2033 (Fig. 2a) and for Mlm80 (Fig. 2b). The common markers in these two maps, Xwggc4655, Xwgrc253, Xwggc4659, and Xmag2185, showed perfect synteny. Mlm80 was flanked by Xwggc4655 and Xwgrc253; while Mlm2033 were flanked by Xwggc4659 and Xgwm344, both distal to Xwggc4655 and Xwgrc253. Since WGGC4655, WGRC253, and WGRC271 were single-copy sequence–based markers, it was concluded that Mlm2033 and Mlm80 might be two genes at different loci. Physical Mapping of Mlm2033 To construct the physical map for Mlm2033, TA2033 BAC clones were first screened with MAG2185 and MAG9060. Three positive BAC clones, H138G2, H194P1, and H130P6, were obtained using MAG2185 and assembled into a contig based on fingerprinting with single-enzyme digestion of HindIII, BamHI, and EcoRI, respectively, and double-enzyme digestion of NotI and PstI. MAG6385 and MAG7310 were two markers designed based on the overhanging end sequences of BAC H194P1 liang et al .: fine m apping of the pm1 cluster and H138G2. Walking using MAG6385 identified a 60-kb BAC (B89H15). Unable to design specific markers because of repetitive end sequences of this BAC, low-pass sequencing was performed, which allowed us to design the repetitive junction marker MAG6546 located ~9 kb apart from one of its ends and consequently identify the BAC clone B588P8. All the three markers cosegregated with Mlm2033. MAG8220, designed based on one end sequence of B588P8, detected polymorphism between the parents and a recombination event between Xmag6546 and Xmag8220 in one of the recombinants. These five BAC clones were therefore ordered and oriented as in Fig. 3. Sequencing B588P8 revealed a hypothetical gene contained nodulin-like domain, which cosegregated with Xmag8220 when converted to the marker MAG8415. Screening of the BAC library using MAG9060 yielded BAC clones B270E13 and B460F16. These two BACs overlap with each other but not with the MAG2185-derived contig. The end-sequence-derived marker MAC8223 detected a locus 0.06 cM proximal to Mlm2033/Xmag9060. Thus, Mlm2033 was delineated into a 0.12-cM interval defined by Xmag8415 and Xmag8223. To orient these two BACs, markers MAG7914 and MAG8626 were designed with the lowpass sequencing data of B460F16. Expected PCR products were obtained with MAG8626 when either one of these two BACs were used as template and with MAG7914 only when B460F16 was used as template. Genotyping the recombinants with these two markers indicated that Xmag8626 cosegregated with Mlm2033, while Xmag7914 was 0.12 cM proximal to the powdery mildew resistance gene, indicating one end of B460F16 was further away from Mlm2033 (Fig. 3). Attempt was made, but was currently not successful, to close the gap between the MAG2185-derived contig and the B270E13/B460F16 contig using BACs. All the newly developed markers based on the BAC sequences were also applied to the Mlm80 population. Of them, MAG8626, MAG8415, and MAG8220 detected polymorphism between Mlm80 and TA2725. These three marker loci all cosegregated with Xmag2185 and were 1.57 cM from Mlm80 (Fig. 2). 5 of 9 Table 2. List of the putative genes in the sequenced bacterial artificial chromosome (BAC) clones in Mlm2033 region No. Position (BAC) Similarity as revealed by BLASTx (accession number) E-value 1 2 3 4 5 6 7 8 9 10 11 12 13 B460F16 B460F16 B460F16 B460F16 B460F16 H194P1/H138G2 H194P1/H138G2 H194P1/H138G2 H194P1/H138G2 H194P1/H138G2 B588P8 B588P8 B588P8 Unknown Putative disease resistance protein RGA3 [Aegilops tauschii] (EMT21940.1) PREDICTED: putative disease resistance RPP13-like protein 3 [Brachypodium distachyon] (XP_003569090.1) PREDICTED: putative disease resistance protein RGA3 [Brachypodium distachyon] (XP_010239445.1) PREDICTED: putative disease resistance protein RGA3 [Brachypodium distachyon] (XP_010239445.1) PREDICTED: putative disease resistance protein RGA3 [Brachypodium distachyon] (XP_010239445.1) Disease resistance RPP8-like protein 3 [Aegilops tauschii] (EMT05779.1) Predicted protein [Hordeum vulgare subsp. vulgare] (BAK03994.1) PREDICTED: putative disease resistance protein RGA3 [Brachypodium distachyon] (XP_010239445.1) PREDICTED: putative disease resistance RPP13-like protein 3 [Brachypodium distachyon] (XP_003569090.1) Hypothetical protein F775_07944 [Aegilops tauschii] (EMT08425.1) PREDICTED: 2’-deoxymugineic-acid 2’-dioxygenase-like [Brachypodium distachyon] (XP_003577107.1) Unknown – 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 5 ´ 10 −173 0.0 5 ´ 10−114 – Fig. 4. Schematic representation of the genes in the Mlm2033-syntenic region of the bacterial artificial chromosome contigs and T. urartu scaffold7153. Resistance gene analogs are shown in red bars; unknown genes are shown in black bars; others are in light blue bars. Homologous genes are connected with dotted lines. All BACs in the Mlm2033 contig were sequenced. A total of 13 putative genes were predicted excluding those encoding transposition-related proteins (Table 2), nine of which were R-gene analogs. Obviously, Mlm2033 contig contains a RGA cluster. Of particular interest were genes 4, 5, and 6; they were highly similar to each other and shared an overall identity of 94.0 to 99.7%, suggesting gene duplication is part of the reason of multiple RGAs. Comparison of the Mlm2033 Region with Brachypodium distachyon, Triticum urartu, and LDN133-865 Sequences Among the 13 genes, only two genes, gene 9 and gene 11, have homologs with >80% identity in B. distachyon. Gene 9, corresponding to MAG2185, was homologous to Bradi1g29670 and Bradi1g29658, and gene 11 was homologous to three B. distachyon genes (Bradi3g56870, Bradi1g54990, and Bradi4g07330). However, none of these B. distachyon genes were located in the Mlm2033-syntenic region. 6 of 9 Search of the T. urartu genome scaffold database identified one scaffold7153 (KD005974.1; 874,486 bp) that could be syntenic to the Mlm2033 region. The predicted genes A, K, and L of this scaffold were homologous, with 89 to 97% identities, to gene 1, gene 11, and gene 13 (a single-copy gene) of the Mlm2033 contigs, respectively, and delimited a 194-kb region containing 12 predicted genes (Fig. 4). Two of the predicted genes, C and D, were RGAs, but only C was similar to one RGA (gene 8) of the Mlm2033 region with 82% identity. We also compared them with LDN133865, a sequence scaffold of the durum wheat ‘Langdon’ (T. turgidum ssp. durum [Desf.] Husn, and T. urartu). The LDN133-865 was only similar to gene 2 and the MAG9060 region of the Mlm2033 region and to gene E, a MATE protein, and gene D of the T. urartu scaffold. To close the Mlm2033 contig gap, attempt was made to develop more markers for BAC library screen using the predicted genes in scaffold7153 but failed. It appeared that the Mlm2033 region has undergone large structural changes by insertion of the RGA clusters between gene 1 and gene 11. the pl ant genome ju ly 2016 vol . 9, no . 2 Mlm2033 was Located in an Interval Subjected to Severe Recombination Suppression The construction of genetic and physical maps of Mlm2033 allowed us to investigate the relationship between physical and genetic distances in this region. Of 832 susceptible F2 plants, there were two recombinant events that occurred in the 10-kb region delimited by Xmag7914 and Xmag8626, and one recombinant event occurred in the 30-kb region delimited by Xmag6546 and Xmag8415. However, between Xmag8626 and Xmag6546, a region of at least 320 kb regardless of the gap, no recombinant event was identified between all the markers including Mlm2033 mapping to this interval. This result indicated that Mlm2033 was located in an interval subjected to severe recombination suppression flanked by regions with much higher recombination rate. Discussion Up to now, more than 12 powdery mildew R genes from different resistance germplasms have been mapped to the region surrounding Pm1. However, it is not really known if these R genes are allelic or in close physical proximity. In this study, two of these genes, Mlm2033 and Mlm80 (Yao et al., 2006), were precisely mapped to a 0.12-cM interval and a 0.86-cM interval, respectively, through marker development and highresolution mapping using recombinants from large segregating populations. Moreover, a physical map for Mlm2033 was established with BAC clones and known sequences. In the primary mapping studies, Pm1a (Ma et al., 1994; Neu et al., 2002), PmU (Qiu et al., 2005), MlIW72 (Ji et al., 2008), HSM1 (Li et al., 2014), MlIW172 (Ouyang et al., 2014), and PmTb7A.2 (Chhuneja et al., 2012, 2015) were mapped to the same marker interval as Mlm2033 or Mlm80. We showed, through high-resolution mapping, that Mlm2033 and Mlm80 turned out to be two different genes and genetically adjacent to each other. This was in conflict with our previous conclusion that the two genes were allelic to each other (Yao et al., 2006). A possible explanation is that the target region is subjected to severe recombination suppression in the allelic test population. Indeed, the Mlm2033 was just located in such a region. Xwggc4655, Xwggc4665, and Xwggc4659 were three markers in common in the MlIW172, Mlm2033, and Mlm80 maps and shared the same relative order (Fig. 2; Ouyang et al., 2014). Based on the mapping positions of the three resistance genes, it was clear that MlIW172 was different from Mlm2033 and Mlm80, since it was mapped to the interval flanked by Xwggc4665 and Xwggc4659 distally adjacent to the Mlm80 interval flanked by Xwggc4655 and Xwgrc253 and proximally adjacent to the Mlm2033 interval flanked by Xwggc4659 and Xgwm344. The mapping position of Xmag8626 in the Mlm80 map that cosegregated with Mlm2033 also supported this conclusion. These results pointed to the possibility that the Pm1 marker interval contains a resistance cluster, which was indirectly supported by the existence of multiple RGAs in the incomplete Mlm2033 contig. Thus, the Pm1 locus could be different from some R gene loci, such as Pm3 locus of liang et al .: fine m apping of the pm1 cluster wheat (Yahiaoui et al., 2004, 2006, 2009; Srichumpa et al., 2005; Bhullar et al., 2009, 2010) and L locus of flax (Linum usitatissimum L.) (Lawrence et al., 1995), that contain a single R gene and have multiple alleles with functions altered, but similar to other R gene loci, such as the M locus of flax (Anderson et al., 1997; Lawrence et al., 2010), the Rp1 locus of maize (Collins et al., 1999) and RGC2 family of lettuce (Meyers et al., 1998), that consist of R-gene clusters with tandem duplication being a likely cause. It would be interesting to find out if all the R genes mapped in the Pm1 interval were different position-wise. The size and complexity of wheat genome impose a great obstacle on map-based gene isolation. To make things worse, the recombination rates in some regions are far lower than the average, which make fine and physically mapping genes within them even more difficult. Mlm2033 is just located in such a region with the recombination rate at least 10 and 64 times lower than the two adjacent regions, respectively. Recombination suppression appears to be quite common in R-gene-containing regions. This has been illustrated in mapping and characterization of tomato (Solanum lycopersicum L.) root-knot nematode R gene Mi (Van Daelen et al., 1993), barley powdery mildew R gene Mla (Wei et al., 1999), wheat leaf rust R gene Lr10 (Stein et al., 2000), rice blast R gene Pi5 (Jeon et al., 2003) and Pi37 (Lin et al., 2007), common bean (Phaseolus vulgaris L.) resistance I locus (Vallejos et al., 2006), peanut (Arachis hypogaea L.) root-knot nematode R gene Rma (Nagy et al., 2010), and so on. Factors suggested to be responsible for recombination suppression include genetic background, high level of sequence diversity, chromosomal rearrangements, hemizygous state, among others (Wei et al., 1999, Casselman et al., 2000; Goel et al., 2003; Akiyama et al., 2005; Lin et al., 2007). Owing to the coevolution of host resistance and pathogen virulence, it is reasonable to conceive that the host R genes and their associated regions are subjected to fast evolution for the hosts to accommodate to hostile environments, which leads to divergence. Indeed, the dominant state of four (Xmag9060, Xmag7310, Xmag6385, and Xmag6546) of the six marker loci cosegregating with Mlm2033 implied that the Mlm2033 region is highly heterogeneous among the parents. Comparison of TA2033 BAC contig sequences surrounding Mlm2033 with the syntenic tetraploid wheat BAC sequence and T. urartu scaffold also revealed substantial structural changes and a high level of polymorphism. Transfer of this type of R-gene-containing chromatin to wheats different in ploidy levels could lead to more severe recombination suppression. Comparative genomics could reveal the evolutionary mechanisms of related genomes. In species without full genome sequence information, it also helps development of markers based on homology and conservation (e.g., Liu et al., 2012; Xue et al., 2012; Gasperini et al., 2012; Saintenac et al., 2013; Bansal et al., 2014) and even gene cloning (Yan et al., 2006). Using this approach, perfect macrocolinearity was shown between wheat chromosome 7A and Bd1, which allowed us to saturate the genomic region containing Mlm2033 and Mlm80 by developing more markers detecting polymorphism. However, it was noted that the 7 of 9 perfect colinearity was disrupted in the regions approaching Mlm2033. Based on the flanking sequences or genes, the Mlm2033 interval was corresponding to the Langdon sequence scaffold LDN133-865, but they lack sequence similarity except for a few short sequence stretches and differ greatly in size. Comparison of the 13 putative genes in BAC contigs of Mlm2033 region with the genes present in the collinear regions in B. distachyon genome and T. urartu scaffold7153 also imply disruption of synteny in the Mlm2033 region. Similar circumstances in wheat have been reported for Lr10 (Feuillet et al., 2003), Lr34 (Bossolini et al., 2007), and Pm3 (Yahiaoui et al., 2004). The breakdown of microcolinearity in the R-gene-containing regions between different species could result from frequent deletions and translocations of R genes (Leister et al., 1998; Quraishi et al., 2009, Luo et al., 2012). The microcolinearity in the Mlm80-containing region might have also been broken down, since this interval corresponds to a physical stretch of 30 kb from Bradi1g29693 to Bradi1g29730 in B. distachyon that has no known R gene-like gene sequences. Thus, even though the comparative genomics approach is justified for fine mapping, one has to be cautious to predict the existence of R genes based on colinearity. The fine mapping of Mlm2033 and Mlm80 in this study established the foundation for their map-based cloning and interpretation of the R-gene evolution in the Pm1 cluster. To reach these goals, we are expanding the mapping populations to get higher mapping resolution and increasing the BAC library volume to close the contig gap of Mlm2033. The newly developed markers will provide great benefit for cloning and marker-assisted selection of the linked genes. Acknowledgments: This project was partially supported by ‘973’ program 2009CB118300, NSFC Funds 30025030, 30771344 and 30771165, ‘863’ program (2012AA101105 and ‘Deep sequencing of A, D wheat genome and resistance functional genomics’). References: Akiyama, Y., W.W. Hanna, and P. Ozias-Akins. 2005. 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