Powdery mildew is one of the most widespread diseases of wheat. The development and deployment of resistant varieties are one of the most economical and effective methods to manage this disease. Our previous study showed that the gene(s) at 2Mb in Chinese Spring (CS)-Aegilops biuncialis 2Mb disomic addition line TA7733 conferred a high level of resistance to powdery mildew of wheat. In this study, resistance spectrum of TA7733 was assayed by using 15 Blumeria graminis f. sp. tritici (Bgt) isolates prevalent in different regions of China. The result indicated that TA7733 was highly resistant to all tested Bgt isolates and the gene(s) on chromosome 2Mb conferred broad-spectrum resistance to powdery mildew. In order to characterize mechanism of powdery mildew resistance by identifying candidates R-genes derived from Ae. biuncialis chromosome 2Mb and develop 2Mb-specific molecular markers, we performed RNA-seq analysis on TA7733 and CS. In total we identified 7,278 unigenes that showed specific expression in TA7733 pre and post Bgt-infection when compared to CS. Of these 7,278 unigenes, 295 were annotated as putative resistance (R) genes. Comparatively analysis of R-gene sequences from TA7733 and CS and integration CS Ref Seq v1.0 were used to develop R-gene specific primers. Of 295 R-genes we identified 53 R-genes were specific to 2Mb and could be involved in powdery mildew resistance. Functional annotation of majority of the 53 R-genes encoded nucleotide binding leucine rich repeat (NLR) protein. The broad-spectrum resistance to powdery mildew in TA7733 and availability of 2Mb-derived putative candidate R-gene specific molecular markers identified in this study will lay foundations for transferring powdery mildew resistance from 2Mb to common wheat by inducing CS-Ae. biuncialis homoeologous recombination. Our study also provides useful candidates for further isolation and cloning of powdery mildew resistance gene(s) from Ae. biuncialis chromosome 2Mb.
Citation: Li H, Dong Z, Ma C, Tian X, Xiang Z, Xia Q, et al. (2019) Discovery of powdery mildew resistance gene candidates from Aegilops biuncialis chromosome 2Mb based on transcriptome sequencing. PLoS ONE 14(11): e0220089. https://doi.org/10.1371/journal.pone.0220089
Editor: Aimin Zhang, Institute of Genetics and Developmental Biology Chinese Academy of Sciences, CHINA
Received: July 2, 2019; Accepted: October 23, 2019; Published: November 11, 2019
Copyright: © 2019 Li et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper and its Supporting Information files.
Funding: This work was financially supported by the National Natural Science Foundation of China (Grant No. 31801361 to HL and Grant No. 31571658 to WL), the Scientific and Technological Research Project of Henan Province of China (Grant No. 192102110011 to HL), the Henan Province Special Major Science and Technology Project (Grant No. 161100110400 to WL) and the State Key Laboratory of Wheat and Maize Crop Science at Henan Agricultural University, China (Grant No. 39990022 to WL). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Common wheat (Triticum aestivum L., 2n = 6x = 42, AABBDD), one of the most widely planted crops in the world provides 20% of the calories and 25% of its protein consumed by human [1,2]. Wheat production plays an important role in food security and social stabilization. However, wheat yields and quality are severely threatened by various diseases, such as rusts, Fusarium head blight (FHB) and powdery mildew. Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is one of the most destructive diseases all over the world, with severe yield losses ranging from 13% to 50% [3,4]. In recent years, certain agronomic practices to increase yields, such as popularization of high planting density, high inputs of irrigation and fertilization have accelerated the spread and severity of powdery mildew [5,6]. Though spraying fungicides can reduce the damage caused by this disease to some extent, it can also result in side effects such as drug resistant of powdery mildew fungus, environment pollution, and high production inputs . Breeding disease-resistant varieties is currently recognized as one of the most effective and economical ways to control powdery mildew.
Wild relatives of common wheat contain a large number of Bgt resistance genes for wheat improvement. Up to now, the number of designated genes of powdery mildew resistance (Pm) was more than 80 at 54 loci [8,9], of which approximately half of Pm genes were derived from wild relatives of wheat. However, some Pm genes had been defeated by new virulent Bgt races or by races that were previously present at very low frequencies in the pathogen population [10,11], and some were difficult to use in wheat breeding because of linkage drags [12,13]. Therefore, ongoing efforts to explore and identify new powdery mildew resistance genes are needed for wheat breeding programs.
Aegilops biuncialis (2n = 4x = 28, UbUbMbMb) is a tetraploid wild relative of wheat, belonging to the section Polyeides of the genus Aegilops. The Ub genome of it was derived from Ae. umbellulata (2n = 2x = 14, UU), and Mb genome from the diploid Ae. comosa (2n = 2x = 14, MM) [14,15]. Aegilops biuncialis owns many desired agronomic traits for wheat improvement, such as resistance to yellow rust , brown rust , powdery mildew and barley yellow dwarf virus , tolerance to drought and salt [19–21], high micronutrients contents , and special high molecular weight glutenin subunits . Successful attempts have been made to cross Ae. biuncialis with wheat, develop a series of wheat-Ae. biuncialis addition lines, and transfer desired genes from Ae. biuncialis into wheat [20,24,25]. In previous study, we identified that CS-Ae. biuncialis 2Mb disomic addition line TA7733 conferred high resistance to powdery mildew compared with its recipient parent CS .
The isolation and cloning of plant disease resistance genes had great significance for both plant disease resistance breeding and the study on molecular mechanisms of disease resistance. Map-based cloning is currently an important method to isolate novel genes. However, it is very challenging to perform fine mapping and map-based cloning of alien genes derived from wild relatives of wheat due to the strict control of homoeologous recombination by pairing homoeologous (Ph) genes in hexaploid wheat backgrounds [27–29]. Furthermore, molecular markers of alien chromosome-specificity were limited for fine mapping of alien genes. Regardless, with the rapid development of high-throughput sequencing, sequencing-based technologies such as RNA-seq have been frequently used to develop molecular markers [1,30,31], detect expression pattern and level of genes responded to pathogens , exploit new genes and identify gene function without prior information of the particular reference genome sequences [33,34]. RNA-seq is very helpful to explore disease-resistant genes derived from wild relatives. For example, Li et al. (2016) obtained eight powdery mildew resistance-related genes from Thinopyrum intermedium by RNA-seq analysis . Zou et al. (2018) successfully isolated a powdery mildew resistance gene Pm60 from T. urartu by combining genetic mapping and RNA-seq analysis .
In this study, we report the assays of a broad-spectrum resistance gene(s) on chromosome 2Mb derived from Ae. biuncialis, discovery of 2Mb-specific candidate genes of powdery mildew resistance, and development of molecular markers of 2Mb specificity based on transcriptome sequencing of CS-Ae. biuncialis 2Mb disomic addition line TA7733. This study will provide foundations for the transfer and cloning of resistance gene(s) from chromosome 2Mb, as well as further understanding of the molecular and genetic mechanisms of disease resistance conferred by Ae. biuncialis chromosome 2Mb.
Materials and methods
Common wheat landrace CS (2n = 6x = 42, AABBDD), Ae. comosa TA2102 (2n = 2x = 14, MM), and CS-Ae. biuncialis 2Mb disomic addition line TA7733 (2n = 44) where a pair of 2Mb chromosomes derived from Ae. biuncialis were added into CS genetic background were used in this study. All the materials were kindly provided by the Wheat Genetics Resource Center at Kansas State University, USA and maintained at the experimental station of Henan Agricultural University, China.
Chromosome spreads were prepared from root tip cells as described by Huang et al. (2018) . The cytological observations were performed using a BX51 Olympus phase contrast microscope (Olympus Corporation, Tokyo, Japan).
Genomic DNA (gDNA) was extracted from fresh leaves using a modified hexadecyl trimethyl ammonium bromide (CTAB) method . The concentration and purity of DNA were measured with the Nanophotometer P360 (Implen GmbH, München, Germany).
Genomic in situ hybridization (GISH) was applied to analyze the chromosomal composition of TA7733. Genomic DNA of Ae. comosa accession TA2102 (genome Mb donor of Ae. biuncialis) and wheat CS were respectively used for probe labeling with fluorescein-12-dUTP and blocking at a ratio of 1:130 to distinguish Ae. biuncialis 2Mb chromosome. GISH was carried out as described by Liu et al. (2017) . Hybridization signals were observed under an OLYMPUS AX80 (Olympus Corporation, Tokyo, Japan) fluorescence microscope, captured with a CCD camera (Diagnostic Instruments, Inc., Sterling Heights, MI, USA) and processed with Photoshop CS 3.0.
After GISH, the hybridization signals were washed off with phosphate-buffered saline (PBS). Eight single-strand oligonucleotides were then used as probes for dual-color nondenaturing fluorescence in situ hybridization (ND-FISH) [36,39]. The eight oligonucleotides includes Oligo-pAs1-1, Oligo-pAs1-3, Oligo-pAs1-4, Oligo-pAs1-6, Oligo-AFA-3, Oligo-AFA-4, Oligo-pSc119.2–1 and Oligo-(GAA)10. The first six were labeled with 6-carboxytetramethylrhodamine (TAMRA) generating red signals, and the last two being labeled with 6-carboxyfuorescein (FAM) generating green signals. All the oligonucleotides were synthesized at Sangon Biological Technology, Shanghai, China.
Evaluation of powdery mildew resistance
A mixture of prevailing Bgt isolates collected in Henan Province were used to evaluate the resistance of CS and CS-Ae. biuncialis 2Mb disomic addition line TA7733. Fifteen prevalent Bgt isolates collected from different regions of China were chosen to evaluate the resistance spectrum of TA7733 at the seedling stage by using CS as a susceptible control. The 15 Bgt isolates were provided by Prof. Pengtao Ma, Yantai University, China. They were all single-pustule-derived powdery mildew virulent isolates by separate artificial inoculation. The infection type (IT) were scored 7–10 days post-inoculation using a 0 to 4 rating scale , with 0 as immune, 0; as nearly immune, 1 as highly resistant, 2 as moderately resistant, 3 as moderately susceptible, and 4 as highly susceptible. IT 0 to 2 were considered as resistance, while IT 3 to 4 were being susceptible.
At 10 days post-Bgt inoculation, the first leaves of TA7733 and CS were cut into 2 cm segments and stained with coomassie brilliant blue following Li et al. (2016)  for further microscopic observation of Bgt development on the leaves.
Illumina library construction and sequencing
Seeds of CS and TA7733 soaking in water for 24 h at 23°C were transferred into a mixture of nutrient soil and vermiculite (1:1). Seedlings with full extended first leaf were dusted using fresh conidiophores of Bgt isolates. Leaves at 0, 12, 24, 48 and 72 hours post-inoculation (hpi) were respectively collected, rapidly frozen in liquid nitrogen and stored at -80°C for RNA extraction.
Total RNA of ten samples (0, 12, 24, 48 and 72 hpi for CS and TA7733, each) were extracted for transcriptome sequencing. Then equal amounts of RNA samples 12–72 hpi from TA7733 and CS were mixed to generate RNA-seq sample RI and SI, respectively. RNA at 0 hpi from TA7733 and CS were accordingly represented as RNA-seq sample RC and SC. Two biological replicates were performed in this study, forming a total of eight RNA samples (RI1, RI2, RC1, RC2, SI1, SI2, SC1 and SC2). The designations 1 and 2 are used to represent replicates 1 and 2, respectively. Libraries with an average insert size of 200 bp constructed from these eight samples were then sequenced using the Illumina HiSeqTM 2500 by the Beijing Genomics Institute.
Reads processing, assembly, and sequence annotation
Prior to assembly, sequencing raw reads were pre-processed using a Perl script dynamic-Trim.pl to remove the adaptor sequences, low-quality sequences, low complexity sequences, short reads and empty reads. Reads data with a quality score (Qphred) ≥ 50 (Q50: ratio of an error rate of 0.01%) were then merged and input into the data assembly software Trinity for assembling into transcripts. The generated unigenes were annotated by a Blastx alignment search (E-value<10−5) against the NCBI non-redundant (NR) protein, SWISSPROT, gene ontology (GO), eukaryotic orthologous groups (KOG), kyoto encyclopedia of genes and genomes (KEGG) and plant resistance gene (PRG) databases.
Amplification and analyses of candidate disease resistance genes
R gene-specific primer sets were designed based on their transcriptome sequences to perform PCR amplification using gDNA from TA7733 and CS as templates to verify 2Mb specific genes. PCR amplification were conducted in 15 μl reaction volumes containing 2 μl template gDNA (100 ng/μl), 0.25 μl forward primer (10 μmol/l), 0.25 μl reverse primer (10 μmol/l), 7.5 μl Taq MasterMix (CW Bio Inc., China) and 5 μl ddH2O. PCR cycling conditions were as follows: 94°C for 5 min followed by 35 cycles of 94°C for 30 s, 50–66°C for 30 s, and 72°C for 1 min, followed by a final 10-min extension at 72°C. The PCR products were digested with four base-restriction enzymes. Five microliters of a restriction enzyme mixture containing 2.8 μl of ddH2O, 2.0 μl of CutSmart buffer, and 0.2 μl of an enzyme stock solution was added to 15 μl of PCR products and incubated for 3.5 h at 65°C. The PCR or restricted PCR products were separated on a 2.0% agarose gel-electrophoresis stained with ethidium bromide and visualized by UV light.
Mapping candidate disease resistance genes onto chromosome 2Mb
Genome sequences of wheat landrace CS (CS Ref Seq v1.0) were used as references in Blastn searches to obtain position information for R genes from Ae. biuncialis chromosome 2Mb. Comparative maps of 2Mb-specific R genes were made using MapDraw software referring homoeologous chromosome locations of CS Ref Seq v1.0.
Cytogenetic analysis of CS-Ae. biuncialis 2Mb disomic addition line TA7733
GISH and ND-FISH were respectively performed to confirm the chromosome composition of CS-Ae. biuncialis 2Mb disomic addition line TA7733 by using fluorescein-labeled gDNA from M genome donor Ae. comosa as a probe and wheat CS DNA as blocker. As shown in Fig 1, there were 44 chromosomes including 42 wheat chromosomes and plus a pair of Ae. biuncialis 2Mb chromosomes in TA7733, confirming the disomic addition of chromosome 2Mb.
(A) GISH patterns of CS-Ae. biuncialis 2Mb disomic addition line TA7733. Total gDNA of Ae. comosa was labelled with fluorescein-12-dUTP and visualized with green fluorescence. (B) ND-FISH patterns of CS-Ae. biuncialis 2Mb disomic addition line TA7733. Blue color indicated chromosomes counterstained with DAPI. Red color showed signals from oligos pAs1-1, pAs1-3, pAs1-4, pAs1-6, AFA-3 and AFA-4. Green color showed signals from oligos pSc119.2–1 and (GAA)10. The arrows indicated Ae. biuncialis chromosome 2Mb.
Assay of powdery mildew resistance of CS-Ae. biuncialis 2Mb disomic addition line TA7733
A mixture of prevalent Bgt isolates collected in Henan Province was used to inoculate seedlings with fully-extended first leaves of TA7733 and its recipient parent CS in the greenhouse. Ten days post-inoculation, the leaves of CS were covered with a large number of Bgt hyphae, with ITs of 3–4, whereas TA7733 showed only stunted spores, with ITs 0 to 1 (Fig 2A). Microscopic observation of first leaf segments stained with coomassie brilliant blue displayed that leaves of susceptible CS were covered with hyphae and spores had formed, while TA7733 only had a few blue spores on leaves (Fig 2B), further confirming that TA7733 was high resistance to powdery mildew. Since CS forms the genetic background TA7733 and is susceptible, the gene(s) conferring resistance to powdery mildew was therefore mapped to chromosome 2Mb derived from Ae. biuncialis.
(A) Disease symptoms of the first leaf of TA7733 and CS at 10 days post Bgt inoculation. (B) Microscopic observation of Bgt development on leaves of TA7733 and CS after staining with coomassie brilliant blue-R-250.
The resistance spectrum of TA7733 was further assayed at the seedling stage by inoculation of 15 prevalent Bgt isolates collected from different regions of China. As shown in Table 1, CS-Ae. biuncialis 2Mb disomic addition line TA7733 showed high level of resistance (IT = 0 or 1) to all the 15 Bgt isolates tested, whereas its recipient parent CS was highly susceptible (IT = 4 or 3) to all tested Bgt isolates. These results indicated that the chromosome 2Mb in TA7733 conferred broad-spectrum resistance to powdery mildew of wheat.
Transcriptome sequencing, de novo assembly and functional annotation
RNA-seq of CS-Ae. biuncialis 2Mb disomic addition line TA7733 and its recipient parent CS were respectively conducted pre and post Bgt-infection. A total of 158,953 unigenes were assembled with a total length of 198,364,757 bp. The average unigene size was 1247.95 bp ranging from 301 to 19,496 bp (Fig 3). Gene function annotation with Blastx to the six public databases (NCBI NR protein, SWISSPROT, GO, KOG, KEGG and PRG databases) using a cutoff E-value of 10−5 resulted in 86,196 (54.23%), 48,724 (30.65%), 40,543 (25.51%), 37,008 (23.28%), 13,414 (8.44%) and 10,969 (6.92%) annotated unigenes, respectively (Table 2). Of which, 86,862 (54.65%) unigenes matched to at least one of the databases.
GO is an international classification system for standardized gene functions, which have three categories: biological process, molecular function and cellular component. A total of 40,543 (25.51% of 158,953) unigenes were assigned to one or more GO term annotations (Fig 4 and S1 Table), of which, “cellular process” (27,404; 67.59% of 40,543), “metabolic process” (24,470; 60.35% of 40,543), and “single-organism process” (20,606; 50.82% of 40,543) were the cardinal terms in the biological process category. In the cellular component category, “cell” (30,742; 75.82% of 40,543), “cell part” (30,695; 75.71% of 40,543), and “organelle” (24,100; 59.44% of 40,543) were the most abundant terms. “Binding” (24,379; 60.13% of 40,543) and “catalytic activity” (21,879; 53.96% of 40,543) were the most representative terms in the molecular function category. Instead, only a few unigenes assigned into the terms of “extracellular matrix part” (9; 0.02% of 40,543), “protein tag” (8; 0.02% of 40,543) and “receptor regulator activity” (1; 0.0024% of 40,543).
The KEGG database was used to systematically describe the pathway where the unigenes involved. Out of a total 158,953 annotated unigenes, 26,589 unigenes were assigned to 23 KEGG pathways (Fig 5 and S2 Table). The most representative pathways in which unigenes involved were the metabolic pathways (11,920, 44.83%), genetic information processing (5,456, 20.52%), environmental information processing (5,095, 19.16%) and cellular processes (4,118, 15.49%).
Analyses of genes involved in responses to Bgt infection from TA7733
One of the objectives of this study was to explore putative R genes specific to Ae. biuncialis chromosome 2Mb, which should be only expressed in TA7733 other than in CS. Based on pairwise comparison of unigenes of TA7733 vs CS, a total of 7,278 genes were uniquely expressed in TA7733, of which 4,382 unigenes were significantly differentially expressed post vs before Bgt-inoculation, and the remaining 2,896 unigenes had insignificantly different expression levels. In consideration of the fact that expression levels of some cloned resistance genes did show no significant difference before and after pathogen infection [9,41], these 2,896 unigenes of TA7733 specificity were also taken for a further selection of chromosome 2Mb-derived R-genes involved in powdery mildew resistance.
To analyze the biological pathways of these 4,382 unigenes, the statistical enrichment of differentially expressed genes (DEGs) in KEGG pathways were tested using the KOBAS software. In consequence, 399 out of 4,382 DEGs were allocated to 162 KEGG pathways (S3 Table). The most representative pathways included the phenylpropanoid biosynthesis (28, 7.02%), plant hormone signal transduction (23, 5.76%), flavonoid biosynthesis (15, 3.76%), stilbenoid, diarylheptanoid and gingerol biosynthesis (14, 3.51%) and MAPK signaling pathway-plant (14, 3.51%), then followed by glutathione metabolism (13, 3.26%), protein processing in endoplasmic reticulum (13, 3.26%), and metabolism of xenobiotics by cytochrome P450 (12, 3.00%). These annotations provided valuable clues in the investigation of the specific processes and identification of the genes involved in powdery mildew resistance conferred by Ae. biuncialis 2Mb chromosome.
Screening and verification of disease resistance gene of chromosome 2Mb specificity
Based on transcriptome data analysis, 7,278 unigenes were uniquely expressed in TA7733. Only 295 of 7,278 unigenes were annotated as putative R-genes by Blastx alignment against the PRG and NCBI databases. However, of 295 R-genes sequences when blastn searched against CS Ref Seq v1.0, only 61 (20.68%) R-genes mapped to wheat homoeologous group 2, and the remaining 234 (79.32%) R-genes to none of the homoeologous group 2 chromosome of wheat.
In order to verify whether these 61 R genes which had an orthologous copy on wheat homoeologous group 2 were derived from Ae. biuncialis chromosome 2Mb, a total of 61 sets of PCR primer pairs were designed based on transcriptome sequences of these R genes. PCR amplification of gDNA of CS and TA7733 confirmed 40 R genes to be specific to chromosome 2Mb, which producing unique amplification in CS-Ae. biuncialis 2Mb disomic addition line TA7733 (S1 Fig and S4 Table). So these 40 R genes were considered as putative candidate genes involved in powdery mildew resistance conferred by Ae. biuncialis 2Mb chromosome.
Chromosome structure variation such as translocation occurs during biological evolution process, whether it occurred in wheat or its wild relatives, will lead to the changes of homoeologous groups. Among the remaining 234 putative R genes of TA7733 specificity which were mapped to none of the homoeologous group 2 chromosome, additional 13 R genes were verified to be also chromosome 2Mb specific by PCR analysis using primer pairs designed based on their transcriptome sequences (S1 Fig and S4 Table). These 13 R genes were also considered as candidate genes involved in powdery mildew resistance conferred by Ae. biuncialis 2Mb chromosome, which adding the number of candidate R genes of 2Mb specificity to a total of 53 (Tables 3 and S5 and S6).
Alignment of these 53 R genes to PRG database assigned 33 putative genes, of which 14 R genes were in CNL class which contains a predicted coiled-coil (CC) structures, a central nucleotide-binding (NB) subdomain and a leucine-rich repeat (LRR) domain, 12 in NL class containing NBS and LRR domains, but lack of CC domain, five in class RLP which contains leucine-rich receptor-like repeat, a transmembrane region of 25AA, and a short cytoplasmic region, each one for TNL class which contains a central NB subdomain, a LRR domain, a interleukin-1 receptor (1L-1R) domain, and N class only containing NBS domain (Table 4). The remaining 20 putative R genes aligned to NCBI database, were predicted encoding protein kinase, disease resistance protein RGA, disease resistance protein RP and cytochrome P450, respectively.
Comparative mapping was carried out by using MapDraw software based on alignment of sequences of these 53 R genes of Ae. biuncialis chromosome 2Mb specificity with those in CS Ref Seq v1.0 (Fig 6). The maps showed that 21, 16 and three R genes were located to the terminal of the long arms and the short arms, and close to the centromeres of wheat homoeologous group 2 chromosomes, respectively. Whereas the remaining 13 R genes were mapped to none-homoeologous group 2, which included wheat chromosomes 3B, 4A, 5B, 6A, 6B, 7B and 7D (Fig 6).
Development of resistant wheat varieties is the most important and environment-friendly way to control Bgt-caused damages. The genes with broad spectrum and durability resistance make them highly valuable in wheat breeding programs . Wild relatives of common wheat harbored considerable genetic diversity for powdery mildew resistance. For example, the wild relatives of common wheat, Secale cereale, Dasypyrum villosum and Ae. searsii conferred powdery mildew resistance gene Pm7, PmJZHM2RL, Pm62 and Pm57 from homoeologous group 2 [38, 42–44]. In this study, resistance assay by using 15 Bgt isolates collecting from different regions in China, verified that Ae. biuncialis 2Mb chromosome in TA7733 conferred broad-spectrum resistance to powdery mildew of wheat. Currently no any other catalogued Pm genes were reported to be derived from Ae. biuncialis homoeologous group 2. Therefore the resistance gene(s) on Ae. biuncialis 2Mb chromosome should be a new Pm gene(s).
Previous studies have generally focused on the significantly differentially expressed genes in interactions between plant and pathogens to explore disease resistance-related genes by transcriptome sequencing [45–47]. However, it was reported that expressions of some cloned genes of disease resistance in plants were not significantly up-regulated after pathogens infection. For example, Zou et al. (2017) reported that the transcription levels of Pm60, a map-based cloned powdery mildew gene, showed no significant differences at various time points after Bgt E09 infection based on qRT-PCR analysis . Li et al. (2017) also discovered that the expression levels of broad-spectrum blast resistance gene bsr-d1 in rice were not significantly up-regulated after blast infection . So, the opportunity to discover disease resistance gene candidates might be undermined if only significantly regulated genes were chosen. In this study we explored Bgt-resistance related candidate genes from all specifically expressed R genes in TA7733 regardless of significance of their expression level changes post vs before Bgt-infection. After PCR verification by using R gene sequence-based primer sets and integrating transcriptome sequences blastn against CS Ref Seq v1.0, we finally verified 53 R genes candidates of chromosome 2Mb specificity, which included 47 unregulated, four up-regulated and two down-regulated genes.
Isolation of plant resistance (R) gene is greatly helpful to breed resistant varieties and elucidates resistance molecular mechanisms. Conventional map-based cloning proved to be a effective method to clone R genes , however, it is time consuming and difficult to fine map R genes from wild relatives of common wheat due to lack of exchange and recombination between the alien chromatin and wheat homoeologous counterpart. Of more than 70 R genes against diverse pathogens currently isolated from various plants by using map-based cloning , nearly three quarters of R genes encoded NBS-LRR protein, which reportedly recognized pathogens and initiated defense responses subsequently . To date, five out of 89 Pm genes including Pm2 , Pm3 , Pm8 , Pm21 [28,52] and Pm60 , have been cloned, all these genes encoded CC-NBS-LRR proteins. In this study, 14 out of 53 2Mb-specific R genes were predicted to encode CC-NBS-LRR protein. These 14 R genes should be considered as the most promising candidate genes for further isolating and cloning Pm genes carried by Ae. biuncialis chromosome 2Mb.
GISH is a popular visual method to identify alien chromosome or chromatin in wheat background. Whereas GISH is expensive and time-consuming especially used to screen a large population derived from distant crossing between wheat and its wild relatives . In contrast, molecular markers are not affected by environmental conditions, tissue or developmental stage and gene expression, and possess high genetic polymorphism [54,55]. So the development and application of molecular markers have been considered as new and low-cost ways to quickly identify alien chromosomes or chromatin. High-throughput RNA-seq technology can generate large amounts of transcriptome sequences and has been widely used to develop molecular markers specific to chromosomes of wild relatives of wheat, especially those with limited genomic sequence references. For example, Li et al. (2017) developed 25 D. villosum 6V#4S-specifc markers using transcriptome data . Wang et al. (2018) developed 134 Ae. longissima chromosome-specific markers by RNA-seq . Li et al. (2019) developed 76 molecular markers specific to the chromosome 1V to 7V of D. villosum#4 based on transcriptome data . Furthermore, the transcription sequences were highly conserved and might be associated with the genes that related to a definite trait . Therefore, the markers developed by transcriptome sequencing will accelerate the identification of candidate functional genes, and increase the efficiency of marker-assisted selection [57,58]. In this study, 53 functional molecular markers of R genes based on transcriptome data analyses were verified to be specific to Ae. biuncialis chromosome 2Mb. These markers will be useful to assist the transfer resistance gene(s) from 2Mb into common wheat by inducing CS-Ae. biuncialis 2Mb homoeologous recombination for wheat disease breeding in the future.
In summary, powdery mildew resistance gene(s) on Ae. biuncialis chromosome 2Mb was verified to be board-spectrum in this study. It could be a valuable disease-resistance resource for wheat breeding programs. Fifty-three disease resistance gene candidates of 2Mb specificity, which were selected based on transcriptome sequencing analyses, will be greatly helpful to further isolate and clone Pm gene(s) derived from chromosome 2Mb and provide the insights into molecular mechanism of 2Mb-conferred powdery mildew resistance. Furthermore, 53 R gene sequence-based functional molecular markers of 2Mb specificity in this study will facilitate the transfer of resistance gene(s) from 2Mb to common wheat by inducing CS-Ae. biuncialis homoeologous recombination.
S1 Fig. Amplification patterns of 53 candidate Ae. biuncialis chromosome 2Mb-specific primers.
(M) 100 bp DNA Ladder. (1, 3) CS. (2, 4) TA7733. (A) CL119404Contig1. (B) CL88277Contig1. (C) CL82670Contig1. (D) 82789Contig1. (E) CL82700Contig1. (F) CL85355Contig1. (G) CL66003Contig1. (H) CL89405Contig1. (I) CL106750Contig1. (J) CL119216Contig1. (K) CL19981Contig2. (L) CL93721Contig1. (M) CL84424Contig1. (N) CL88613Contig1. (O) CL91022Contig1. (P) 96221Contig1. (Q) comp19533_c0_seq1_6. (R) CL85258Contig1. (S) CL113949Contig1. (T) CL86521Contig1. (U) CL105879Contig1. (V) CL90029Contig1. (W) CL84846Contig2. (X) CL87530Contig1. (Y) CL29910Contig1. (Z) CL92547Contig1. (AA) CL75219Contig1. (AB) CL108886Contig1. (AC) CL113652Contig1. (AD) CL80063Contig1. (AE) CL89447Contig1. (AF) CL93169Contig1. (AG) CL114224Contig1. (AH) CL116612Contig1. (AI) CL67241Contig1. (AJ) CL119539Contig1. (AK) CL90483Contig1. (AL) CL91742Contig1. (AM) comp84147_c0_seq1_6. (AN) CL124Contig7. (AO) CL100654Contig1. (AP) CL104996Contig1. (AQ) CL107524Contig1. (AR) CL107607Contig1. (AS) CL465Contig5. (AT) CL66266Contig1. (AU) CL72629Contig1. (AV) CL75868Contig1. (AW) CL86319Contig1. (AX) CL79458Contig1. (AY) comp121700_c0_seq1_5. (AZ) comp80277_c0_seq1_7. (BA) comp93868_c0_seq1_7.
S1 Raw_image. (original image of S1 Fig): Raw images of amplification patterns of 53 candidate Ae. biuncialis chromosome 2Mb-specific primers.
Lanes: M, 100 bp Ladder DNA Marker; 1, common wheat CS; 2, CS-Aegilops biuncialis 2Mb disomic addition line TA77333; 3, common wheat CS; 4, CS-Ae. biuncialis 2Mb disomic addition line TA77333. (A) CL119404Contig1. (B) CL88277Contig1. (C) CL82670Contig1. (D) 82789Contig1. (E) CL82700Contig1. (F) CL85355Contig1. (G) CL66003Contig1. (H) CL89405Contig1. (I) CL106750Contig1. (J) CL119216Contig1. (K) CL19981Contig2. (L) CL93721Contig1. (M) CL84424Contig1. (N) CL88613Contig1. (O) CL91022Contig1. (P) 96221Contig1. (Q) comp19533_c0_seq1_6. (R) CL85258Contig1. (S) CL113949Contig1. (T) CL86521Contig1. (U) CL105879Contig1. (V) CL90029Contig1. (W) CL84846Contig2. (X) CL87530Contig1. (Y) CL29910Contig1. (Z) CL92547Contig1. (AA) CL75219Contig1. (AB) CL108886Contig1. (AC) CL113652Contig1. (AD) CL80063Contig1. (AE) CL89447Contig1. (AF) CL93169Contig1. (AG) CL114224Contig1. (AH) CL116612Contig1. (AI) CL67241Contig1. (AJ) CL119539Contig1. (AK) CL90483Contig1. (AL) CL91742Contig1. (AM) comp84147_c0_seq1_6. (AN) CL124Contig7. (AO) CL100654Contig1. (AP) CL104996Contig1. (AQ) CL107524Contig1. (AR) CL107607Contig1. (AS) CL465Contig5. (AT) CL66266Contig1. (AU) CL72629Contig1. (AV) CL75868Contig1. (AW) CL86319Contig1. (AX) CL79458Contig1. (AY) comp121700_c0_seq1_5. (AZ) comp80277_c0_seq1_7. (BA) comp93868_c0_seq1_7.
S1 Table. Gene ontology of transcriptome of CS-Ae. biuncialis 2Mb disomic addition line TA7733.
S2 Table. The KEGG pathway classification of transcriptome of CS-Ae. biuncialis 2Mb disomic addition line TA7733.
S3 Table. KEGG pathway classification of DEGs of CS-Ae. biuncialis 2Mb disomic addition line TA7733.
S4 Table. The 53 Ae. biuncialis chromosome 2Mb-specific markers developed in this study based on unigenes annotated as R genes.
S5 Table. The expression levels of 53 candidate disease resistance genes from Ae. biuncialis chromosome 2Mb.
We sincerely thank Dr. Yuli Song (Institute of Plant Protection, Henan Academy of Agricultural Sciences) for providing prevailing Bgt isolates collected in Henan Province. We would like to thank Dr. Sunish Sehgal (Department of Agronomy, Horticulture and Plant Science, South Dakota State University) for critical reading and amendment of the manuscript.
- 1. Ramirez-Gonzalez RH, Segovia V, Bird N, Fenwick P, Holdgate S, Berry S, et al. RNA-Seq bulked segregant analysis enables the identification of high-resolution genetic markers for breeding in hexaploid wheat. Plant Biotechnol J. 2015; 13: 613–624. pmid:25382230
- 2. Fu DL, Uauy C, Distelfeld A, Blechl A, Epstein L, Chen XM, et al. A kinase-START gene confers temperature-dependent resistance to wheat stripe rust. Science. 2009; 323: 1357–1360. pmid:19228999
- 3. Griffey C, Das M, Stromberg EJ. Effectiveness of adult-plant resistance in reducing grain yield loss to powdery mildew in winter wheat. Plant Dis. 1993; 77: 618–622.
- 4. Li GQ, Carver BF, Cowger C, Bai GH, Xu XY. Pm223899, a new recessive powdery mildew resistance gene identified in Afghanistan landrace PI223899. Theor Appl Genet. 2018; 131: 2775–2783. pmid:30327847
- 5. Ma PT, Xu HX, Luo QL, Qie YM, Zhou YL, Xu YF, et al. Inheritance and genetic mapping of a gene for seedling resistance to powdery mildew in wheat line X3986-2. Euphytica. 2014; 200: 149–157.
- 6. Morgounov A, Tufan HA, Sharma R, Akin B, Bagci A, Braun HJ, et al. Global incidence of wheat rusts and powdery mildew during 1969–2010 and durability of resistance of winter wheat variety Bezostaya 1. Eur J Plant Pathol. 2012; 132: 323–340.
- 7. Huang J, Zhao ZH, Song FJ, Wang XM, Xu HX, Huang Y, et al. Molecular detection of a gene effective against powdery mildew in the wheat cultivar Liangxing 66. Mol Breed. 2012; 30: 1737–1745.
- 8. Zhang DY, Zhu KY, Dong LL, Liang Y, Li GQ, Fang TL, et al. Wheat powdery mildew resistance gene Pm64 derived from wild emmer (Triticum turgidum var. dicoccoides) is tightly linked in repulsion with stripe rust resistance gene Yr5. 2019; https://doi.org/10.1016/j.cj.2019.03.003.
- 9. Zou SH, Wang H, Li YW, Kong ZS, Tang DZ. The NB-LRR gene Pm60 confers powdery mildew resistance in wheat. New Phytol. 2018; 218: 298–309. pmid:29281751
- 10. McDonald BA, Linde C. Pathogen population genetics, evolutionary potential, and durable resistance. Annu Rev Phytopathol. 2002; 40: 349–379. pmid:12147764
- 11. Xiao MG, Song FJ, Jiao JF, Wang XM, Xu HX, Li HJ. Identification of the gene Pm47 on chromosome 7BS conferring resistance to powdery mildew in the Chinese wheat landrace Hongyanglazi. Theor Appl Genet. 2013; 126: 1397–1403. pmid:23429903
- 12. Ma PT, Xu HX, Zhang HX, Li LH, Xu YF, Zhang XT, et al. The gene PmWFJ is a new member of the complex Pm2 locus conferring unique powdery mildew resistance in wheat breeding line Wanfengjian 34. Mol Breed. 2015; 35: 210.
- 13. Summers R, Brown J. Constraints on breeding for disease resistance in commercially competitive wheat cultivars. Plant Pathol. 2013; 62: 115–121.
- 14. Resta P, Zhang HB, Dubcovsky J, Dvořák J. The origins of the genomes of Triticum biunciale, T. ovatum, T. neglectum, T. columnare, and T. rectum (Poaceae) based on variation in repeated nucleotide sequences. Am J Bot. 1996; 83: 1556–1565.
- 15. Badaeva E, Amosova A, Samatadze T, Zoshchuk S, Shostak N, Chikida N, et al. Evolution, Genome differentiation in Aegilops. 4. Evolution of the U-genome cluster. Plant Sys Evol. 2004; 246: 45–76.
- 16. Damania AB, Pecetti L. Variability in a collection of Aegilops species and evaluation for yellow rust resistance at two locations in northern Syria. J Genet Breed. 1990; 44: 97–102.
- 17. Dimov A, Zaharieva M, Mihova S. Rust and powdery mildew resistance in Aegilops accessions from Bulgaria. In: Damania AB, editors. 1993. pp. 165–169.
- 18. Makkouk K, Ghulam W, Comeau A. Resistance to barley yellow dwarf luteovirus in Aegilops species. Can J Plant Sci. 1994; 74: 631–634.
- 19. Zhao H, Zhang W, Wang J, Li F, Cui F, Ji J, et al. Comparative study on drought tolerance of wheat and wheat-Aegilops biuncialis 6Ub addition lines. J Food Agric Environ. 2013; 11: 1046–1052.
- 20. Molnár I, Gáspár L, Sárvári É, Dulai S, Hoffmann B, Molnár-Láng M, et al. Physiological and morphological responses to water stress in Aegilops biuncialis and Triticum aestivum genotypes with differing tolerance to drought. Funct Plant Biol. 2004; 31: 1149–1159.
- 21. Colmer TD, Flowers TJ, Munns, R. Use of wild relatives to improve salt tolerance in wheat. J Exp Bot. 2006; 57: 1059–1078. pmid:16513812
- 22. Farkas A, Molnár I, Dulai S, Rapi S, Oldal V, Cseh A, et al. Increased micronutrient content (Zn, Mn) in the 3Mb(4B) wheat-Aegilops biuncialis substitution and 3Mb.4BS translocation identified by GISH and FISH. Genome. 2014; 57: 61–67. pmid:24702063
- 23. Zhou JP, Yao CH, Yang EN, Yin MQ, Liu C, Ren ZL. Characterization of a new wheat-Aegilops biuncialis addition line conferring quality-associated HMW glutenin subunits. Genet Mol Res. 2014; 13: 660–669. pmid:24615031
- 24. Schneider A, Linc G, Molnár I, Molnár-Láng M. Molecular cytogenetic characterization of Aegilops biuncialis and its use for the identification of 5 derived wheat-Aegilops biuncialis disomic addition lines. Genome. 2005; 48: 1070–1082. pmid:16391676
- 25. Schneider A, Molnar-Lang M. Detection of various U and M chromosomes in wheat-Aegilops biuncialis hybrids and derivatives using fluorescence in situ hybridisation and molecular markers. Czech J Genet Plant Breed. 2012; 48: 169–177.
- 26. Xia Q, Mai YN, Dong ZJ, Liu WX. Identification of powdery mildew resistance resources from wheat-wild relative disomic addition lines and development of molecular markers of alien chromosome-specialty. J Henan Agric Sci. 2018; 47: 64–69.
- 27. Wang ZZ, Li HW, Zhang DY, Guo L, Chen JJ, Chen YX, et al. Genetic and physical mapping of powdery mildew resistance gene MlHLT in Chinese wheat landrace Hulutou. Theor Appl Genet. 2015; 128: 365–373. pmid:25471672
- 28. He HG, Zhu SY, Zhao RH, Jiang ZN, Ji YY, Ji J, et al. Pm21, encoding a typical CC-NBS-LRR protein, confers broad-spectrum resistance to wheat powdery mildew disease. Mol Plant. 2018; 11: 879–882. pmid:29567454
- 29. Cao AZ, Xing LP, Wang XY, Yang XM, Wang W, Sun YL, et al. Serine/threonine kinase gene Stpk-V, a key member of powdery mildew resistance gene Pm21, confers powdery mildew resistance in wheat. Proc Natl Acad Sci. 2011; 108: 7727–7732. pmid:21508323
- 30. Wang KY, Lin ZS, Wang L, Wang K, Shi QH, Du LP, et al. Development of a set of PCR markers specific to Aegilops longissima chromosome arms and application in breeding a translocation line. Theor Appl Genet. 2018; 131: 13–25. pmid:28887628
- 31. Li SJ, Wang J, Wang KY, Chen JN, Wang K, Du LP, et al. Development of PCR markers specific to Dasypyrum villosum genome based on transcriptome data and their application in breeding Triticum aestivum-D. villosum#4 alien chromosome lines. BMC Genomics. 2019; 20: 289. pmid:30987602
- 32. Rubio M, Rodríguez-Moreno L, Ballester AR, Moura MC, Bonghi C, Candresse T, et al. Analysis of gene expression changes in peach leaves in response to Plum pox virus infection using RNA-Seq. Mol Plant Pathol. 2015; 16: 164–176. pmid:24989162
- 33. Zhang H, Fu Y, Guo H, Zhang L, Wang CY, Song WN, et al. Transcriptome and proteome-based network analysis reveals a model of gene activation in wheat resistance to stripe rust. Int J Mol Sci. 2019; 20: 1106.
- 34. Zhang H, Yang YZ, Wang CY, Liu M, Li H, Fu Y, et al. Large-scale transcriptome comparison reveals distinct gene activations in wheat responding to stripe rust and powdery mildew. BMC genomics. 2014; 15: 898. pmid:25318379
- 35. Li QQ, Niu ZB, Bao YG, Tian QJ, Wang HG, Kong LR, et al. Transcriptome analysis of genes related to resistance against powdery mildew in wheat-Thinopyrum alien addition disomic line germplasm SN6306. Gene. 2016; 590: 5–17. pmid:27265028
- 36. Huang XY, Zhu MQ, Zhuang LF, Zhang SY, Wang JJ, Chen XJ, et al. Structural chromosome rearrangements and polymorphisms identified in Chinese wheat cultivars by high-resolution multiplex oligonucleotide FISH. Theor Appl Genet. 2018; 131: 1967–1986. pmid:29947816
- 37. Li HH, Jiang B, Wang JC, Lu YQ, Zhang JP, Pan CL, et al. Mapping of novel powdery mildew resistance gene(s) from Agropyron cristatum chromosome 2P. Theor Appl Genet. 2017; 130: 109–121. pmid:27771744
- 38. Liu WX, Koo DH, Xia Q, Li CX, Bai FQ. Song YL, et al. Homoeologous recombination-based transfer and molecular cytogenetic mapping of powdery mildew-resistant gene Pm57 from Aegilops searsii into wheat. Theor Appl Genet. 2017; 130: 841–848. pmid:28116459
- 39. Du P, Zhuang LF, Wang YZ, Yuan L, Wang Q, Wang DR, et al. Development of oligonucleotides and multiplex probes for quick and accurate identification of wheat and Thinopyrum bessarabicum chromosomes. Genome. 2016; 60: 93–103. pmid:27936984
- 40. Li GQ, Fang TL, Zhang HT, Xie CJ, Li HJ, Yang T, et al. Molecular identification of a new powdery mildew resistance gene Pm41 on chromosome 3BL derived from wild emmer (Triticum turgidum var. dicoccoides). Theor Appl Genet. 2009; 119: 531–539. pmid:19471905
- 41. Li WT, Zhu ZW, Chern M, Yin JJ, Yang C, Ran L, et al. A natural allele of a transcription factor in rice confers broad-spectrum blast resistance. Cell. 2017; 170: 114–126. pmid:28666113
- 42. Zhuang L, Sun L, Li AX, Chen TT, Qi ZJ. Identification and development of diagnostic markers for a powdery mildew resistance gene on chromosome 2R of Chinese rye cultivar Jingzhouheimai. Mol Breed. 2011; 27: 455–465.
- 43. Jiang J, Friebe B, Gill BS. Recent advances in alien gene transfer in wheat. Euphytica. 1993; 73: 199–212.
- 44. Zhang RQ, Fan YL, Kong LN, Wang ZJ, Wu JZ, Xing LP, et al. Pm62, an adult-plant powdery mildew resistance gene introgressed from Dasypyrum villosum chromosome arm 2VL into wheat. Theor Appl Genet. 2018; 131: 2613–2620. pmid:30167758
- 45. Tan GX, Liu K, Kang JM, Xu KD, Zhang Y, Hu LZ, et al. Transcriptome analysis of the compatible interaction of tomato with Verticillium dahliae using RNA-sequencing. Front Plant Sci. 2015; 6: 428. pmid:26106404
- 46. Xiao J, Jin XH, Jia XP, Wang HY, Cao AZ, Zhao WP, et al. Transcriptome-based discovery of pathways and genes related to resistance against Fusarium head blight in wheat landrace Wangshuibai. BMC Genomics. 2013; 14: 197. pmid:23514540
- 47. Hao YB, Wang T, Wang K, Wang XJ, Fu YP, Huang LL, et al. Transcriptome analysis provides insights into the mechanisms underlying wheat plant resistance to stripe rust at the adult plant stage. PLoS One. 2016; 11: e0150717. pmid:26991894
- 48. Wang DF, Wang XB, Mei Y, Dong HS. The wheat homolog of putative nucleotide-binding site-leucine-rich repeat resistance gene TaRGA contributes to resistance against powdery mildew. Funct Integr Genomics. 2016; 16: 115–126. pmid:26815536
- 49. Sánchez-Martín J, Steuernagel B, Ghosh S, Herren G, Hurni S, Adamski N, et al. Rapid gene isolation in barley and wheat by mutant chromosome sequencing. Genome Biol. 2016; 17: 221. pmid:27795210
- 50. Yahiaoui N, Srichumpa P, Dudler R, Keller B. Genome analysis at different ploidy levels allows cloning of the powdery mildew resistance gene Pm3b from hexaploid wheat. Plant J. 2004; 37: 528–538. pmid:14756761
- 51. Hurni S, Brunner S, Buchmann G, Herren G, Jordan T, Krukowski P, et al. Rye Pm8 and wheat Pm3 are orthologous genes and show evolutionary conservation of resistance function against powdery mildew. Plant J. 2013; 76: 957–969. pmid:24124925
- 52. Xing LP, Hu P, Liu JQ, Witek K, Zhou S, Xu JF, et al. Pm21 from Haynaldia villosa encodes a CC-NBS-LRR protein conferring powdery mildew resistance in wheat. Mol Plant. 2018; 11: 874–878. pmid:29567451
- 53. Du WL, Wang J, Lu M, Sun SG, Chen XH, Zhao JX, et al. Characterization of a wheat-Psathyrostachys huashanica Keng 4Ns disomic addition line for enhanced tiller numbers and stripe rust resistance. Planta. 2014; 239: 97–105. pmid:24085532
- 54. King I, Purdie K, Rezanoor H, Koebner R, Miller T, Reader S, et al. Characterization of Thinopyrum bessarabicum chromosome segments in wheat using random amplified polymorphic DNAs (RAPDs) and genomic in situ hybridization. Theor Appl Genet. 1993; 86: 895–900. pmid:24193994
- 55. Song LQ, Lu YQ, Zhang JP, Pan CL, Yang XM, Li XQ, et al. Physical mapping of Agropyron cristatum chromosome 6P using deletion lines in common wheat background. Theor Appl Genet. 2016; 129: 1023–1034. pmid:26920547
- 56. Li SJ, Lin ZS, Liu C, Wang K, Du LP, Ye XG. Development and comparative genomic mapping of Dasypyrum villosum 6V#4S-specific PCR markers using transcriptome data. Theor Appl Genet. 2017; 130: 2057–2068. pmid:28653149
- 57. Gupta P, Rustgi S. Molecular markers from the transcribed/expressed region of the genome in higher plants. Funct Integr Genomics. 2004; 4: 139–162. pmid:15095058
- 58. Zhang HY, Wei LB, Miao HM, Zhang TD, Wang CY. Development and validation of genic-SSR markers in sesame by RNA-seq. BMC Genomics. 2012; 13: 316. pmid:22800194