Skip to main content

Candidate powdery mildew resistance gene in wheat landrace cultivar Hongyoumai discovered using SLAF and BSR-seq

Abstract

Background

Wheat powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is an important disease affecting wheat production. Planting resistant cultivars is an effective, safe, and economical method to control the disease. Map construction using next-generation sequencing facilitates gene cloning based on genetic maps and high-throughput gene expression studies. In this study, specific-locus amplified fragment sequencing (SLAF) was used to analyze Huixianhong (female parent), Hongyoumai (male parent) and two bulks (50 homozygous resistant and 50 susceptible F2:3 segregating population derived from Huixianhong × Hongyoumai to determine a candidate gene region for resistance to powdery mildew on the long arm of chromosome 7B in wheat landrace Hongyoumai. Gene expressions of candidate regions were obtained using bulked segregant RNA-seq in 10 homozygous resistant and 10 susceptible progeny inoculated by Bgt.. Candidate genes were obtained using homology-based cloning in two parents.

Results

A 12.95 Mb long candidate region in chromosome 7BL was identified, and five blocks in SLAF matched the scaffold of the existing co-segregation marker Xmp1207. In the candidate region, 39 differentially expressed genes were identified using RNA-seq, including RGA4 (Wheat_Chr_Trans_newGene_16173)—a disease resistance protein whose expression was upregulated in the resistant pool at 16 h post inoculation with Bgt. Quantitative reverse transcription (qRT)-PCR was used to further verify the expression patterns in Wheat_Chr_Trans_newGene_16173 that were significantly different in the two parents Hongyoumai and Huixianhong. Two RGA4 genes were cloned based on the sequence of Wheat_Chr_Trans_newGene_16173, respectively from two parent and there was one amino acid mutation: S to G in Huixianhong on 510 loci.

Conclusion

The combination of SLAF and BSR-seq methods identified a candidate region of pmHYM in the chromosome 7BL of wheat landrace cultivar Hongyoumai. Comparative analysis between the scaffold of co-segregating marker Xmp1207 and SLAF-seq showed five matching blocks. qRT-PCR showed that only the resistant gene Wheat_Chr_Trans_newGene_16173 was significantly upregulated in the resistant parent Hongyoumai after inoculation with Bgt, and gene cloning revealed a difference in one amino acid between the two parent genes, indicating it was involved in the resistance response and may be the candidate resistance gene pmHYM.

Peer Review reports

Background

Powdery mildew, caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), is an important disease of wheat [1]. Although wheat powdery mildew can be controlled by fungicides, wheat resistance is the safest and most effective means to prevent or slow the spread of the disease. However, co-evolution and the use of single or few Pm genes can lead to rapid development of matching virulence genes in the pathogen population, resulting in breakdown of cultivar resistance [2, 3]. Therefore, it is important to discover, identify, and clone powdery mildew resistance (PMR) genes.

Mapping and homologous cloning are the major methods used to discover resistance genes; however, gene mapping is the basic of the work. Before the development of high-throughput sequencing, many types of markers, such as RFLP, RAPD, SCAR, STS, CAPs, AFLP, and SSR were used in gene marker identification. This method involved screening primers and fine mapping, which was time-consuming and tedious, thereby limiting studies on candidate genes. Single nucleotide polymorphism (SNP) refers to a single nucleotide mutation that causes DNA sequence diversity at the genome level. SNP markers are becoming major molecular markers, with high distribution density, good genetic stability, and a diallelic genotype, which is easy to detect through high-throughput automated sequencing. The increasing throughput of next-generation sequencing and de novo and reference-based SNP discovery has been applied to several species and is gradually becoming the major method for functional gene mining [4,5,6]. SLAF-seq, based on automated methods, has a significant advantage of high throughput screening [7]. It is an important method for excavating genomic SNP and a high-resolution strategy for large-scale de novo SNP discovery and genotyping. This technology has been made available for haplotype mapping, genetic mapping, linkage mapping, and polymorphism mapping. In previous studies, bulk segregant analysis (BSA), a classical method of marker identification in preliminary screening of primers [8,9,10,11,12,13,14,15], selects 6–10 individuals that represent an extreme phenotype. Due to the small sample sizes, markers identified via this method are usually not closely linked. SLAF uses a minimum of 50 individuals to construct a phenotypic pool, which greatly improves the accuracy of the identified candidate region. Large specific markers of any density that exist throughout the whole genome can be developed based on SLAF-seq to implement fine positioning for functional candidate regions. SLAF-seq has been used for fine mapping and screening of candidate genes in many animals and plants [16,17,18,19].

At present, Pm1 ~ Pm68 have been named as powdery mildew resistance genes, and Pm1 (Pm1a-1e), Pm2 (Pm2a-2c), Pm3 (Pm3a-3j), Pm4 (Pm4a-4d), Pm5 (Pm5a-5e), Pm8 (Pm8 and Pm17), and Pm24 (Pm24a-24b) contain alleles of these resistance genes [20]. However, there has been little progress in whole wheat sequencing because it is difficult to clone genes from polyploid organisms. Until now only a few Pm genes have been cloned, including Pm3 [21], Pm38/Yr18/Lr34/Sr57 [22], Pm8 [23], Pm46/Yr46/Lr67/Sr55 [24], Pm2 [25], Pm60 [26], Pm17 [27], Pm21 [28, 29], Pm24 [30], Pm5e [31], and Pm41 [32].

Of all the identified genes, 30 are derived from common wheat and the remainder are from wild wheat relative species. Exogenous resistance sources were usually used in resistance breeding, sometimes good resistance (R) genes exist in the far-edge materials and are difficult to incorporate. Wheat landraces in China are the products of natural and artificial selection and have better adaptability and important value in the practice of resistance breeding. Previous studies elucidated Pm gene composition in some wheat landraces in China and showed that the resistance to wheat powdery mildew in Mazamai, Xiabbaidongmai, Youbailan, Hongjuanmang, Aiganmangmai, Hongtoumai, Dahongtou, Hongyanglazi, Jiantouhong, and Bensanyuehuang were attributable to a single recessive gene [33,34,35,36,37]. These studies enrich our understanding of wheat landraces.

Wheat landrace Hongyoumai was collected and conserved by the Germplasm Resources Bank of the Wheat Research Center at Henan Academy of Agricultural Sciences. This landrace was identified to be highly resistant to wheat powdery mildew at both the seedling and adult stages [38]. Xu constructed a Chancellor*Hongyoumai F2 hybrid cross, and genetic analysis indicated that Hongyoumai carries one dominant resistance gene to powdery mildew [39]. Wang constructed F2:3 segregating population derived from Yumai13*Hongyoumai and reported that the resistance of Hongyoumai to the Bgt isolate GY was controlled by one dominant resistance gene, temporarily named PmHYM, which was located on chromosome 7BL near SSR markers [40]. Fu constructed F2:3 segregating population derived from Huixianhong*Hongyoumai, and genetic analysis indicated that the resistance of Hongyoumai to the isolate E09 was controlled by one recessive gene, named pmHYM, and performed fine mapping to identify co-segregating marker Xmp1207 of gene pmHYM using a 90 k SNP chip [41]. Anti-spectral analysis of 19 wheat powdery mildew pathogen isolates showed that pmHYM was different from the Pm5 gene and its reported allele, and it was a new Pm5 allele or another gene [41]. The virulence frequencies of wheat powdery mildew isolates collected from different areas of Henan for ten years were lower than 40% according to Li [42] and our unpublished data, indicating that it can still be used in breeding for Pm resistance. Transcript sequencing was conducted using Yumai13*Hongyoumai F2:3 [43]. However, no candidate resistance genes in Hongyoumai have been identified yet.

In previous research, we have constructed the hybrid combinations of Huixianhong (female parent) × Hongyoumai (male parent) and done the identification and analysis of the resistance of the susceptible parents Huixianhong, resistant parent Hongyoumai, and F2:3 individuals dirived from Huixianhong ×Hongyoumai. In this study, we used the SLAF-seq approach to analyze the susceptible parent Huixianhong, resistant parent Hongyoumai, resistant pools (Rp) of 50 F2:3 homolugous resistant progeny, and susceptible pools (Sp) of 50 F2 susceptible progeny derived from the Huixianhong*Hongyoumai F2:3 population. We identified differentially expressed genes (DEGs) between Rp of 10 F2:3 and Sp of 10 F2 using RNA-seq. Screening and expression patterns of target genes were analyzed in the two parents Hongyoumai and Huixianhong, using gene clone and qRT-PCR, respectively. The aim was to find the candidate region of gene pmHYM and further discover candidate PMR regions in Hongyoumai.

Results

SNPs accessed using SLAF

SLAF-seq was conducted using an Illumina HiSeq 2500 at Biomarker Technologies, and 194.02 Mb reads were obtained. Two indicators of sequencing quality, Q30 value and guanine-cytosine (GC) content, were 88.09 and 43.44%, respectively (Table 1). Evaluation and monitoring of sequencing data from the control Oryza sativa was used to validate enzymatic digestion in this term, and 388,044,970 pair-end reads with average comparison efficiency more than 89.08% using wheat genome as a reference were obtained, along with 614,983 SLAF tags with an average depth of 52.18 in parents and 169.11 in mixed pools (Table 2 and Table 3).

Table 1 Evaluation of sample sequencing data
Table 2 Comparison Statistics with Reference Genomes
Table 3 Statistics of SLAF tags

A total of 292,753 SNPs were obtained from two parents, containing 851 non-synonymous-coding regions, as well as 392,556 SNPs in two bulks, containing 1283 non-synonymous-coding regions (Table 4). The distribution of all SNPs on wheat chromosomes is shown in Fig. 1.

Table 4 SNP annotation result
Fig. 1
figure 1

SNP Distribution on wheat Genome

A total of 210,495 high-quality SNPs were obtained by filtering out loci with multi-alleles, a read support degree < 4, genotype consistency in mixed pools and the parent filtered site. Euclidean distance associate analysis was used to identify a region with a length of 12.95 Mb on chromosome 7BL (Fig. 2 and Table 5).

Fig. 2
figure 2

Distribution integration graph of ED correlation values on all chromosomes (a) and resolution graph on the chromosome 7B (b). Abscissa refers chromosome name;dots represent ED value of every SNP locus;full line represents ED value after fitting; dotted line represents Significant correlation threshold; the higher ED value represent the better the association effect

Table 5 Statistical list of associated region information based on ED associate analysis

SNP-index analysis

SNP-index and ΔSNP-index distribution of the two bulks is shown in Fig. 3. According to separation ratio theory of progeny in the term, the association threshold was 0.667. The region above the threshold was screened and 22 regions were identified, 40.51 Mb in length and containing 649 genes; of these, one was a non-synonymous-coding gene. The region on chromosome 7BL is shown in Table 6. From the intersection of the Euclidean distance and SNP-index, one candidate region, 12.9547 Mb in length, was identified on 7BL from 237,210,580 to 250,165,241, which contained 253 candidate genes. The average distance between the two SLAF markers was 0.0514 cM. The region located in the SSR markers range, corresponding to the region of co-segregating marker Xmp1207 derived from TA_TGACv1.30.dna.genome|TGACv1_scaffold_578754_7BL was in five blocks: 237371494–241,751,552; 240,463,691–243,321,483; 241,760,892–24,326,939; 241,453,352–243,128,561; 241,721,380–244,424,206.

Fig. 3
figure 3

Distribution integration graph of SNP-index correlation values on all chromosomes (a) and resolution graph on chromosome 7B (b). Abscissa refers chromosome name, color dots represent SNP-index or ΔSNP-index by calculating, black line refers fitting SNP-index or ΔSNP-index. Above figure refers SNP-index value distribution in Rp; middle figure refers SNP-index value distribution in ab pools; bottom figure refers ΔSNP-index value distribution and red line refers theoretical correlation threshold line

Table 6 Statistical list of associated region information based on SNP-index

BSR-seq analysis

Sequence assembly and analysis

Over 93 million reads were generated, with 51 million reads from T01 (R-pool) and 42 million from T02 (S-pool). A total of 23.62 Gb clean data were obtained, with a Q30 value of 86.69% and GC content of 55.96% (Table 7). The comparison efficiency of reads from two samples T01 and T02 were 70.59 and 71.76%. A total of 11,233 new genes were excavated and 7887 genes were annotated.

Table 7 Evaluation of sample sequencing data by RNA-seq

Differential expression analysis and candidate gene analysis

A total of 1311 DEGs were identified, including 1080 upregulated and 231 downregulated with a Fold-Change ≥2.0 and FDR < 0.01 criterion. Combining SLAF-BSA and RNA-seq, there were 39 known DEGs, including 11 upregulated and 28 downregulated in the candidate region (Table 8). One gene, transcript Wheat_Chr_Trans_newGene_16173, which codes one putative disease resistance protein RGA4, was identified in co-segregating candidate area 243,334,027–243,335,287, and 24.67-fold upregulated genes were expressed in the R-pool than in the S-pool.

Table 8 Differential expressed genes in candidate region

The expression of Wheat_Chr_Trans_newGene_16173 (gene annotation: Putative disease resistance protein RGA4) in Hongyoumai were 23.4, 23.08, 23.29, 22.38 at 0, 24, 48, 72 h post inoculation (hpi), respectively, which were higher than those in Huixianhong, indicating that it may be involved in the resistance to wheat powdery mildew (Fig. 4). However, no SNPs or InDels in corresponding regions of gene Wheat_Chr_Trans_newGene_16173 were found in the SLAF results, maybe because the sequences in the corresponding region were not identified by SLAF.

Fig. 4
figure 4

Expression analysis of Wheat_Chr_Trans_newGene_16173. Relative expression level of Wheat_Chr_Trans_newGene_16173 in parent Hongyoumai than Huixianhong at the same time point after inoculation

Gene cloning and bioinformation analysis

Integrated genes 3780 bp in length were obtained in Huixianhong and Hongyoumai blastx analysis (http://www.ncbi.nlm.nih.gov/), and showed that ORF fragments of Hongyoumai were the same as disease resistance protein RGA4 (Accession no. XM_020324610.1) and one amino acid mutation: S to G in Huixianhong on 510 loci. SMART analysis (http://smart.embl-heidelberg.de/) revealed that coded genes contained seven resistance structure domains: one NB-ARC (179 ~ 463), five leucine-rich repeat (LRR) (597–626; 627–649; 650–673; 1149–1172; 1173–1196), and one LRR_3 (627–940) (Fig. 5).

Fig. 5
figure 5

Conserved Structure domain of gene RGA4 and mutation loci in Huixianhong. Bottom areas refers the Conserved Structure domain of gene RGA4; Framed area represents mutation aa

Discussion

SLAF is based on SNPs and can identify more abundant polymorphisms than other markers. BSA analysis uses 50 individuals of extreme phenotype. Together they improve the accuracy of mapping and narrow down the candidate regions. An exact resistance region 12.95 Mb in length on chromosome 7BL in wheat landrace Hongyoumai was identified in this study using these methods. Further, five blocks were identified by combining the matching scaffold of co-segregating marker Xmp1207 and reducing the scope.

Transcriptome sequencing is an efficient method of screening differential expression transcripts, and is a primary method for obtaining some character-related genes combined with subsequent function analysis. In this study, BSR-seq was used to identify possible candidate genes and identified one candidate resistance transcript Wheat_Chr_Trans_newGene_16173, which was differentially expressed in the two parent types, in the co-segregating region. Blastx results showed that the homologous gene of Wheat_Chr_Trans_newGene_16173 belonged to the RGA4-like resistance gene and integrated ORFs were cloned in Hongyoumai and Huixianhong based on reported disease resistance protein RGA4 [Aegilops tauschii subsp. tauschii] (Accession no. XM_020324610.1).

Resistance genes with LRR domains are widely accepted as playing major roles in plant resistance to pathogens. The LRR domains are thought to interact directly or indirectly with their avirulent effectors to specifically recognize the pathogen. A change in one acid amino in LRR domains may delete or weaken the resistance reaction [44]. Nucleotide-binding site leucine-rich repeat (NBS-LRR) proteins are a current hot topic in plant disease resistance gene research. Genes with NBS-LRR domains are an important part of known resistance genes. NBS conserved domains in resistance genes may have specific relationships with pathogen elicitors and downstream proteins of resistance signal transduct. The NBS domain in NBS-LRR proteins is proposed to function as a molecular switch that adjusts the conformation changes of plant resistance proteins through binding ADP or ATP to regulate resistance signal conduction downstream. There are many NBS-LRR resistance genes and many cloned genes of this kind, including Pm3 [21], Pm21 [28, 29], Pm60 [26], Pm5e [31], Pm41 [32], Yr10 [44], Lr1 [45], Lr10 [46], Lr21 [47], Sr33 [48], Sr35 [49], Cre1, and Cre3 [50]. Lr24 [51] and Lr35 [52] are also confirmed NBS genes.

In this study, one wheat powdery mildew resistance gene, a RGA4-like gene, which has one NB-ARC, five LRR domains, and one LRR_3 was identified and cloned and belonged to the typical NBS-LRR-type gene. Also, one amino acid mutation was observed in the susceptible parent Huixianhong and was differentially expressed in two parents. This suggests that a RGA4-like gene from Hongyoumai may be the candidate Pm gene or one part plays an important role in resistance to wheat powdery mildew. The amino acid mutation locus was outside the conserved domains, the aa change indicated likely presence of another gene or variation in other regulatory elements, so further study is necessary to discover the mechanism of action of RGA4-like gene in Hongyoumai.

Conclusions

A candidate region for pmHYM, 12.95 Mb in length, was located on chromosome 7BL in wheat landrace cultivar Hongyoumai using SLAF-BSA. There were five matching blocks between the scaffold of co-segregating marker Xmp1207 and SLAF-seq. Using RNA-seq, 11 upregulated and 28 downregulated transcripts were observed in which a disease resistance protein RGA4 (Wheat_Chr_Trans_newGene_16173) was upregulated and expressed in the resistant pool at 16 hpi. Further, qRT-PCR results showed Wheat_Chr_Trans_newGene_16173 was aslo obviously higher expression in Hongyoumai (resistant parent) than in Huixianhong (susceptible parent), indicating it maybe play a role in the resistance response.

Methods

Plant materials and treatments

All plant materials, including cultivar Huixianhong (female parent) and wheat landrace Hongyoumai (male parent) F2:3 segregating population were derived from Huixianhong × Hongyoumai. Wheat cultivar Jinfeng 1 code was selected and maintained in the wheat disease research laboratory at the Institute of Plant Protection, Henan Academy of Agricultural Sciences, China. The Bgt E09 race was provided by Prof. Yilin Zhou (Institute of Plant Protection, Chinese Academy of Agricultural Sciences).

Huixianhong, Hongyoumai 50 homozygous resistant and 50 susceptible progeny were planted in nutrition pots and placed in a climate chamber at 16–18 °C, with a light/dark photoperiod of 16/8 h, seedlings at the two-leaf and 1 stem stage were collected and mailed to Biomarker Technologies for DNA extraction and SLAF analysis. Two bulk DNA of the F2:3 segregating population were developed using Huixianhong as susceptible and Hongyoumai as resistance donors. BSA was conducted on 50 resistant and 50 susceptible progeny.

Ten susceptible progeny and ten F2:3 resistant progeny were planted in nutrition pots and maintained in a growth chamber at 16–18 °C, with a light/dark photoperiod of 16/8 h. Inoculation with the Bgt E09 race (maintained on susceptible wheat cultivar Jinfeng 1 code) was performed by shaking conidia onto wheat leaves when plants were at the three-leaf stage. At 16 h post inoculation (hpi), leaves were individually collected, wrapped in tin foil, frozen in liquid nitrogen, and immediately stored at − 80 °C. Then, samples were sent in dry ice to Biomaker Technologies for RNA-sequencing.

Hongyoumai and Huixianhong were planted and inoculated with the Bgt E09 race. The inoculated and control wheat leaves were collected at 0, 24, 48, and 72 hpi, and were immediately frozen in liquid nitrogen and stored at − 80 °C prior to extraction of total RNA.

SLAF library construction and sequencing

Total genomic DNA was extracted from wheat leaves according to the cetyltrimethylammonium bromide (CTAB) method. DNA quality and concentration were estimated using an ND-1000 spectrophotometer (Nanodrop, Wilmington, DE, USA) and electrophoresis in 0.8% agarose gels with a lambda DNA marker. Wheat genome sequences were constructed as a reference to calculate electronic enzyme digestion using restriction enzymes RsaI. The SLAF library was constructed as described by Sun et al [7], with steps as follows: Genomic DNA of each sample was digested using RsaI and a single nucleotide (A) overhang was added to the digested fragments. Dual-index sequencing adapters were ligated to the A-tailed fragments using T4 ligase. Then, the fragments were amplified using PCR and the products were separated using 2.0% agar electrophoresis. Fragments of 364–394 bp in length were excised, purified, and sequenced to obtain target fragments. Pair-end sequencing was conducted using an Illumina HiSeq 2500 system (Illumina, Inc., San Diego, CA, USA) according to the manufacturer’s recommendations (Beijing Biomarker Company).

SLAF-seq data analysis

SLAF-seq identification and genotyping were performed as described by Sun [7] and Zhang [53]. To assure the quality of sequence analysis, two read lengths of 100 bp were used for data evaluation and analysis. The first 100 bp and last 100 bp in one sequence were both evaluated with a quality score Q30 (indicating a 0.1% chance of error, and 99.9% confidence). Reads <Q30 were filtered out and the ratio of the number of high-quality reads with quality scores >Q30 (indicating a 0.10% chance of error and 99.90% confidence) were added to the total number of raw reads and the GC content was calculated. After the barcodes and the terminal 5 bp positions were trimmed, the high-quality reads were mapped onto the reference genome sequence using SOAP software [54]. Sequences mapping to the same position with over 95% identity were grodowned into one SLAF locus [53]. The sequence error rate was estimated using the Oryza sativa genome sequence data as the control. SLAF-tags were identified in parents and mixed pools, and the SLAF-tags that were polymorphic in the female (Hongyoumai) and male (Huixianhong) parents were considered as SLAF markers for subsequent analysis.

SNP accession and SNP-index analysis

SNP is the major form of polymorphic SLAF. SNP detection was conducted using GATK software and SAM tools [55]. SNPs with common variant loci identified by the two methods were considered valid. SnpEff [55, 56] was used to note and forecast the resulting variation (SNP, Small InDel). Variation regions and effects were identified according to the location and gene information of various loci on the reference genome. High-quality SNPs were obtained as follows. First, SNP loci with many genotypes and reads with a downport degree < 4 bp were filtered out. Second, SNP loci that were coincident between Rp and Sp and genes of Rp did not come from the resistant parent were also filtered out. Third, credible SNPs were obtained. Association analysis was conducted using two methods – Euclidean distance and SNP-index [55, 57, 58]. A candidate region was identified from the intersection of the ED and SNP-index analysis. Genes in the candidate region were annotated using BLAST and the databases NR, Swiss-Prot, GO, KEGG, and COG [57,58,59,60].

The ΔSNP-index was calculated using significant differences between bulk genotype frequency. Δ(SNP-index values approaching 1 indicated more SNP markers linked to the target trait.

$$\mathrm{SNP}\_\mathrm{index}\ \left(\mathrm{Rp}\right)=\mathrm{MRp}/\left(\mathrm{PRp}+\mathrm{MRp}\right)$$
$$\mathrm{SNP}\_\mathrm{index}\ \left(\mathrm{Sp}\right)=\mathrm{MSp}/\left(\mathrm{PSp}+\mathrm{MSp}\right)$$
$$\Delta \left(\mathrm{SNP}\_\mathrm{index}\right)=\mathrm{SNP}\_\mathrm{index}\ \left(\mathrm{Rp}\right)-\mathrm{SNP}\_\mathrm{index}\ \left(\mathrm{Sp}\right),$$

where MRp indicates the depth of Rp in the male parent, PRp indicates the depth of Rp in the female parent, MSp indicates the depth of Sp in the male parent, and PSp indicates the depth of Sp in the female parent. False positive loci were filtered out by fitting the ΔSNP-index value of SNP markers from the same chromosome.

BSR-seq analysis

RNA-seq was conducted using Illumina HiSeq sequencing with two pooled samples of resistance F2:3 and susceptible offspring. Each pooled sample contained equal amounts of RNA collected from 10 individuals. Ten resistant plants and 10 susceptible plants were used individually for RNA extracts following the instruction manual of the Trizol Reagent (Life technologies, California, USA). RNA integrity and concentration were checked using a Nanodrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Equal amounts of RNA of qualified samples constituted T01 (R-pool) and T02 (S-pool) for library construction as follows. First, mRNA was isolated using NEBNext Poly (A) mRNA Magnetic Isolation Module (NEB, E7490) and the enriched mRNA was fragmented into approximately 200 nt RNA inserts, which were used to synthesize the first-strand cDNA and the second cDNA. The double-stranded cDNA was end-repaired, de-A-tailed and adaptor ligated. The suitable fragments were isolated using Agencourt AMPure XP beads (Beckman Coulter, Inc.), and enriched using PCR to obtain the cDNA library. Qubit 2.0 and Agilent 2100 were used to detect the concentration and insert size of the cDNA library, respectively, and qPCR was used to detection the exact quantity for effective concentration of the library. Finally, the qualified cDNA libraries were sequenced using an Illumina HiSeq 2500 sequencing platform.

Reads containing adapter sequences, unknown nucleotides > 5%, and low-quality reads of the raw sequencing data, were removed to obtain high-quality clean data (Q30 > 85%). Then, clean reads were mapped to the wheat genome (ftp://ftp.ensemblgenomes.org/pub/plants/release-30/fasta/triticum_aestivum/dna/) using TopHat2. Mapping data, alternative splicing analysis, gene-structure optimization analysis, and the discovery of new genes were performed and gene expression levels were calculated using the reads per kilobase per million mapped reads (RPKM) method. Clean Reads from two samples were used for comparative analysis with the reference genome using TopHat2 Transcripts were filtered for a fold-change threshold of ≥2.0 and ≤ 0.5. The False Discovery Rate (FDR) < 0.01 was applied to identify significantly differentially expressed genes.

qRT-PCR verification

Total RNA was isolated from wheat leaf extracts using Trizol reagent (TAKARA Biomedical Technology) according to the manufacturer’s instructions. The first strand of cDNA was synthesized from 2 μg of the total RNA using RT-PCR (TAKARA). The cDNA samples were diluted × 10 and used as the templates for qRT-PCR, which was performed on an ABI PRISM 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). According to the sequence of the candidate gene Wheat_Chr_Trans_newGene_16173 (Putative disease resistance protein RGA4), forwards primer GGGGTCCTCCATCTTC and reverse primer TGGTGCCCAGCCGTT were synthesized to analyze the transcript levels in Hongyoumai and Huixianhong. Wheat 26S rRNA (GenBank accession no. Z11889.1) was used as a control normalization parameter, with primers as follows: 26S-F: GAAGAAGGTCCCAAGGGTTC; 26S-R: TCTCCCTTTAACACCAACGG. The reaction system was as follows: 2 × qPCR Mix 10 μL, 2 μL cDNA, and 1 μL/10 μmol L− 1 of each primer, with H2O to a total volume of 20 μL. The temperature cycle program was 95 °C for 10 min, 95 °C for 10 s, 55 °C for 30 s, and 72 °C for 34 s for 40 cycles. All reactions were performed in triplicate, including three non-template controls. Dissociation curves were generated for each reaction to ensure specific amplification. Transcript concentration was calculated using 2-△△Ct [61].

Gene cloning

Primers were synthesized based on the ORF of the sequence of putative disease resistance protein RGA4 [Aegilops tauschii subsp. tauschii] (Accession no. XM_020324610.1) to amplify full length gene sequences of Hongyoumai and Huixianhong. Forwards primer: 5840-F: ATGGCGGCGACGGT; reverse primer: 5840-R:CTAATCTCTACGGATGGCACATTTCC. Reaction volume: 2 × fast pfu master mix 25 μL, 1 μL 5840-F, 1 μL 5840-R, 3 μL cDNA, with H2O up to 50 μL reaction volume. The PCR amplification cycle was 94 °C for 3 min, 94 °C for 30 s, 58 °C for 1 min, 72 °C for 1 kb/30 s for 33 cycles, followed by 72 °C for 10 min. Target gene fragments were isolated using 1.0% agarose gel electrophoresis recovery, purification, and sequencing.

Abbreviations

Bgt:

Blumeria graminis f. sp. tritici

NGS:

Next-Generation Sequencing

SLAF:

Specific-Locus Amplified Fragment Sequencing

BSA:

Bulked segregant analysis

hpi:

hours post inoculation

DEGs:

Differential expressed genes

qRT-PCR:

Quantitative reverse transcription-Polymerase Chain Reaction

aa:

amino acid

PMR:

powdery mildew resistance

SNP:

Single nucleotide polymorphism,

NBS-LRR:

Nucleotide binding site-leucine-rich repeats

GC:

guanine-cytosine

ED:

Euclidean Distance

RPKM:

the reads per kilobase per million mapped reads

FDR:

False Discovery Rate

References

  1. Xie H, Chen X, Sheng BQ, Xin ZY, Kong FJ, Lin ZS, et al. Identification of a wheat line YW243 resistance to powdery mildew and genetic analysis. Acta Agron Sin. 2001;27-6:715–21.

    Google Scholar 

  2. Li H, Chen X, Xin ZY, Ma YZ, Xu HJ. Development and identification of wheat-Haynaldia villosa 6DL/6VS translocation lines with powdery mildew resistance (in Chinese). Sci Agric Sin. 1999;32(5):9–15.

    CAS  Google Scholar 

  3. Liu JY, Liu DJ, Tao WJ, Li WL, Chen PD. Study on the conversion of RFLP markers co-segregated with Pm4a to sequenced-tagged-site markers. J Agri Biotech. 1999;7(2):113–6.

    CAS  Google Scholar 

  4. Schneeberger K, Weigel D. Fast-forward genetics enabled by new sequencing technologies. Trends Plant Sci. 2011;16:282–8.

    CAS  PubMed  Google Scholar 

  5. Shi W, Eli M, John KM, Mikhail VM. 2b-RAD:a simple and flexible method for genome-wide genotyping. Nat Methods. 2012;9:808–10.

    Google Scholar 

  6. Mokry M, Nijman IJ, van Dijken A, Benjamins R, Heidstra R, Scheres B, et al. Identification of factors required for meristem function in Arabidopsis using a novel next generation sequencing fast forward genetics approach. BMC Genomics. 2011;12:256.

    PubMed  PubMed Central  Google Scholar 

  7. Sun XW, Liu DY, Zhang XF, Li WB, Liu H, Hong WG, et al. SLAF-seq: an efficient method of large-scale De novo SNP discovery and genotyping using high-throughput sequencing. PLoS One. 2013;8:e57900.

    Google Scholar 

  8. Ruyter-Spira CP, Gu Z, Van der PJ, Groenen M. Bulked segregant analysis using microsatellites: mapping of the dominant white locus in the chicken. Poult Sci. 1997;76:386–91.

    CAS  PubMed  Google Scholar 

  9. Becker A, Chao DY, Zhang X, Salt DE, Baxter I. Bulk segregant analysis using single nucleotide polymorphism microarrays. PLoS One. 2011;6:e15993.

    PubMed  PubMed Central  Google Scholar 

  10. Suo GL, Huang ZJ, He CF, Shen YZ, Wang J. Identification of the molecular markers linked to the salt-resistance locus in the wheat using RAPD-BSA technique. Acta Bot Sin. 2001;43:598.

    CAS  Google Scholar 

  11. Dussle C, Quint M, Melchinger A, Xu M, Lübberstedt T. Saturation of two chromosome regions conferring resistance to SCMV with SSR and AFLP markers by targeted BSA. Theor Appl Genet. 2003;106:485–93.

    CAS  PubMed  Google Scholar 

  12. Lee BY, Penman D, Kocher T. Identification of a sex-determining region in Nile tilapia (Oreochromis niloticus) using bulked segregant analysis. Anim Genet. 2003;34:379–83.

    CAS  PubMed  Google Scholar 

  13. Asnaghi C, Roques D, Ruffel S, Kaye C, Hoarau JY, Telismart H, et al. Targeted mapping of a sugarcane rust resistance gene (Bru1) using bulked segregant analysis and AFLP markers. Theor Appl Genet. 2004;108:759–64.

    CAS  PubMed  Google Scholar 

  14. Cheema KK, Grewal NK, Vikal Y, Sharma R, Lore JS, Das A, et al. A novel bacterial blight resistance gene from Oryza nivara mapped to 38 kb region on chromosome 4L and transferred to Oryza sativa L. Genet Res. 2008;90:397.

    CAS  Google Scholar 

  15. Zhao SH, Zhu MJ, Chen HB. Immunogenomics for identification of disease resistance genes in pigs: a review focusing on gram-negative bacilli. J Anim Sci. 2012;3(34):1–13.

    CAS  Google Scholar 

  16. Chen S, Huang Z, Dai Y, Qin S, Gao Y, Zhang L, et al. The development of 7E chromosome-specific molecular markers for Thinopyrumelongatum based on SLAF-seq technology. PLoS One. 2013;8:e65122.

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Zhang Y, Wang L, Xin H, Li D, Ma C, Ding X, et al. Construction of a high-density genetic map for sesame based on large scale marker development by specific length amplified fragment (SLAF) sequencing. BMC Plant Biol. 2013;13:141.

    PubMed  PubMed Central  Google Scholar 

  18. Hu MJ, Zhang HP, Liu K, Cao JJ, Wang SX, Jiang H, et al. Cloning and characterization of TaTGW-7A gene associated with grain weight in wheat via SLAF-seq-BSA. Front Plant Sci. 2016;7:1902. https://doi.org/10.3389/fpls.

    Article  PubMed  PubMed Central  Google Scholar 

  19. Yin JL, Fang ZW, Sun C, Zhang P, Zhang X, Lu C, et al. Rapid identification of a stripe rust resistant gene in a space-induced wheat mutant using specific locus amplified fragment (SLAF) sequencing. Sci Rep-UK. 2018;8:3086.

    Google Scholar 

  20. McIntosh RA, Dubcovsky J, Rogers WJ, Morris C, Xia XC. Catalogue of gene symbols for wheat: 2017 supplement (KOMUGI Wheat Genetic Resource Database). https://shigen.nig.ac.jp/wheat/komugi/genes/symbolClassList.jsp.

  21. 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. J Mol Cell Biol. 2004;37:528–38.

    CAS  Google Scholar 

  22. Krattinger SG, Lagudah ES, Spielmeyer W, Singh RP, Huerta-Espino J, McFadden H, et al. A pu-tative ABC transporter confers durable resistance to multiplefungal pathogens in wheat. Science. 2009;323:1360–3.

    CAS  PubMed  Google Scholar 

  23. 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;76:957–69.

    CAS  PubMed  Google Scholar 

  24. Moore JW, Herrera-Foessel S, Lan C, Schnippenkoetter W, Ayliffe M, Huerta-Espino J, et al. A recently evolved hexose transporter variant confers resistance to multiple pathogens in wheat. Nat Genet. 2015;47:1494–8.

    CAS  PubMed  Google Scholar 

  25. Sanchez-Martin 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.

    PubMed  PubMed Central  Google Scholar 

  26. Zou SH, Wang H, Li YW, Kong ZS, Tang DZ. The NB-LRR gene Pm60 confers powdery mildew resistance in wheat. New Phytol. 2017;218:298–309.

    PubMed  Google Scholar 

  27. Singh SP, Hurni S, Ruinelli M, Brunner S, Sanchez-Martin J, Krukowski P, et al. Evolutionary divergence of the rye Pm17 and Pm8 resistance genes reveals ancient diversity. Plant Mol Biol. 2018;98:249–60.

    CAS  PubMed  Google Scholar 

  28. Xing LP, Hu P, Liu JQ, Witek K, Zhou S, Xu JF. Pm21 from Haynaldiavillosa encodes a CC-NBS-LRR protein conferring powdery mildew resistance in wheat. Mol Plant. 2018;11:874–8.

    CAS  PubMed  Google Scholar 

  29. 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;11:879–82.

    CAS  PubMed  Google Scholar 

  30. Lu P, Guo L, Wang ZZ, Li BB, Li J, Li YH, et al. A rare gain of function mutation in a wheat tandem kinase confers resistance to powdery mildew. Nat Commun. 2020;11:680.

    CAS  PubMed  PubMed Central  Google Scholar 

  31. Xie JZ, Guo GH, Wang Y, Hu TZ, Wang LL, Li JT, et al. A rare single nucletide variant in Pm5e confers powdery mildew resistance in common wheat. New Phytol. 2020;228(3):1011–26.

    CAS  PubMed  Google Scholar 

  32. Li MM, Dong LL, Li BB, Wang ZZ, Xie JZ, Qiu D, et al. A CNL protein in wild emmer wheat confers powdery mildew resistance. New Phytol. 2020;228(3):1027–37.

    CAS  PubMed  Google Scholar 

  33. Xiong EH, Zhu W, Cao Y, Chai SB, Fang XW. A genetic analysis of powdery mildew resistance in three native wheat varieties. J Jiangsu Agri College. 1995;16(2):47–50.

    CAS  Google Scholar 

  34. Xue F, Duan XY, Zhou YL, Ji WQ. Postulation of powdery mildew resistant genes carried in some chinese wheat landraces and the genetic diversity analysis. J Triticeae Crops. 2009;29:228–35.

    CAS  Google Scholar 

  35. Xiao MG. Identification of the genes conferring resistance to powdery mildew in the Chinese landraces. PhD thesis, Chinese Academy of Agricultural Sciences. Institute of Plant Protection; 2013.

  36. Xu HX, Xu YF, Geng LG, An DG. Resistance of Chinese wheat landraces and relatives at seedling stage to powdery mildew. Chin J Eco-Agric. 2011;19:1210–4.

    Google Scholar 

  37. Xu XD. Identification and mapping of powdery mildew resistance genes from 5 wheat landraces. PhD thesis. China Agricultural University. College of Plant Protection; 2017.

  38. Wang XF, Zhang ZS, Liu HY, He WL. Evaluation of resistance and slowmildewing of some wheat varieties onHenan province. Acta Agriculture Universitatis Henanensis. 1996;30:160–3.

    Google Scholar 

  39. Xu HX. Development of molecular markers linked to five genes for the powdery mildew resistance in wheat. MS thesis. Henan University. School of life sciences; 2004.

  40. Wang JM, Liu HY, Kang ZS. Genetic analysis and location of a powdery mildew resistance gene in wheat landrace Hongyoumai by SSR marker. Acta Phytopathologica Sinica. 2009;39:285–9.

    Google Scholar 

  41. Fu BS, Zhang ZL, Zhang QF, Wu XY, Wu JZ, Cai SB. Identification and mapping of a new powdery mildew resistance allele in the Chinese wheat landrace Hongyoumai. Mol Breed. 2017;37:133.

    Google Scholar 

  42. Li YH, Wang JM, Xu F, Yang GQ, Song YL, Liu LL. Virulence structure of the wheat powdery mildew population in Henan Province in China during 2011~2014. Acta Phytopathologica Sinica. 2016;4:573–6.

    Google Scholar 

  43. Wang JM, Liu HY, Xu HM, Li M, Kang ZS. Analysis of differential transcriptional profiling in wheat infected by Blumeria graminis f. sp. tritici using genechip. Mol Biol Rep. 2012;39:381–7.

    CAS  PubMed  Google Scholar 

  44. Liu W, Frick M, Huel R, Nykiforuk CL, Wang XM, Gaudet DA, et al. The stripe rust resistance gene Yr10 encodes an evolutionary-conserved and unique CC-NBS-LRR sequence in wheat. Mol Plant. 2014;7:1740–55.

    CAS  PubMed  Google Scholar 

  45. Cloutier S, Mccallum BD, Loutre C, BanksTW WT, Feuillet C, et al. Leaf rust resistance gene Lr1, isolated from bread wheat (Triticum aestivum L.) is a member of the large psr567 gene family. Plant Mol Biol. 2007;65:93–106.

    CAS  PubMed  Google Scholar 

  46. Sela H, Spiridon LN, Petrescu AJ, Akerman M, Mandel-Gutfreund Y, Nevo E, et al. Ancient diversity of splicing motifs and protein surfaces in the wild emmer wheat (Triticum dicoccoides) LR10 coiled coil (CC) and leucine-rich repeat (LRR) domains. Mol Plant Pathol. 2012;13:276–87.

    CAS  PubMed  Google Scholar 

  47. Huang L, Brooks SA, Li W, Fellers JP, Trick HN, Gill BS. Map-based cloning of leaf rust resistance gene Lr21 from the large and polyploid genome of bread wheat. Genet. 2003;164:655–64.

    CAS  Google Scholar 

  48. Periyannan S, Moore J, Ayliffe M, Clemence M, Zhang JP, Zhang P, et al. The gene Sr33, an ortholog of barley Mla genes, encodes resistance to wheat stem rust race Ug99. Sci. 2013;341:786–8.

    CAS  Google Scholar 

  49. Saintenac C, Zhang W, Salcedo A, Rouse MN, Trick HN, Akhunov E, et al. Identification of wheat gene Sr35 that confers resistance to Ug99 stem rust race group. Sci. 2013;341:783–6.

    CAS  Google Scholar 

  50. De Majnik J, Ogbonnaya FC, Moullet O, Lagudah E. The cre1and cre3 nematode resistance genes are located at homeologous loci in the wheat genome. Mol Plant Microbe In. 2003;16:1129–34.

    Google Scholar 

  51. Zhang LR, Yang WX, Liu DQ. Diversity and evolutionary relationship of NBS-type resistance gene analogues in wheat. Acta Agriculturae Boreali Sinica. 2011;26(4):23–6.

    Google Scholar 

  52. Gao Q, Wang HY, Liu DQ, Li X, Yang WX. RGA analysis of the wheat leaf rust resistance gene Lr35. Acta Agriculturae Boreali Sinica. 2008;23(6):50–3.

    Google Scholar 

  53. Zhang J, Zhang QX, Cheng TR, Yang WR, Pan HT, Zhong JJ, et al. High-density genetic map construction and identification of a locus controlling weeping trait in an ornamental woody plant (Prunus mume Sieb. Et Zucc). DNA Res. 2015;22:1–9.

    Google Scholar 

  54. Li R, Li Y, Kristiansen K, Wang J. SOAP:short oligonucleotide alignment program. Bioinformatics. 2008;24:713–4.

    CAS  PubMed  Google Scholar 

  55. Hill JT, Demarest BL, Bisgrove BW, Gorsi B, Su YC, Yost HJ. MMAPPR: mutation mapping analysis pipeline for pooled RNA-seq. Genome Res. 2013;23:687–97.

    CAS  PubMed  PubMed Central  Google Scholar 

  56. Cingolani P, Platts A, Wang LL, Coon N, Wang L, et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff:SNPs in the genome of Drosophila melanogaster strain w118; iso 2; iso 3. Fly. 2012;6:80–92.

    CAS  PubMed  PubMed Central  Google Scholar 

  57. Fekih R, Takagi H, Tamiru M, Abe A, Natsume S, Yaegashi H, et al. MutMap+:Genetic Mapping and Mutant Identification without Crossing in Rice. PLoS One. 2013;8:1–10.

    Google Scholar 

  58. Takagi H, Abe A, Yoshida K, Kosugi S, Natsume S, Mitsuoka C, et al. QTL-seq:Rapid mapping of quantitative trait loci in rice by whole genome resequencing of DNA from two bulked populations. Plant J. 2013;74:174–83.

    CAS  PubMed  Google Scholar 

  59. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–402.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. Deng YY, Li JQ, Wu SF, Zhu YP, Chen YW, He FC. Integrated nr database in protein annotation system and its localization. Comput En. 2006;32:71–4.

    Google Scholar 

  61. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△Ct method. Methods. 2001;25:402–8.

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

Thank Professor Zhou Yilin (Institute of Plant Protection, Chinese Academy of Agricultural Sciences) kindly provides us with Bgt E09.

Ethical statement for experimental research and field studies on plants

Experimental research and field studies on plants complies with relevant institutional, national, and international guidelines and legislation.

Funding

This study was financially supported by the National Key Research and Development Project (grant no.

2016YFD0300705) and Henan Wheat Industry Technology System (no. S2010–01-G05).

Author information

Authors and Affiliations

Authors

Contributions

WJ and SY were responsible for the data analysis and experimental design. WJ wrote the manuscript. WJ, LY, XF, XH, HZ, LL and SY were responsible for the programs and the manuscript revision. All authors have commented, read and approved the final manuscript.

Corresponding author

Correspondence to Yuli Song.

Ethics declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Additional information

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, J., Li, Y., Xu, F. et al. Candidate powdery mildew resistance gene in wheat landrace cultivar Hongyoumai discovered using SLAF and BSR-seq. BMC Plant Biol 22, 83 (2022). https://doi.org/10.1186/s12870-022-03448-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s12870-022-03448-5

Keywords