Introduction

Wheat powdery mildew, caused by the biotrophic fungus Blumeria graminis f. sp. tritici (Bgt), is a destructive foliar disease that threatens wheat production, especially in regions with humid and cool climates (Juroszek and von Tiedemann 2013). It is more severe in dense crops of semi-dwarf cultivars grown under irrigated conditions with high inputs of fertilizer (Singh et al. 2016). In China, the areas affected by powdery mildew range from 6 to 8 million hectares leading to grain losses of 300,000 metric tons each year in the past decade (Ma et al. 2016a; Sun et al. 2018; Chen et al. 2019). The most effective, economical and environment-friendly method to control powdery mildew is resistant cultivars. However, new introductions with race-specific resistance are usually rapidly overcome by virulent races of the pathogen, especially when a single resistance gene is deployed across a wide area (Luo et al. 2009; Li et al. 2020a). Consequently, a search for new sources of resistance for breeding is an ongoing task.

To date, more than 100 Pm resistance genes/alleles, including 68 that are formally named, have been identified and mapped on all wheat chromosomes except for 3D and 4D (https://shigen.nig.ac.jp/wheat/komugi/genes/symbolClassList.jsp; He et al. 2020; Kang et al. 2020). Plant resistance genes tend to be clustered rather than evenly distributed across wheat genome (McHale et al. 2006). For instance, several genes are located in wheat chromosome arm 2BS, indicating a complex cluster that includes pm26 (Rong et al. 2000), pm42 (Hua et al. 2009), MlIW170 (Liu et al. 2012), Ml5323 (Piarulli et al. 2012), PmL962 (Shen et al. 2015), pmWE99 (Ma et al. 2016b) and Pm68 (He et al. 2020).

Wild emmer wheat (Triticum turgidum ssp. dicoccoides, WEW, AABB, 2n = 4x = 28), the progenitor of both cultivated tetraploid and hexaploid wheats (Nevo et al. 2002), carries a significant number genes for resistance to powdery mildew (Xie and Nevo 2008). Some of these genes have been transferred to durum and/or common wheat, including Mlzec1 (Mohler et al. 2005) and MlAB10 (Maxwell et al. 2010) on chromosome arm 2BL; PmG16 (Ben-David et al. 2010), MlIW72 (Ji et al. 2008) and MlIW172 (Ouyang et al. 2014) on 7AL; Ml3D232 (Zhang et al. 2010) on 5BL; MlIW30 (Geng et al. 2016) and MlNFS10 (Yin et al. 2021) on 4AL; and PmG3M (Xie et al. 2012) on 6BL. Other WEW derived Pm genes include Pm16 (Reader and Miller 1991) and Pm30 (Liu et al. 2002) that are possibly allelic on chromosome arm 5BS (Chen et al. 2005), Pm36 (Blanco et al. 2008) on 5BL, Pm41 (Li et al. 2009) on 3BL and Pm64 (Zhang et al. 2019) on 2BL. Among all these genes, only Pm41 has been cloned and it encodes a protein with a coiled-coil, nucleotide-binding site and leucine-rich repeat structure (CNL) (Li et al. 2020b), which is typical of many disease resistance genes in the plant kingdom.

Molecular markers, especially the simple sequence repeat (SSR), are highly efficient for gene localization (Zhang et al. 2019). However, SSR markers are not uniformly distributed on chromosomes and their numbers are insufficient for fine mapping of target genes (https://wheat.pw.usda.gov/GG3/). The releases of reference genome sequences of common wheat (Triticum aestivum cv. Chinese Spring RefSeq v.1.0) (IWGSC 2018) and whole-genome-shotgun assembly of WEW accession Zavitan (Avni et al. 2017) have greatly assisted marker development and map-based cloning of important genes. In addition to SSR markers, re-sequencing of mapping parents allows identification of single nucleotide polymorphisms (SNP) and insertion/deletion (InDel) variations in target regions providing further relevant markers for fine mapping (Li et al. 2020c).

In the present study, we report: (1) identification and fine mapping of powdery mildew resistance gene MlIW39 that was introgressed from WEW accession IW39 into common wheat, and (2) development of co-segregating markers allowing efficient selection of the resistance allele in genetic studies and wheat breeding.

Materials and methods

Plant materials

Powdery mildew resistant line 8D49 (87-1/IW39//2*87-1) is a second backcross selection from a wild emmer wheat (WEW) accession IW39 and susceptible Chinese common wheat line 87-1. WEW accession IW39 was kindly provided by Dr. T. Fahima, University of Haifa, Israel. 8D49 was highly resistant to Bgt isolate E09 both at the seedling and adult growth stages (all-stage resistance). 8D49 was crossed with the susceptible spring wheat Apogee and 152 F2:3 lines were used for genetic analyses and mapping. To fine map the target gene, three populations of 2303 F2 plants were generated from crosses of 8D49 with susceptible cultivars Shi4185, Liaochun10 and Apogee, respectively. Xuezao, a highly susceptible common wheat line, was used as the susceptible control and to multiply Bgt isolate E09, which was provided by Dr. Xiayu Duan, Institute of Plant Protection, Chinese Academy of Agriculture Sciences, Beijing, China. Five hundred and forty-five Chinese varieties/landraces and 279 accessions from other countries were used to test the efficacy of the markers co-segregating with MlIW39 in further research and breeding (genotype list not shown).

Powdery mildew evaluations

The parental lines 8D49, Apogee, Shi4185, Liaochun10, and the corresponding F1, F2, F2:3 materials and recombinant families from the fine mapping populations were evaluated for response to powdery mildew under greenhouse conditions. In brief, the F2 seeds were planted in rectangular seedling trays with 128 cells (8 × 16), 1 seed per cell. Thirty seeds for each F2:3 family were planted in 50-cell trays (5 × 10) and each family was sown in two cells (15 seeds per cell). The resistant and susceptible parents, as well as susceptible control, were planted in the middle of each tray as the resistant and the susceptible controls, respectively. Seedlings with unfolded first leaves were inoculated with Bgt E09 by dusting and brushing of conidiospores. Infection types (ITs) were evaluated after 15 days and re-confirmed two days later on a scale of 0–4, in which, 0, 0; 1, 2, 3, 4 represented immune, necrotic flecks, high resistance, moderate resistance, moderate susceptibility and high susceptibility, respectively. Phenotypes were classified into two groups, resistant (R, IT 0–2) and susceptible (S, IT 3–4) (Liu et al. 1999). 8D49 was also challenged by 25 Bgt isolates collected from different regions of China, and the susceptible Zhongzuo9504 was the control.

Bulked segregant analysis

The leaves of individual F2 plants were used to extract the genomic DNA by using a modified cetyltrimethylammonium bromide (CTAB) method (Devi et al. 2013). Based on F2 genotypes deduced from powdery mildew responses of corresponding F3 families, resistant and susceptible DNA pools were constructed by bulking 10 homozygous resistant and 10 homozygous susceptible F2 plants, respectively. The DNA pools were genotyped with the Illumina 90 K iSelect SNP Chip by China Golden Marker Biotech Co. (CGMB, Beijing, China).

Marker development and genotyping

SSR markers were designed according to the Chinese Spring reference genome sequence (RefSeq v1.0) (https://wheat-urgi.versailles.inra.fr/) using BatchPrimer3 with minim of 12, 10, 8, 6 and 4 di-nucleotide, tri-nucleotide, tetra-nucleotide, penta-nucleotide and hexa-nucleotide SSR pattern repeats, respectively (http://batchprimer3.bioinformatics.ucdavis.edu/). To improve the efficiency of marker development, the parental lines 8D49 and Apogee were re-sequenced. Briefly, high quality genomic DNA of the parents was used to construct paired-end-sequencing libraries with insert sizes of approximately 500 bp (Chai et al. 2018). The average sequencing depth was 6× of the Chinese Spring reference genome. Sequence reads were generated using the Illumina HiSeq X Ten platform with 150-bp paired-end reads and mapped to the Chinese Spring reference genome (RefSeq v1.0) with the Burrows-Wheeler Aligner program (BWA,ver.0.7.15) (Li and Durbin 2009). The HaplotypeCaller module of the Genome Analysis Toolkit (GATK) was used to identify single-nucleotide polymorphisms (SNPs) and insertion/deletions (InDels) sequences. We searched the InDel variations between pairs of parents in the target interval to design InDel markers as described in Qiu et al (2020). The SSR and InDel markers used to fine map the powdery mildew resistance locus are listed in Table S3.

The PCR system contained 5 μl 2 × Taq PCR StarMix, 1.5 μl primer (mixture of left and right primers, 2 μM), 1.5 μl DNA template (50–100 ng/μl) and 2 μl ddH2O. PCR cycling was as follows: 94 °C for 5 min; 35 cycles of 94 °C denaturation for 30 s, 56 °C primer annealing for 30 s, 72 °C extension for 30 s; and a final extension of 72 °C for 5 min. PCR products were separated in 10% non-denaturing polyacrylamide gels (39 acrylamide:1 bisacrylamide), that were silver stained and photographed after electrophoresis.

Data analysis

SSR and InDel markers polymorphic between the resistant and susceptible pools, along with the two parents, were used to genotype the 152 F2 individuals derived from the cross Apogee × 8D49 for linkage analysis. The linkage map was constructed with JoinMap4.0 (Van Ooijen 2006) and the Kosambi mapping function was used to convert recombination values into map distance (Kosambi 1943). Mapdraw was used to draw the linkage map (Liu and Meng 2003). Chi-squared tests for goodness-of-fit were used to evaluate the deviations of observed segregation ratios from the theoretical Mendelian ratios.

Comparative genomics analysis

The nearest flanking markers were used as queries in BLASTn search against the genomes of durum wheat cv. Svevo (https://www.interomics.eu/durum-wheat-genome; Maccaferri et al. 2019) and hexaploid wheat cv. Chinese Spring (http://www.wheat-urgi.versailles.inra.fr/; IWGSC 2018), Norin61, Mace, CDC Stanley, Jagger, Julius_MAGIC3 and spelt wheat PI 190962 (https://webblast.ipk-gatersleben.de/wheat_ten_genomes/; Walkowiak et al. 2020) to confirm the corresponding physical interval in these genomes and the annotated genes in the target interval (Tables S4, S5). Collinearity analysis among different genomes was performed on Triticeae-Gene Tribe (TGT) (http://wheat.cau.edu.cn/TGT/; Chen et al. 2020).

Sequence comparison and RT-PCR analysis of annotated genes in the target interval

Annotated genes within the mapped interval were amplified from the resistant and susceptible parents 8D49 and Shi4185 using TKS Gflex™ DNA polymerase (TAKARA, Dalian, China) using the primers listed in Table S6. Primers were designed using Primer3Plus (http://www.primer3plus.com/cgi-bin/dev/primer3plus.cgi) according to the information in the WEW accession Zavitan reference genome. PCR products were separated by 1% agarose gel electrophoresis, and target DNA fragments were recycled from the gel and sequenced by the TsingKe Biological Technology in Beijing. The sequencing results were analyzed with the DNAMAN version 6.0 (Gao et al. 2019).

Leaves of 8D49 and Shi4185 at two-leaf seedling stage were collected at 24 hours post inoculation (hpi) with Bgt isolate E09 for total RNA isolation with TRIzol reagent (Invitrogen, Shanghai, China). First-strand cDNA was synthesized from 1 ug total RNA using a FastKing RT Kit following the manufacturer’s instructions (TIANGEN Biotech Co. Ltd, Beijing, China). Specific primers used for RT-PCR analysis are listed in Table S6.

Results

Genetic analysis of powdery mildew resistance in 8D49

The parental lines, F1 and F2 populations derived from crosses Apogee × 8D49, Liaochun10 × 8D49 and Shi4185 × 8D49 were tested for response to Bgt isolate E09. 8D49 was highly resistant (IT = 0;), whereas Apogee, Liaochun10 and Shi4185 were highly susceptible (IT = 4) (Fig. 1). F1 plants were resistant (IT = 0;), indicating dominance of resistance. Resistant plants produced a very thin layer of mycelia on infected leaves, then the infection sites necrosed with expansion over time, whereas the leaves of susceptible plants remained green with heavy sporulation. The segregations of resistant and susceptible F2 plants as well as the pooled data fitted 3:1 ratio (Table 1). These results indicate that the powdery mildew resistance in 8D49 is governed by a single dominant gene, provisionally designated as MlIW39.

Fig. 1
figure 1

Phenotypes of resistant parent 8D49 and susceptible parents, including Apogee, Shi4185 and Liaochun10, at two weeks post-inoculation with Bgt isolate E09

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Table 1 Responses of F2 plants from crosses 8D49 with three susceptible wheat varieties to the Bgt isolate E09
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Bulked segregant analysis (BSA) and molecular mapping of the MlIW39 locus

To determine the genomic position of the gene for the powdery mildew resistance, we constructed DNA pools from 10 homozygous resistant and 10 homozygous susceptible F2 plants and genotyped them with the 90 K iSelect SNP Chip. One hundred and ninety-two SNPs were polymorphic between the pools of the detected 79,058 SNP markers, 67 of them (about 34.90%) were anchored on chromosome 2B; 62 (92.54%) were concentrated in the 0–40 Mb chromosomal region (Fig. 2; Tables S1, S2), suggesting the resistance gene was distally located on the short arm of chromosome 2B.

Fig. 2
figure 2

Number of SNP markers between the resistant and susceptible pools of the F2 population derived from cross 8D49 × Apogee. a Distribution of SNPs per chromosome; b The physical positions of SNPs on chromosome 2B based on the Chinese Spring reference sequence v1.0

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To map MlIW39 more precisely, we designed 82 SSR primers in the candidate region based on Chinese Spring reference genome sequence (RefSeq v1.0) and screened the polymorphic markers in the two pools and parents. Six polymorphic loci, IWB12-4, QB-3-16, SSR12, SSR34, SSR43, SSR48, were identified and used to genotype the 152 F2 individuals derived from Apogee × 8D49 and a genetic linkage map (Fig. 3a) was constructed from the data. To further saturate the genetic linkage map, an additional 23 InDel primers were developed according to re-sequenced data from the parents. This added a further four markers, 7Seq247, 7Seq546, 7Seq270, 7Seq288, to the linkage map (Fig. 3a). Finally, MlIW39 locus was localized to a 0.7 cM genetic interval between flanking markers 7Seq546 and QB-3-16 at genetic distances of 0.4 cM (distal) and 0.3 cM (proximal) (Fig. 3a).

Fig. 3
figure 3

Fine mapping of the powdery mildew resistance gene MlIW39. a Genetic linkage map of MlIW39. Numbers above the line are genetic distances between adjacent markers in cM. b Physical positions of molecular markers used for fine mapping of MlIW39 according to the WEW accession Zavitan reference genome v1.0, r indicates the number of recombinants. c Genotypes and phenotypes of the six relevant recombinant events. Recombinant events and phenotypes are indicated at the left and right, respectively. Black, white and gray blocks indicate homozygous segments from 8D49, homozygous segments from susceptible parents and heterozygous segments, respectively. d Annotated genes in the target region of the MlIW39 locus. Red pentagons represent genes related to disease resistance

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Fine mapping of powdery mildew resistant gene MlIW39

To further narrow down the genomic region harboring the MlIW39 locus, 48 InDel markers were developed according to the InDel polymorphisms in the MlIW39 genetic interval between 8D49 and Apogee; 10 InDel markers were polymorphic between the resistant and three susceptible parents. The flanking markers 7Seq546 and QB-3-16, also polymorphic between 8D49 and Shi4185 and Liaochun10, were used to genotype all 2,303 F2 individuals from the three mapping populations, and 38 recombinants were identified (Fig. 3b). The 10 InDel markers located between markers 7Seq546 and QB-3-16 were used to genotype all recombinants which were classified into 20 categories (Table S7). Genotyping of the 38 recombinants was performed by progeny testing with Bgt isolate E09. Finally, MlIW39 was placed in an approximate 460.3 kb interval flanked by markers 7Seq610 and 7Seq705 (Fig. 3c and d). According to the WEW Zavitan (v1.0) reference genome sequence (https://wheat.pw.usda.gov/jb/?data=/ggds/whe-zavitan2017), this genomic region contained six high-confidence protein-coding genes (Fig. 3d), among which four were related to disease resistance genes. These included TRIDC2BG003930 which encodes putative disease resistance protein homologous to At3g14460, TRIDC2BG003950 encoding a G-type lectin S-receptor-like serine/threonine-protein kinase B120, TRIDC2BG003970 encoding RPM1-like disease resistance protein and TRIDC2BG003990 encoding a putative disease resistance RPP13-like protein 1.

Comparative mapping of MlIW39 among durum and hexaploid wheat genomes

The 7Seq610 and 7Seq705 markers flanking MlIW39 were used to search against the durum wheat cv. Svevo reference genome and hexaploid wheat genomes, including Chinese Spring, Norin61, Mace, CDC Stanley, Jagger, Julius_MAGIC3 and spelt wheat PI 190962 to define the interval in these genomes (Table S4). Protein-coding genes annotated in the corresponding interval were used to perform collinearity analysis between the tetraploid and hexaploid genomes (Table S5). Sequence analysis revealed that three genes, TRIDC2BG003930, TRIDC2BG004000 and TRIDC2BG004010, were syntenic among different genomes, whereas three disease resistance-related genes, namely, TRIDC2BG003950, TRIDC2BG003970 and TRIDC2BG003990, were present on chromosome arm 2BS only in WEW. On the other hand, there were three added genes in the durum and hexaploid wheat genomes and a further two only in hexaploid wheat (Fig. 4).

Fig. 4
figure 4

Collinearity of the genomic region of MlIW39 between durum and hexaploid wheat. Orthologous genes are linked by lines; solid and dashed lines indicate match scores of 70–100 and 50–70, respectively. Za-1, TRIDC2BG003930; Za-2, TRIDC2BG003950; Za-3, TRIDC2BG003970; Za-4, TRIDC2BG003990; Za-5, TRIDC2BG004000 and Za-6, TRIDC2BG004010; Sv-1, TRITD2Bv1G010240; Sv-2, TRITD2Bv1G010260; Sv-3, TRITD2Bv1G010300; Sv-4, TRITD2Bv1G010330; Sv-5, TRITD2Bv1G010390 and Sv-6, TRITD2Bv1G010400; CS-1, TraesCS2B02G045700; CS-2, TraesCS2B02G045800; CS-3, TraesCS2B02G045900; CS-4, TraesCS2B02G046000; CS-5, TraesCS2B02G046100; CS-6, TraesCS2B02G046300; CS-7, TraesCS2B02G046400 and CS-8, TraesCS2B02G046500; PI-1 to PI-8; No-1 to No-8; Ma-1 to Ma-8; CD-1 to CD-8; Ja-1 to Ja-8 and Ju-1 to Ju-8 represent genes TraesTSP2B01G048000 to TraesTSP2B01G048700; TraesNOR2B01G051100 to TraesNOR2B01G051800; TraesMAC2B01G050700 to TraesMAC2B01G051400; TraesSTA2B01G053300 to TraesSTA2B01G054000; TraesJAG2B01G047100 to TraesJAG2B01G047800 and TraesJUL2B01G046900 to TraesJUL2B01G047600 in the genomes of spelt wheat PI 190962 and common wheat cultivars Norin 61, Mace, CDC Stanley, Jagger and Julius_MAGIC3, respectively

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Analysis of the annotated genes in the candidate interval

To compare the six annotated genes in the candidate interval, we amplified TRIDC2BG003930, TRIDC2BG004000 and TRIDC2BG004010 from the resistant and susceptible parents. However, we obtained TRIDC2BG003950, TRIDC2BG003970 and TRIDC2BG003990 only from the resistant parent (Fig. S1a), a result that coincided with the comparative mapping showing these three genes present only in the WEW genome. Sequences alignment revealed that there were 7 and 10 SNPs between 8D49 and Shi4185 in genes TRIDC2BG003930 and TRIDC2BG004010, respectively (Table S8). There was considerable variation in TRIDC2BG004000 between 8D49 and Shi4185 (data not shown). RT-PCR analysis showed that all 6 predicted genes were expressed in seedlings of 8D49 after Bgt infection (Fig. S1b). Further research is needed to identify the candidate gene for MlIW39.

Marker assessment and response of 8D49 to a panel of Bgt isolates

To evaluate the usefulness of the markers flanking MlIW39 in wheat research and breeding, two markers 7Seq622 and 7Seq727 that co-segregated with the resistance allele were tested in 545 Chinese cultivars/landraces and 279 accessions from other countries. The PCR product sizes of the two markers in 8D49 were unique and not detected in any of the 824 varieties (data not shown). Hence, these markers should be diagnostic for marker-assisted selection of MlIW39.

When inoculated with 25 additional Bgt isolates collected from different regions in China line 8D49 was resistant to almost all the isolates showing IT 0 or 0; to 6 isolates, IT 1 to 15 isolates, IT 2 to 3 isolates and IT 2+ to 1 isolate (Table 2; Fig. S2). This result indicates that MlIW39 confers a wide spectrum resistance and thus is a potentially valuable source of resistance for wheat breeding.

Table 2 Infection types produced by 8D49 and control Zhongzuo9504 to 25 Bgt isolates collected from different locations in China
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Discussion

Comparison of MlIW39 with other powdery mildew resistance genes on 2BS

In this study, we identified and fine mapped a dominant powdery mildew resistance allele MlIW39, transferred to common wheat from wild emmer to a 460.3 kb genomic region on short arm of chromosome 2B according to the reference genome of WEW accession Zavitan (v1.0) (https://wheat.pw.usda.gov/jb/?data=/ggds/whe-zavitan2017) (Fig. 3d). To date, a total of seven powdery mildew resistance genes had been mapped on wheat chromosome arm 2BS, including pm26 (Rong et al. 2000), pm42 (Hua et al. 2009), MlIW170 (Liu et al. 2012), Ml5323 (Piarulli et al. 2012), PmL962 (Shen et al. 2015), pmWE99 (Ma et al. 2016b) and Pm68 (He et al. 2020). Among these genes, pm26, pm42 and MlIW170 were from T. dicoccoides (Rong et al. 2000; Hua et al. 2009; Liu et al. 2012), pm26 and MlIW170 were reported to be allelic or closely linked loci (Liang et al. 2015). PmL962 and pmWE99 were from Thinopyrum intermedium (Shen et al. 2015; Ma et al. 2016b), Ml5323 from T. turgidum ssp. dicoccum and Pm68 from T. turgidum ssp. durum (Piarulli et al. 2012; He et al. 2020). MlIW39 differs from the three known genes pm26, pm42 and MlIW170 derived from WEW according to their difference in genomic positions and effects. Based on the genetic maps of these four genes, MlIW39 was located on the distal side of pm26, MlIW170 and pm42 (Fig. 5a, d, e, f). For their effects on resistance phenotype, MlIW39 is a dominant gene, whereas pm26 and pm42 were recessive and MlIW170 was incomplete dominant. The genes Ml5323 and PmL962 are proximal to marker Xcau516, and the genetic distances between the markers and the genes were 7.2 cM and 16.06 cM, respectively (Fig. 5g, i), while MlIW39 was positioned at the distal part to marker Xcau516. The gene pmWE99 was 10.4 cM distal to SSR marker Xgwm148, which mapped to the 107.9 Mb region in the WEW accession Zavitan reference assembly. The genetic distance between marker 7Seq288 and MlIW39 was 15.5 cM, and the marker was anchored at the position 27.8 Mb in the reference assembly (Fig. 5a, b, h), indicating the pmWE99 is far from MlIW39. Moreover, pmWE99 gene was recessive, originated from Th. intermedium. Based on above analysis, we concluded that MlIW39 was different from these six genes.

Fig. 5
figure 5

Comparison of the current linkage map of MlIW39 (a) with those reported for Pm68 (c), MlIW170 (d), pm26 (e), pm42 (f), Ml5323 (g), pmWE99 (h) and PmL962 (i) using the linked markers Xcau516/BF202540 and Xgwm148 as anchors. b Physical map of the MlIW39 region based on the WEW accession Zavitan reference genome sequence (v1.0), physical locations in Mb are shown at the right

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He et al. (2020) reported a powdery mildew resistance gene Pm68 in durum wheat and located it on the physical interval 21.59–23.37 Mb in the reference genome of durum wheat cv. Svevo, which corresponded to 17.69–19.56 Mb in the reference assembly of WEW accession Zavitan (v1.0) (Fig. 5b and c). The physical interval of Pm68 overlaps the region harboring MlIW39 (18.7–19.1 Mb) (Fig. 5a and b), suggesting the two genes might be at the same locus or closely linked loci. The origin of the two genes was different. Pm68 was identified from durum wheat, while MlIW39 was from WEW. However, genetic maps are not entirely reliable and dominance might vary with background, we can’t rule out the possibility that MlIW39 may be identical or allelic to these Pm genes. Only after cloning of some of the genes can we determine their relationships.

Candidate genes for MlIW39

MlIW39 was delimited to a 460.3 kb genomic region containing 6 high-confidence protein-coding genes (TRIDC2BG003930 to TRIDC2BG004010). Among the six predicted loci, four (TRIDC2BG003930, TRIDC2BG003950, TRIDC2BG003970 and TRIDC2BG003990) encode disease resistance-related proteins. Moreover, the latter three genes TRIDC2BG003950, TRIDC2BG003970 and TRIDC2BG003990 were only present in the WEW genome (Fig. 4). To date, the most of cloned powdery mildew resistance genes are nucleotide-binding leucine-rich repeat (NLR) genes, including Pm3 (Yahiaoui et al. 2004), Pm8 (Hurni et al. 2013), Pm2 (Sánchez-Martín et al. 2016), Pm17 (Singh et al. 2018), Pm60 (Zou et al. 2018), Pm21 (He et al.2018; Xing et al. 2018), Pm5e (Xie et al. 2020), Pm41 (Li et al. 2020b) and Pm1a (Hewitt et al. 2020). One of these four genes might be the candidate gene for MlIW39 and cloning of the gene is underway to identify the real candidate gene.

Potential of MlIW39 for wheat breeding

Many genes for resistance to different pathogens have been identified in wild emmer wheat, including genes for resistance to stripe rust (Klymiuk et al. 2018), leaf rust (Moseman et al. 1985), stem rust (Anikster et al. 2005), powdery mildew (Nevo et al. 1985) and Fusarium head blight (Buerstmayr et al. 2003). Yr15 for resistance to stripe rust has been successfully used in agriculture (Yaniv et al. 2015; Chen and Kang 2017). Further explorations of WEW will unravel additional favorable genes useful for wheat improvement (Xie and Nevo 2008).

Up to now, more than 100 powdery mildew resistance genes have been reported (https://shigen.nig.ac.jp/wheat/komugi/genes/symbolClassList.jsp), nearly half of them are derived from wheat wild relatives. Some resistance genes in wild relatives are associated with undesirable traits and a time-consuming procedure is required to reduce or eliminate the linkage drag (Summers and Brown 2013; Li et al. 2016). Since WEW and cultivated wheat are closely related, the chromosomes from WEW can cross over readily with their homologues in common wheat; therefore, the linkage drag will not be a problem for the utilization of genes from WEW (Nevo 2014). In this study, a powdery mildew resistance gene MlIW39 was transferred from WEW into common wheat and resulted in an all-stage resistant line 8D49. In addition to its excellent powdery mildew resistance, 8D49 showed longer spike and larger seed size than its recurrent parent 87-1 (data not shown), indicating no detrimental effects with MlIW39. 8D49 is highly effective against some Bgt isolates collected from different regions of China (Table 2; Fig. S2). Because the powdery mildew pathogen is highly variable, one major resistance gene will not be enough to control the disease if deployed alone. MlIW39 must be used in combination with other Pm genes to broaden the genetic basis and to increase the durability of any resistance that is deployed. The co-segregating molecular markers 7Seq622 and 7Seq727 of MlIW39 should be useful in building effective combinations of resistance genes by marker-assisted selection.