Introduction

Wheat (Triticum aestivum L.) is widely grown as a staple crop in many temperate regions of the world. Wheat production, in terms of yield and stability, is constantly challenged by many diseases, and wheat powdery mildew, caused by the fungus Blumeria graminis f. sp. tritici (DC.) Speer, Bgt, is an epidemic foliar disease in many maritime or semi-continental climates (Morgounov et al. 2012). Reported yield reductions caused by powdery mildew range from 5 to 40% and can be as high as 62% in severely infected fields (Singh et al. 2016). Decreases in quality due to powdery mildew infection have also been reported (Samobor et al. 2006). In China, powdery mildew has affected an area of around 6–8 million hectares in recent years (http://cb.natesc.gov.cn/sites/cb/). Fungicides such as triadimefon are often used by farmers to prevent powdery mildew in fields, but fungicide resistance has been detected in the pathogen population (Shi et al. 2015). This, together with the environmental pollution concern, discourages the continuous application of fungicides in protection of wheat from the disease. The improvement of powdery mildew resistance is the preferred method for limiting disease epidemics and minimizing the economic losses caused by the disease.

Breeding for powdery mildew-resistant cultivars relies on the availability of resistant resources. Currently, designated powdery mildew (Pm) resistance genes or alleles from Pm1 to Pm60 have been mapped on specific chromosomes (https://shigen.nig.ac.jp/wheat/komugi/genes/symbolClassList.jsp). Among them, some were identified in T. aestivum and the rest originated from close or distant relatives of wheat (Guo et al. 2017). Additionally, many temporarily designated Pm or Ml resistance genes have been located on different chromosomes.

Some resistance genes can be effective against powdery mildew only for a period in agriculture because of virulence shift in the pathogen populations (Hsam and Zeller 2002). Others are not useful in cultivar development due to the linkage drag caused by association between powdery mildew resistance and certain deleterious traits (Summers and Brown 2013). Identification of new resistance genes is a continuous objective in breeding programs. Since the 1990s, various classes of molecular markers have been used to saturate genetic maps and identify powdery mildew resistance genes (Huang and Röder 2004; McIntosh et al. 2013). The wheat consensus SSR map has integrated 3700 loci (http://wheat.pw.usda.gov). Many designated powdery mildew resistance genes were initially identified with the aid of SSR markers. However, the wheat SSR markers are scattered on chromosomes and due to the huge size of the wheat genome (~ 17 Gb), they are not numerous enough for the fine mapping of target genes. Many wheat bin-mapped ESTs have been used to develop STS and SSR markers for locating the chromosomal bins of the resistance genes (Qi et al. 2004). The EST sequences are suitable for comparative genomics analysis due to the consensus that exists among the EST sequences of grass species.

Conserved synteny exists between wheat and its close relatives; and, the wheat EST database (Mochida et al. 2006; Coordinators 2016), genomic sequences of Brachypodium distachyon L. (International Brachypodium Initiative 2010), rice (Oryza sativa L.) (International Rice Genome Sequencing Project 2005), and sorghum [Sorghum bicolor (L.) Moench] (Paterson et al. 2009) are available. So, comparative genomics analysis has become an effective method to develop more molecular markers for the genetic mapping or fine mapping of Pm genes. For example, saturated linkage maps have been developed for Pm6 (Qin et al. 2011), MlIW172 (Ouyang et al. 2014), Pm41 (Wang et al. 2014), MlHLT (Wang et al. 2015), MlWE4 (Zhang et al. 2015), and PmTm4 (Xie et al. 2017) using this strategy. The release of genomic sequences of T. aestivum cv. Chinese Spring (AABBDD genome) (Belova et al. 2013; Choulet et al. 2014; International Wheat Genome Sequencing Consortium 2014; Zimin et al. 2017), Aegilops tauschii (DD genome) (Jia et al. 2013; Zhao et al. 2017; Luo et al. 2013, 2017), T. urartu (AA genome) (Ling et al. 2013, 2018), and wild emmer wheat (T. turgidum ssp. dicoccoides) (AABB genome) (Avni et al. 2017) makes comparative genomics analysis and map-based cloning in wheat more informative.

Wheat landraces from China have provided several powdery mildew resistance genes. The first gene, Pm5e, was identified on chromosome arm 7BL in Fuzhuang 30, which was selected from a cross between the two landraces Liquan Heshangtou and Huaxian Qisifeng (Huang et al. 2003). Another allele in this locus, Pm5d, was identified in IGV1-556, which was derived from the accession CI 10904 that was introduced from Jinling University, Nanjing (Hsam et al. 2001). The provisionally designated genes PmH (Hongquanmang, Zhou et al. 2005), PmTm4 (Tangmai 4, Hu et al. 2008), Mlmz (Mazhamai, Zhai et al. 2008), Mlxbd (Xiaobaidong, Xue et al. 2009), pmHYM (Hongyoumai, Fu et al. 2017), PmBYYT (Baiyouyantiao, Xu et al. 2018a), and PmSGD (Shangeda, Xu et al. 2018b) were also localized in the chromosomal region around the Pm5 locus. PmTm4 was believed to have originated from the landrace Laozaomai (Hu et al. 2008). There are two alleles on locus Pm24 on chromosome arm 1DS, Pm24a in Chiyacao (Huang et al. 2000) and Pm24b in Baihulu (Xue et al. 2012). Pm47 was located on chromosome arm 7BS in Hongyanglazi (Xiao et al. 2013). PmX in Xiaohongpi (Fu et al. 2013), MlHLT in Hulutou (Wang et al. 2015), Pm2c in Niaomai (Xu et al. 2015), and Pm45 in D57 (Wuzhaomai) (Ma et al. 2011) were mapped on chromosome arms 2AL, 1DS, 5DS, and 6DS, respectively.

A landrace, Xuxusanyuehuang (XXSYH), collected from Fengdu County, Sichuan province, appeared to be highly resistant against different Bgt isolates. The aims of this study were to examine (1) the effectiveness of the XXSYH gene(s) to Bgt isolates from wheat-producing regions of China; and (2) the inheritance and molecular mapping of the Pm gene(s) in XXSYH by means of comparative genomics analysis.

Materials and methods

Plant materials

The F1, F2, and F2:3 populations, and F5 recombinant inbred lines (RILs) were developed by crossing XXSYH to the susceptible cultivar Mingxian 169 for the genetic analysis and molecular detection of the Pm gene in XXSYH. Chromosome arm assignment of the target resistance gene-linked markers was performed using the Chinese Spring (CS) nullisomic–tetrasomic, ditelosomic, and deletion lines. Twenty-five wheat accessions that carry known Pm genes or gene combinations were used to differentiate the Bgt isolates. Zhongzuo 9504 was included in this study for maintaining and increasing Bgt isolates, and it was used as the susceptible control in all assessments of the powdery mildew reactions.

Powdery mildew evaluations

Fifteen single-colony cultures of Bgt isolates, collected from different wheat fields in China, were used to evaluate the resistance of XXSYH to powdery mildew (Table 1). Isolate E09 was used to phenotype the mapping populations and the two parents for genetic analysis of the target resistance gene. Evaluations of powdery mildew reactions to the Bgt isolates at the seedling stage were conducted in a greenhouse set at 22 °C day/18 °C night with 60% relative humidity and a 12-h light/12-h dark photoperiod. Xuxusanyuehuang, Mingxian 169, the F1, 286 F2 plants, 159 F2:3 families, and 200 F5 RILs were tested. At least 15 plants from each F2:3 family and F5 line were examined. Two independent tests were conducted for the RIL population. Seedlings at the one leaf stage were artificially inoculated with Bgt isolates by dusting conidiospores that were multiplied on the susceptible plants of Zhongzuo 9504. Infection types (ITs) of all plants were rated on a 0–4 scale 15 days after inoculation (Liu et al. 1999). The inoculated plants were divided into either a resistant group (IT 0–2) or a susceptible group (IT 3–4).

Table 1 Reactions of Xuxusanyuehuang, Mingxian 169, wheat entries possessing known powdery mildew resistance genes or gene combinations, and the susceptible control Zhongzuo 9504 after inoculation with 15 isolates of Blumeria graminis f. sp. tritici (Bgt)
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Molecular marker analysis

Genomic DNA was extracted from the young leaves using the cetyltrimethylammonium bromide (CTAB) method (Saghai-Maroof et al. 1984). Resistant and susceptible DNA bulks were composed of equal amounts of DNA from the representative plants of 10 homozygous resistant and 10 homozygous susceptible F2:3 families for bulked segregant analysis (BSA) (Michelmore et al. 1991). Polymorphisms of wheat genomic SSRs (i.e., Xgwm, Xwmc, Xbarc, Xcfa, and Xcfd series) and EST markers (http://wheat.pw.usda.gov) were examined. The reaction mixture (10 μl) for DNA amplification was prepared by mixing 50 ng DNA, 0.2 mM dNTPs, 0.2 μM of each primer, 1 U of Taq polymerase, and 1× assay buffer. The following conditions were used for DNA amplification: 94 °C for 5 min; 35 cycles of 94 °C for 30 s, 53–60 °C (depending on primers used) for 30 s, 72 °C for 30 s; and 72 °C for 10 min. The amplification products were visualized on 8% non-denaturing polyacrylamide gels (Acr/Bis = 39:1) after silver staining.

Chromosome arm assignment of the target resistance gene, comparative genomics analysis, and marker development

The resistance gene-linked markers were localized by comparing the banding patterns amplified from the Chinese Spring nullisomic–tetrasomic, ditelosomic, and deletion lines. Assignment of polymorphic markers to chromosome bins was conducted by determining the smallest deletion bin that possesses them.

The EST sequences flanking the target gene on bin 4AL-4-0.80-1.00 were used to search for the orthologous genes in the CDS sequences of Ae. tauschii, B. distachyon (http://mips.helmholtz-muenchen.de/plant/Brachypodium/), rice (http://rice.plantbiology.msu.edu/), and sorghum (http://mips.helmholtzmuenchen.de/plant/sorghum/) genomic sequences. Then, the wheat EST sequences homologous to CDS sequences of Brachypodium within the homologous genomic regions were used to develop EST-SSR markers polymorphic between the two parents and the contrasting DNA bulks. The Brachypodium orthologous gene sequences flanking the polymorphic EST-SSR markers were used as query to search the Chinese Spring genomic sequences released by the International Wheat Genome Sequencing Consortium (IWGSC) (http://www.wheatgenome.org/) to determine the homologous contigs or scaffolds on chromosome arm 4AL. The acquired contig sequences were used to develop SSR markers using the software Batchprimer3 (https://probes.pw.usda.gov/batchprimer3/). Polymorphic EST-SSR and SSR markers were mapped on the F5 RILs to develop the linkage map of the target gene.

Linkage analysis and genetic linkage map construction

The deviations between the observed phenotypic data and the expected segregation ratios in the genetic analysis of the resistance gene using F2, F2:3, or F5 populations were analyzed with the Chi-squared (χ2) test. Linkage relationships and distances between the polymorphic markers and the Pm gene in XXSYH with the F5 population were determined using the software Mapmaker version 3.0 with the Kosambi map function and an LOD threshold of 3.0 (Lincoln et al. 1993). A genetic linkage map for the target gene in XXSYH was developed using the software Mapdraw V2.1 (Liu and Meng 2003).

Results

Reactions of XXSYH to Bgt isolates

In the seedling tests, all the 15 Bgt isolates examined were avirulent on lines carrying genes or gene combinations involving PmH, Pm16, Pm24, Pm52, Pm2 + 6, and Pm5 + 6 (Table 1). Lines with genes Pm1c, Pm2, Pm5e, and Pm13 were resistant against 14 isolates. The virulence frequencies on lines with Pm1a, Pm3b, Pm4a-4c, and Pm33 ranged from 26.7 to 53.3%. The same reaction patterns were observed between lines carrying Pm4a and Pm4b. Most Bgt isolates were virulent on lines with Pm3a, Pm3c, Pm3e, Pm3 g, Pm5a, Pm6, Pm7, and Pm8. Line carrying Pm19 was susceptible to all Bgt isolates tested.

Xuxusanyuehuang was resistant to 11 of the 15 isolates examined, but was susceptible to isolates 1, 3, 6, and 14. Compared to the differential wheat entries, XXSYH differed from the lines carrying the known genes Pm1c in its reaction to 3 isolates, PmH, Pm16, Pm24, Pm52, Pm5 + 6, and Pm2 + 6 to 4 isolates, Pm2, Pm5e, and Pm13 to 5 isolates, Pm4a, Pm4b, and Pm1a to 7 isolates, and Pm4b and Pm4c to 8 isolates (Table 1). Mingxian 169 was as susceptible to all the Bgt isolates as the control cultivar Zhongzuo 9504.

Genetic analysis of the gene for powdery mildew resistance in XXSYH

A genetic analysis was carried out to characterize the inheritance mode of the powdery mildew resistance gene in XXSYH against Bgt isolate E09 using the F1, F2:3, and F5 populations developed from the XXSYH × Mingxian 169 cross (Table 2). The parental cultivars XXSYH (IT 1) and Mingxian 169 (IT 3) showed distinct phenotypic responses to isolate E09. The IT of F1 plants was the same as the susceptible parent Mingxian 169 (Fig. 1). The segregation of resistant and susceptible F2 plants provided a good fit to a 1:3 ratio. The 159 F2:3 families and 200 F5 RILs segregated in ratios of 1:2:1 (homozygous resistant: heterozygous: homozygous susceptible) and 1:1 (resistant: susceptible), respectively (Table 2). These results clearly demonstrate that XXSYH carries a single recessive gene for resistance to Bgt isolate E09.

Table 2 Genetic analysis of resistance to isolate E09 of Blumeria graminis f. sp. tritici in F1, F2, F2:3, and F5 progenies derived from Xuxusanyuehuang × Mingxian 169 cross
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Fig. 1
figure 1

The phenotypic reactions of resistant parent Xuxusanyuehuang, susceptible parent Mingxian 169, and the susceptible control Zhongzuo 9504 to Bgt isolate E09

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Localization of the gene for powdery mildew resistance with SSR markers

By analysis of the polymorphisms of 120 SSR primer pairs randomly distributed on different wheat chromosomes, we identified only one marker, Xgwm160, located on chromosome arm 4AL that was polymorphic between the parental cultivars, as well as the contrasting DNA bulks. Screening of 104 additional pairs of SSR primers mapped on 4AL produced two more polymorphic markers, Xbarc52 and Xbarc327. Genotype analysis of the F5 mapping population revealed that the powdery mildew resistance gene was localized between the co-dominant markers Xgwm160 and Xbarc327 within a 4.64-cM genetic interval (Fig. 2). Because of its unique position on chromosome arm 4AL, this gene was designated Pm61.

Fig. 2
figure 2

Comparative genetic linkage and physical maps of powdery mildew resistance gene Pm61 and the orthologous genomic regions of Triticum aestivum, Aegilops tauschii, T. urartu and wild emmer. The linkage map was constructed using F5 RIL population derived from Xuxusanyuehuang × Mingxian 169 cross

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Molecular marker development for Pm61 through comparative genomics analysis

The SSR markers Xgwm160 and Xbarc52 were mapped to deletion bin 4AL-4-0.80-1.00, so 20 EST-STS markers on this chromosome region (https://wheat.pw.usda.gov/cgi-bin/westsql/bin_candidates.cgi?bin=4AL4-0.80-1.00) were initially screened for their polymorphisms between the parental cultivars and the contrasting bulked segregants. Unfortunately, no polymorphic EST-STS markers between the parental cultivars or the contrasting DNA bulks were identified. An additional 105 wheat EST sequences that were anchored on the bin 4AL-4-0.80-1.00 were compared to the genomic sequence databases of Brachypodium, rice, and sorghum using the batch Blast program hosted at GrainGenes (http://www.graingene/lblgov/cgi-bin/nphblast_interface.cgi). Orthologous genes were found on Brachypodium chromosome 1, rice chromosome 6, and sorghum chromosome 10 (Table S1). The wheat EST sequences with high synteny to the orthologous genes were used to design 125 SSR primer pairs. Three co-dominant EST-SSR markers, namely Xicsx29 (BE490293), Xicsx65 (BE591440), and Xicsx79 (BG604834) (Fig. 3), and one dominant EST-SSR marker, Xicsx73 (BF200736) (Table 3), were polymorphic between the parental cultivars and the contrasting DNA bulks. Pm61 was re-localized in the genetic interval (4.18 cM) between markers Xgwm160 and Xicsx29 (BE490293) using the F5 mapping population (Fig. 2).

Fig. 3
figure 3

Amplification patterns of markers Xicsx436 (SSR), Xicsx79 (EST-SSR), Xgwm160 (SSR), and Xicsx65 (EST-SSR) in Xuxusanyuehuang (PR), Mingxian 169 (PS), homozygous resistant (R), heterozygous resistant (H), and homozygous susceptible (S) F5 recombinant inbred lines derived from Xuxusanyuehuang × Mingxian 169 cross. M: 100 bp DNA ladder

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Table 3 Detailed information on EST-SSR and SSR markers linked to the powdery mildew resistance gene Pm61
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The homologous region on Brachypodium chromosome 1 (Bradi1g50205 to Bragilg52140), corresponding to the genetic interval between markers Xicsx79 (BG604834) and Xicsx73 (BF200736) (Fig. 3), was used to blast the Ae. tauschii and T. turgidum ssp. dicoccoides CDS sequence databases and the rice and sorghum genomic sequence databases to determine the region of collinearity. The coding sequences of collinear Brachypodium genes were used as queries to search the 454 Chinese Spring contigs and the IWGSC individual chromosome survey sequences (http://www.wheatgenome.org/) for identifying the homologous contigs or scaffolds on chromosome arm 4AL. Based on those sequences, 398 pairs of SSR primers were designed. Seven polymorphic markers developed from different Brachypodium orthologous genes, namely Xicsx367 (Bradi1g51960), Xicsx436 (Bradi1g50280), Xicsx511 (Bradi1g52040), Xicsx520 (Bradi1g52050), Xicsx528 (Bradi1g52090), Xicsx530 (Bradi1g52110), and Xicsx538 (Bradi1g51750), were incorporated into the genetic linkage map (Fig. 3, Table 3). Based on their banding patterns, Xicsx436 (Fig. 3), Xicsx511, Xicsx520, Xicsx528, Xicsx530, and Xicsx538 were co-dominant, while Xicsx367 was dominant. Pm61 was placed to a 0.46-cM interval and flanked by markers Xgwm160 and Xicsx79 at genetic distances of 0.23 cM and 0.23 cM at the distal end of chromosome arm 4AL, respectively (Fig. 2).

Comparative genomics analysis of the genetic interval flanking Pm61 and gene prediction

In the Chinese Spring genomic sequence, the genetic interval between the closest flanking markers Xgwm160 and Xicsx79 for Pm61 (0.46 cM) was mapped on chromosome 4AL within a 1.3-Mb genomic region (717963176–719260469), which contained 26 predicted genes (Table S2). A detailed comparative genomics analysis was conducted to search for the conserved collinear orthologous genes among the CDS sequence databases of Ae. tauschii, T. urartu, and wild emmer. The collinear orthologous genomic region corresponding to the genetic interval of Pm61 spanned an 8.2 kb genomic region consisting of 17 predicted orthologous genes (AET7Gv20073500–AET7Gv21099600) on chromosome 7D of Ae. tauschii. This region was collinear with a 1.8-Mb genomic region (Tu7_TuG1812G0716125900.01.T01–Tu7_TuG1812G0716156200.01.T01) consisting of 13 predicted orthologous genes on chromosome 7A of T. urartu. Two collinear genomic regions were detected in the wild emmer genome. One was 2.06 Mb (TRIDC4AG066800–TRIDC4AG067400) with 11 predicted orthologous genes, and the other was 484 kb (TRIDC7AG003210–TRIDC7AG003290) with 4 predicted orthologous genes on chromosome 7A (Fig. 2, Table S2). The annotation of the conserved collinear orthologous genes demonstrated that five (TraesCS4A01G454300.1, TraesCS4A01G454400.1, TraesCS4A01G454900.1, TraesCS4A01G455100.1, and TraesCS4A01G455200.1) in Chinese Spring, three (AET7Gv20074800, AET7Gv20075100, and AET7Gv20119500) in Ae. tauschii, one (Tu7_TuG1812G0716126400.01.T01) in T. uraru, and three (TRIDC4AG067170, TRIDC4AG067180, and TRIDC7AG003280) predicted genes in wild emmer encoded for proteins associated with disease resistance. They included NBS-LRR disease resistance protein, receptor-like kinase family protein, and RPM1-like disease resistance protein (Table S2).

Chromosome bin assignment of Pm61

Because the synteny genomic regions flanking the Pm61 locus were found in chromosomes of both homoeologous groups 4 and 7, the Chinese Spring nullisomic–tetrasomic, ditelosomic, and deletion lines for the chromosomes of these homoeologous groups were used to determine the chromosome and the physical bin location of the markers that were linked to Pm61. The absence of products from markers Xicsx65 and Xicsx79 in the nullisomic–tetrasomic line N4A-T4D, and the deletion lines 4AL-4, 4AL-5, 4AL-12, and 4AL-13 on 4AL-4-0.80-1.00 demonstrated that Pm61 was located in the distal chromosomal bin 4AL-4-0.8-1.00 (Fig. 4a). Xicsx65 and Xicsx79 produced identical products in XXSYH, CS, and the group 7 nullisomic–tetrasomic and ditelosomic lines (Fig. 4b), indicating that Pm61 was not present on any of the homoeologous group 7 chromosomes.

Fig. 4
figure 4

Amplification patterns of the Pm61-linking markers Xicsx65 and Xicsx79 in Xuxusanyuehuang, Mingxian 169, Chinese Spring (CS), and CS homoeologous groups 4 (a) and 7 (b) nullisomic–tetrasomic, ditelosomic, and deletion lines. M: 100 bp DNA ladder

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Comparison of physical positions between Pm61 and MlIW30 identified in wild emmer

MlIW30, a single dominant Pm gene derived from wild emmer, was mapped on chromosome bin 4AL-4-0.8-1.00 (Geng et al. 2016). The homologous genomic region carrying MlIW30 in wheat was collinear with the corresponding Brachypodium genomic region extending from Bradi1g50220 to Bradi1g52230. Polymorphic markers linked to MlIW30 were detected between XXSYH and Mingxian 169, as well as the contrasting DNA bulks. Two SSR markers, XB1g2020.2 and XB1g2070.1, developed from genes Bradi1g52020 and Bradi1g52070, were linked to Pm61, but they were mapped to the proximal side of Pm61 at genetic distances of 2.55 cM and 4.18 cM, respectively (Fig. 2). Pm61 and MlIW30 were located 0.23 cM and 1.8 cM from the common SSR marker Xgwm160 on the proximal side, respectively. However, the two nearest flanking markers XB1g2000.2 and XB1g2020.2 located MlIW30 in a 0.1-cM genetic interval corresponding to a 21 kb (732769506–732790522 on chromosome arm 4AL) physical interval in the genome of Chinese Spring, which was obviously different from the 1.3 Mb physical localization of Pm61 (717963176–719260469) (Table S3).

Discussion

Chinese landrace XXSYH was resistant to some Bgt isolates collected from China in the seedling tests. A recessive gene Pm61 conferred the resistance to powdery mildew in this cultivar. Molecular marker analysis localized Pm61 in a 0.46-cM genetic interval on chromosome arm 4AL. Results of physical mapping of the closest flanking markers Xgwm160 and Xicsx79 assigned Pm61 in a 1.3-Mb physical interval in the chromosome 4AL genomic sequence of Chinese Spring.

More than 13,000 wheat landraces are preserved in the Gene Bank of China in Beijing (Liu et al. 2000). Extensive studies have been conducted to evaluate the resistance to powdery mildew of the Chinese wheat landraces. In the first large scale test, Sheng et al. (1992) identified six immune or highly resistant and 71 moderately resistant landraces in a collection of 3441 accessions from eight provinces in China. Wang et al. (1996) obtained 44 resistant landraces out of 867 accessions indigenous to Henan province. Four cultivars were moderately resistant among 1837 wheat landraces from Jiangsu province (Xiong et al. 1995), and seven landraces were highly resistant in 1152 wheat accessions from Shaanxi province (Hu et al. 2007). Seedling resistance was observed in 46 accessions, and the adult plant resistance was detected in 193 landraces from Gansu province (Cao et al. 2010). Variation in the frequencies of powdery mildew-resistant landraces was observed in different wheat-producing regions (Li et al. 2011). In subsequent studies, more than 20 Pm resistance genes/alleles from the Chinese wheat landraces have been identified, and some of them have been mapped on chromosome arms 2AL (Fu et al. 2013), 7BS (Xiao et al. 2013), 7BL (Hsam et al. 2001; Huang et al. 2003; Zhou et al. 2005; Hu et al. 2008; Zhai et al. 2008; Xue et al. 2009; Fu et al. 2017; Xu et al. 2018a, b), 1DS (Huang et al. 2000; Xue et al. 2012; Wang et al. 2015), 5DS (Xu et al. 2015), and 6DS (Ma et al. 2011). Based on its unique position, Pm61 from XXSYH is a new locus conferring resistance to powdery mildew on chromosome arm 4AL.

Geng et al. (2016) reported a temporarily designated gene MlIW30 on the distal part of chromosome arm 4AL. Although Pm61 and MlIW30 share the same deletion bin 4AL-4-0.8-1.00 on chromosome arm 4AL, they differed obviously in their mode of inheritance, origin, and precise physical localization in the recently released Chinese Spring reference genomic sequence. Pm61 in the Chinese wheat landrace XXSYH exhibited recessive inheritance when tested with Bgt isolate E09, while MlIW30, which originated from an Israeli T. turgidum ssp. dicoccoides accession IW30, showed a dominant mode of inheritance in response to this Bgt isolate. Because of their geographic isolation, these genes evolved independently in different ecotypes even though they are located on the same chromosome. Wild emmer is the tetraploid ancestor of common wheat (Nevo et al. 2013). It has been suggested that wild emmer and common wheat have developed an integrated and stable genetic system during their long-term evolution (Shi et al. 2005). The A genomes of these related species are not completely identical, but are homoeologous. The genomic region of Pm61 that was flanked by the two nearest markers (Xgwm160 and Xicsx79) spans a 1.3 Mb (717963176–719260469) region of chromosome arm 4AL, which is different from the genomic region (732769506–732790522) in which MlIW30 is located.

Two major QTL for resistance to powdery mildew were identified on wheat chromosome 4A. QPm.uga-4A from soft red winter wheat AGS 2000 was located on chromosome arm 4AS (Hao et al. 2015), which is obviously different from Pm61. QPm.tut-4A was detected on chromosome arm 4AL of the wheat-T. militinae introgression line 8.1 (Jakobson et al. 2012). This QTL differed from Pm61 in its origin from T. militinae although they share the common SSR marker Xgwm160.

A translocation in the distal region between 4AL and 7BS had occurred during the evolution of T. aestivum and T. turgidum (Hossain et al. 2004; Miftahudin et al. 2004; Ishikawa et al. 2009; Hernandez et al. 2012). We detected some homoeologous genes around the Pm61 locus on chromosomes 7AS and 7DS in the common wheat genome. Comparative genomics analysis using the recently released genomic sequences of Ae. tauschii (Luo et al. 2017), T. urartu (Ling et al. 2018), and wild emmer (Avni et al. 2017) indicated that the orthologous genomic region of the Pm61 locus was located on chromosome 7D in Ae. tauschii, 7A in T. urartu, and 4A and 7A in wild emmer (Fig. 3, Table S2). The results of chromosomal and physical bin mapping using Chinese Spring aneuploid and deletion lines for the homoeologous groups 4 and 7 chromosomes confirmed the localization of Pm61 on 4AL rather than on any of the homoeologous group 7 chromosomes.

A well-assembled genome sequence of common wheat has recently become available (https://urgi.versailles.inra.fr/download/iwgsc/IWGSC_RefSeq_Assemblies/v1.0/). Because of the high levels of macro- and micro-collinearities between wheat genome and Brachypodium, rice and sorghum genomes, comparative genomics analysis has often been used as an effective means to develop linked molecular markers for gene mapping in common wheat. In the present study, we mapped Pm61 in a small genetic interval using the collinear genomic region on Brachypodium chromosome 1 generated by comparative genomics analysis. Then, the genetic interval flanking Pm61 was used to blast the genomic sequences of Ae. tauschii, T. urartu, and wild emmer to search for collinear regions, which can serve as a framework for fine mapping and map-based cloning of this gene. Further research is in progress to develop closely linked and/or co-segregating markers for the fine mapping of Pm61 in the wheat landrace XXSYH.

In summary, Chinese wheat landrace XXSYH carries a new recessive gene for resistance to powdery mildew, which is designated Pm61. Molecular mapping analysis located Pm61 on the distal end of chromosome arm 4AL. Based on the comparative genomics analysis, four EST-SSR and seven SSR polymorphic markers were developed and incorporated in the genetic linkage map, which mapped Pm61 to a 0.46-cM genetic interval between markers Xgwm160 and Xicsx79, corresponding to a 1.3-Mb interval of the genomic region of 4AL.

Author Contribution Statement

HjL and JL conceived and designed the study. HS, JH, WS, DQ, LC, PW, YL, TL, YQ, and WC conducted the experiments. HZ, HwL, LY, YZ, and ZL analyzed data. JL, HjL, and HS wrote the manuscript with the contributions of ZL.