Mapping of Powdery Mildew Resistance Gene pmCH89 in a Putative Wheat-Thinopyrum intermedium Introgression Line

Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a globally serious disease adversely affecting wheat production. The Bgt-resistant wheat breeding line CH09W89 was derived after backcrossing a Bgt resistant wheat-Thinopyrum intermedium partial amphiploid TAI7045 with susceptible wheat cultivars. At the seedling stage, CH09W89 exhibited immunity or high resistance to Bgt pathotypes E09, E20, E21, E23, E26, Bg1, and Bg2, similar to its donor line TAI7045 and Th. intermedium. No Th. intermedium chromatin was detected based on genomic in situ hybridization of mitotic chromosomes. To determine the mode of inheritance of the Bgt resistance and the chromosomal location of the resistance gene, CH09W89 was crossed with two susceptible wheat cultivars. The results of the genetic analysis showed that the adult resistance to Bgt E09 in CH09W89 was controlled by a single recessive gene, which was tentatively designated as pmCH89. Two polymorphic SSR markers, Xwmc310 and Xwmc125, were linked to the resistance gene with genetic distances 3.1 and 2.7 cM, respectively. Using the Chinese Spring aneuploid and deletion lines, the resistance gene and its linked markers were assigned to chromosome arm 4BL in the bin 0.68–0.78. Due to its unique position on chromosome 4BL, pmCH89 appears to be a new locus for resistance to powdery mildew. These results will be of benefit for improving powdery mildew resistance in wheat breeding programs.


Introduction
Common wheat (Triticum aestivum L.) is the most widely cultivated and important staple food crop in the world, and is constantly challenged by many diseases such as powdery mildew and rusts. Powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is a globally serious disease of wheat. It often occurs in regions with cool and humid climates, resulting in severe yield reductions [1,2]. In China, especially in the southwestern region, powdery mildew is the most frequently occurring disease due to moderate temperatures and rainy conditions during the wheat growing season. Yield losses can surpass 100 million kg per year when powdery mildew epidemics occur [3]. However, powdery mildew pathogen populations are very dynamic due to the continuous appearance of new virulent pathotypes capable of overcoming widely used host resistance genes. As a result, many previously resistant wheat varieties become susceptible. Therefore, new sources of effective and durable resistance genes are required for breeding high-yielding cultivars. Selection of and field deployment of resistant varieties is the most economic, effective, and environmentally friendly approach to controlling the disease [4]. Thus, the discovery and utilization of new powdery mildew resistance genes have become common objectives for wheat geneticists and breeders worldwide.
Fifty-three formally designated major genes (Pm1-Pm53) [5][6][7] and over 100 quantitative trait loci (QTLs) [8,9] for resistance to powdery mildew have been identified on the 21 chromosomes in bread wheat. The majority (39) of them are derived from common wheat, and a minority (18) were transferred from wild relatives of wheat. Thinopyrum intermedium (Host) Barkworth and Dewey (2n = 6x = 42, JJ s S), an uncultivated relative of wheat, has many potentially useful agronomic characteristics that could be used in wheat breeding programs, such as wide adaptability and tolerance to cold, drought, and salinity.
Th. intermedium has been widely studied in research directed toward wheat improvement due to its immunity or resistance to many serious wheat diseases. Hence, a series of addition lines, substitution lines, and translocation lines have been developed over the past years and are available for both agronomic improvement and resistance breeding. To date, many multi-resistant lines have been developed by crossing susceptible wheat cultivars with resistant partial amphiploids as donor parents. So far, two powdery mildew resistance genes (Pm40 [10] and Pm43 [11]), one stem rust resistance gene (Sr44 [12]), and one stripe rust resistance gene (Yr50 [13]) have been transferred from Th. intermedium into common wheat. Pm40, Pm43, Sr44 and Yr50 have been mapped to chromosomes 7BS, 2DL, 7D and 4BL, respectively. CH09W89, a Th. intermedium-derived wheat introgression line, is highly resistant to powdery mildew. It exhibits resistance to powdery mildew under greenhouse conditions in Taiyuan, Shanxi province. We identified a number of wheat genotypes with effective resistance against common Chinese races. The purposes of the present study were to determine the resistance inheritance of and locate the new gene in the Th. intermedium-derived line CH09W89.

GISH Identification of Alien Chromatin in CH09W89
GISH analysis was performed on somatic cells of both the resistant line CH09W89 and the recombinant control (line 03W006) using Th. intermedium genomic DNA as the labeled probe and Chinese Spring (CS) genomic DNA as blocker DNA. A pair of chromosomes revealed green fluorescence signals at the distal regions of their short arms in the positive control ( Figure 2a). No visible hybridization signal could be found in CH09W89 (Figure 2b), indicating that no detectable wheat-Th. intermedium translocation is present in CH09W89. However, the translocated chromosome segment (if present) might be too small to be detected by GISH. Figure 2. GISH pattern of lines 03W006, the wheat-Thinopyrum intermedium recombinant control (a) and CH09W89 (b) at mitosis using Th. intermedium genomic DNA as the probe. Arrows indicate alien chromatin. Scale bar = 10 μm.

Inheritance of the Resistance to Powdery Mildew in CH09W89
At the adult plant stage, when inoculated with race E09, F1 plants from both crosses (SY95-71/CH09W89 and CH09W89/Mianyang) showed infection types (IT 3-4) similar to the susceptible parent, showing that the resistance gene was recessive ( Table 2). Segregation in the F2 and BC1 populations ( Table 2) included many plants with intermediate responses. When the numbers of F2 plants with IT 0-2 and those with IT 3-4 were pooled as separate resistant and susceptible groups, the ratios were consistent with those expected for segregation at a single locus. When tested with the same race, the F2:3 lines from SY95-71/CH09W89 and the BC1 plants from CH09W89/ MY11//CH09W89 segregated one homozygous resistant (HR): two segregating (Seg): one homozygous susceptible (HS), and one resistant: one susceptible, respectively, confirming segregation ratios for a single gene ( Table 2). In addition, in segregating F3 lines from SY95-71/CH09W89, the pooled numbers of resistant and susceptible plants were 1130 susceptible: 373 resistant (χ 2 (3:1) = 0.03, P1df = 0.87). These results suggested that a single recessive gene for resistance to powdery mildew was present in CH09W89, and was provisionally designated pmCH89.

Identification of Microsatellite Markers Linked to pmCH89
A total of 596 SSR markers, covering all 21 pairs of chromosomes, were used to screen polymorphisms between resistant (resistant parent and Br (bulked resistant)) and susceptible materials (susceptible parent and Bs (bulked susceptible)). About 156 (26.2%) of the microsatellite primers chosen for initial screening were polymorphic between resistant and susceptible materials. Four markers, Xbarc193, Xbarc199, Xwmc125 (Figure 3), and Xwmc310 (Figure 3), were associated with pmCH89. Linkage analysis using the four markers on F2 plants and the powdery mildew response genotypes inferred from reactions of the F2:3 families indicated that they were linked to the resistance gene. The three SSR markers, Xwmc125, Xbarc199, and Xwmc310, were located on the long arm of chromosome 4B, and Xbarc193 on the short arm of chromosome 4B [14] (http://wheat.pw.usda. gov/cgi-bin/graingenes), indicating that pmCH89 is located on 4B. The F2 population segregated 1:2:1 for all four markers.
Analyses with Joinmap 4.0 also showed linkage between the markers and pmCH89; Xwmc310 and Xwm125 were close to the resistance gene with genetic distances of 3.1 and 2.7 cM, respectively, and Xbarc199 and Xbarc193 were more distant with respective genetic distances of 7.8 and 28.0 cM (Figure 4).

Chromosome Arm Assignment and Deletion Bin Mapping
To determine the location of pmCH89 on chromosome 4B, the four SSR markers Xwmc125, Xbarc193, Xbarc199, and Xwmc310 (http://wheat.pw.usda.gov/cgi-bin/graingenes) were used to test a set of Chinese Spring (CS) nullitetrasomic and ditelosomic lines. The four microsatellite primer pairs amplified products of the expected sizes in CS and the CS nulli-tetrasomic lines N4AT4B and N4DT4B, but no PCR product was observed for the nulli-tetrasomic N4BT4A and Dt4BS lines (Figure 5a), except Xbarc193. Only Xbarc193 had PCR products in the ditelosomic line Dt4BS. Because amplified products of three markers were absent in the N4BT4A and Dt4BS lines, we confirmed the assignment of the linked microsatellite markers to the long arm of chromosome 4BL.
The deletion lines of CS chromosome 4BL were used to determine the physical bin locations of pmCH89 and its flanking markers. Xwmc310 and Xwmc125 were not detected in 4BL-4 and 4BL-3, and Xwmc125 was also absent in 4BL-1 (Figure 5b). The results indicated that Xwmc310 is located at 4BL bin 0.68-0.71 and that Xwmc125 is at 4BL bin 0.71-0.78. Therefore, the powdery mildew resistant gene pmCH89 could be assigned to 4BL bin 0.68-0.78 ( Figure 4). Based on its origin and map location, the recessive gene pmCH89 is apparently new.

Discussion
Exploitation of genetic variability, especially for genes conferring disease resistance, is essential for the development of new, improved plant varieties. The uncultivated relatives of wheat have provided some of the most important and widely used resistance genes which have been deployed in commercial cultivars around the world. Th. intermedium is an important perennial Triticeae species for wheat improvement due to its resistance to viral and fungal diseases. Some wheat-Th. intermedium derivatives have been found to be highly resistant to the fungus causing powdery mildew. For example, a resistance gene was recently found in partial amphiploids [15] and a substitution line 2E (2D), in which an E-chromosome of Th. intermedium was substituted for chromosome 2D in wheat [16]. CH09W89, a putative derivative of Th. intermedium accession Z1141, was produced by crossing and backcrossing TAI7045, a partial wheat-Th. intermedium amphiploid, with susceptible wheat cultivars and selecting for powdery mildew resistance. Line CH09W89 displayed good agronomic characteristics and also carried resistance to powdery mildew. However, there was no documentation of the chromosome content of this line. As all of the wheat parents in the pedigree of CH09W89 are highly susceptible, the resistance in that line is assumed to derive from Th. intermedium. Genetic segregation data clearly indicate the presence of a single recessive resistance gene in CH09W89 (Table 2). Two other powdery mildew resistance genes, Pm40 [10] and Pm43 [11], previously introgressed into common wheat from this donor species, were dominant. Given its action and chromosomal location, the resistance gene in CH09W89 seems to involve a novel locus.
In general, because of the presence of the Ph1 gene on wheat chromosome 5B, the recovery of a wheat-alien recombinant is difficult due to the lack of homoeologous pairing between the chromosomes of distant wild relatives and those of wheat. However, chromosomes of Th. intermedium and wheat can recombine [17,18], suggesting that the transfer of alien chromosome segments by spontaneous chromosome translocations from this species to wheat chromosomes is possible. A good example of this appears to be the current study in which a small Th. intermedium-derived segmental translocation carrying resistance to powdery mildew has apparently been produced by backcrossing the resistant partial amphiploid TAI7045, derived from Th. intermedium, with a susceptible wheat cultivar [19]. However, GISH analysis of CH09W89 produced no cytological evidence for an alien translocation in this study. No apparent linkage drag was observed in the introgression line CH09W89. The gene pmCH89, based on widespread effectiveness and a unique chromosome location, must be either present in an intercalary cryptic translocation from Th. intermedium or a wheat gene derived from an unknown source. Cryptic alien chromosome transfers have been reported in other studies [20][21][22]. Further studies are needed to confirm the source of pmCH89.
Several QTLs for adult plant resistance with major or minor effects on powdery mildew response were also mapped on chromosome 4BL in previous studies [8]. Among them, QPm.nuls-4BL, a major QTL with resistance contributed by Avocet, accounting for between 21.0% and 40.2% of the total phenotypic variation in a RIL population, was located on the region around Xgwm251 and Xgwm375 [28]. This QTL was at the same position as similar QTLs QPm.sfr-4B, Qpm.ipk-4B, and Qpm.caas-4BL, detected respectively in the Forno/Oberkulmer, Synthetic/Opata, and Fukuho-komugi/ Oligoculm populations [8]. Their location is very close to the centromere of chromosome 4B due to the close linkage to Xgwm149 [14], whereas in this study, pmCH89 was physically mapped on the intercalary bin 0.68-0.78 of chromosome 4BL, about 3 cM proximal to Xwmc125 (Figures 4 and 5). This would seem to be a different locus. Additionally, Marone et al. [9] detected a major QLT, MQTL14-4BL, locus for powdery mildew resistance from T. durum on 4BL between the markers Xbcd110 and Xgwm6a. This gene also has a different chromosomal location from pmCH89 because the distal flanking marker Xgwm6a for MQTL14-4BL is proximal to Xwmc125 with a genetic distance of 16 cM [14] and its physical location is in chromosome 4BL distal bin 0.86-1.00 [29].
In this current work, linkage analysis showed that a putative Th. intermedium-derived powdery mildew resistance gene pmCH89 was flanked by Xwmc310 and Xwm125 (Figure 4). Between the flanking markers Xwmc310 and Xwmc125, a genetic distance of 5.8 cM was found. This indicated that there was recombination between these loci as well as between them and the resistance gene. Interestingly, the markers, which span 5.8 cM in the SY95-71/CH09W89 cross, cover approximately 23 cM in the Synthetic/Opata cross [14]. This shows that the recombination rate in the present study is about three-fold lower than in a cross involving hexaploid wheat. These results suggest that pairing and crossovers between chromosome 4B and an unidentified fragment of Th. intermedium chromosome introgressed into CH09W89 seem to be possible but at a reduced rate. In fact, such suppressed recombination is common in populations segregating for alien introgressions in plants. For example, powdery mildew resistance genes Pm12 and Pm27, introgressed into common wheat chromosome 6B from Aegilops speltoides and Triticum timopheevii, respectively, showed no recombination or low recombination between the alien segments (6S or 6G) and the wheat chromosome 6B [30,31].
It is reported that many of the resistance genes used in agriculture eventually become ineffective with the continual change of the pathogen's virulence structure [32]. Consequently, there is an urgent need to discover and transfer more powdery mildew resistance genes from alien sources, which represent an abundant genetic resource, to commercial cultivars. The present gene pmCH89 identified herein was transferred into a commercial wheat background, and a range of powdery mildew resistant introgression lines have been obtained. Henceforth, these lines could be used in wheat breeding programs. Marker data showed that Xwmc125, Xbarc193, Xbarc199, and Xwmc310 were linked to pmCH89. The locations of four linked microsatellite loci were verified with CS nulli-tetrasomic and ditelosomic stocks and deletion lines, and were further confirmed by assigning linked microsatellite markers to chromosome 4BL in the bin 0.68-0.78. Based on the genetic and physical bin maps, the powdery mildew resistance gene pmCH89 was mapped on the interstitial region of chromosome 4BL and closely flanked by SSR markers Xwmc310 and Xwmc125, which were proximal at 3.1 cM and distal at 2.7 cM, respectively, to the resistance gene. Because resistance to powdery mildew in many Chinese cultivars has been overcome by virulent races of the pathogen, pmCH89 and the identification of closely flanking markers may be beneficial for increasing the overall diversity of available resistance genes with the potential to provide more comprehensive and durable protection against the disease.
Wheat cultivars Jingshuang 16, SY95-71, and Mianyang 11 (MY11) are all susceptible to wheat powdery mildew. To study the powdery mildew resistance of CH09W89, we developed segregating populations (F2, F2:3, and BC1) by crossing CH09W89 with susceptible wheat cultivars SY95-71 and MY11. The F1 was used for determining the dominance of the resistance and F2, F2:3, and BC1 were tested for segregation of powdery mildew resistance. An F2 population and derived F2:3 lines from SY95-71/CH09W89 were used for further microsatellite screening and gene mapping. The mapping population was comprised of 175 F2 plants and 166 derived F2:3 families, the difference being due to insufficient seeds of nine F2 plants.

Cytogenetic Analysis
Genomic in situ hybridization is a powerful molecular cytogenetic technique to identify alien chromatin in the wheat background. GISH analysis was performed using a similar protocol to that described by Han et al. [33]. In this study, Th. intermedium Z1141 genomic DNA was used as a probe and the CS genomic DNA was used as blocker DNA.

Evaluation of Powdery Mildew Responses
Seven isolates of the prevailing local B. graminis f. sp. tritici (Bgt) pathotypes provided by the Plant Protection Institute, Chinese Academy of Agricultural Sciences (Table 1), were used for resistance tests in the seedling stage. Using Jingshuang 16 as a control, CH09W89 and the parental lines were grown in 70 × 45 × 18 cm flat plastic trays. When the first leaves were fully expanded, inoculations were performed by dusting conidiospores from sporulating seedlings of Chancellor onto the test seedlings according to the method described by Xiang [34]. Host infection types (ITs) were scored 2-3 weeks after inoculation, when the susceptible check Jingshuang 16 became heavily infected, using a 0-4 rating scale [35], where 0 = no visible symptoms, 0; = necrotic flecks, 1 = necrosis with low sporulation, 2 = necrosis with moderate sporulation, 3 = no necrosis with moderate to high sporulation, and 4 = no necrosis with full sporulation. Scores of 0-2 were classified as resistant and 3-4 as susceptible.
To determine the genetics of resistance in CH09W89, E09, which is a prevalent pathotype in the Beijing area and avirulent on CH09W89, but is virulent on SY95-71 and MY (Mianyang) 11, was used to test F1, F2, F2:3, and BC1 populations derived from SY95-71/CH09W89 and CH09W89/ MY11//CH09W89 ( Table 2). All seeds from the parents, F1, F2, F2:3, and BC1 populations were planted in the greenhouse. Twenty seeds of each parent and F1, 175 seeds of F2, 78 seeds of BC1, and 20-25 seeds for each of the F2-derived F2:3 families were planted randomly in a 100 cm row, 25 cm apart. Susceptible spreaders of Jingshuang 16 and SY95-71 were planted in every 10th row for each population. The predominant Bgt race E09 was used for adult plant testing and the spreaders were artificially inoculated two times at the seedling stage. Adult plant reactions were scored twice, at the ear emergence stage and at the milky ripe stage, using the 0-4 scale previously described. To determine the genotypes of F2 plants from the cross of CH09W89 with SY95-71, the F2-derived F3 families were tested with the same race used in the F2 tests.

Molecular Marker Analysis
Genomic DNA, extracted from young seedling leaf tissue collected from the F2 individuals from which the F2:3 families originated, were used for molecular analysis. SSR markers linked to the resistance gene were identified by bulked segregant analysis (BSA). Equal amounts of DNA from 10 resistant F2 segregants were pooled into a resistant bulk, and DNA from 10 susceptible F2 segregants were pooled into a susceptible bulk according to Michelmore et al. [36]. The resistant parent, susceptible parent, resistant bulk (Br), and susceptible bulk (Bs) were then screened by genome and chromosome specific markers from across the wheat genome. Markers that were polymorphic between the resistant and susceptible parents and bulks were used to genotype the F2:3 lines and for linkage analysis.
Wheat microsatellite markers were used to detect polymorphism among parents, and resistant and susceptible bulks. The polymorphism markers were genotyped in F2 individuals to determine genetic linkage between the powdery mildew resistance gene(s) and markers.
PCR was performed in a 20 μL solution comprised of 80-100 ng template DNA, 2 μL 10× buffer (10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2), 0.2 mM of each dNTP, 1 unit Taq DNA polymerase, 0.25 μM of each primer. Amplification was performed at 94 °C for 5 min initial denaturation, 35 cycles each consisting of 45 s at 94 °C for denaturation, 45 s at either 50, 55 or 60 °C (based on primer annealing temperature), 1 min at 72 °C for extension; and finally a 10 min extension step at 72 °C before cooling to 4 °C. After PCR amplification, 12 μL of formamide loading buffer (0.4 g/mL sucrose, 1 mg/mL bromophenol blue, and 1 mg/mL xylene cyanol) was added to each PCR product. Then, 4-6 μL of each sample was loaded on 8% non-denaturing polyacrylamide gels (Acr:Bis = 29:1) and separated at 150 V for approximately 2 h, then visualized by silver staining. PCR for each SSR marker was performed in a PTC200 Peltier Thermal Cycler (Bio-Rad Laboratories Inc., Hercules, CA, USA).

Data Analysis and Chromosomal Assignment
The goodness-of-fit of observed phenotypes and expected segregation ratios was determined by χ-square tests (χ 2 ). Linkages between markers and the resistance gene were determined using Joinmap 4.0 software [37] with a LOD threshold 3.0. Map distances were determined by using the Kosambi mapping function.

Conclusions
A new powdery mildew resistance gene, tentatively designated as pmCH89, was found in a putative wheat-Th. intermedium introgression line developed by crossing the resistant partial amphiploid TAI7045 with susceptible cultivars, and the resistance is effective against the existing powdery mildew races in China, including the most widely virulent and predominant pathotypes. The gene was physically mapped on the intercalary bin 0.68-0.78 of chromosome 4BL and closely flanked by markers Xwmc310 and Xwmc125. pmCH89, together with the identified closely linked markers, could be useful in marker-assisted selection for improving powdery mildew resistance in wheat breeding programs.