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Acta Botanica Sinica 植 物 学 报 2004, 46 (7): 867-872 http://www.chineseplantscience.com Identification and Microsatellite Markers of a Resistance Gene to Powdery Mildew in Common Wheat Introgressed from Triticum durum ZHU Zhen-Dong, KONG Xiu-Ying, ZHOU Rong-Hua, JIA Ji-Zeng* (Key Laboratory of Crop Germplasm and Biotechnology, Ministry of Agriculture, Institute of Crop Germplasm Resources, The Chinese Academy of Agricultural Sciences, Beijing 100081, China) Abstract: A powdery mildew resistance gene in a BC3F2 population, derived from a cross made between an amphidiploid of Triticum durum Desf.-Aegilops caudata L. and T. aestivum L. cv. Laizhou 953, was identified. Genetic analysis of resistance to powdery mildew in BC3F2 population and derived BC3F3 families indicated a single dominant gene controlled the resistance. By bulk segregation analysis, two microsatellite markers, Xgwm311 and Xgwm382, were identified to be closely linked to the resistance gene with genetic distance of 5.9 cM and 4.9 cM, respectively. DNA from T. durum accession DR147, Ae. caudata acc. Ae14, and recurrent parent wheat (Triticum aestivum L.) cv. Laizhou 953 were amplified with primer pairs WMS311 and WMS382, the specific bands related to the resistance gene were only present in T. durum acc. DR147. Results showed that the resistance gene originated in T. durum acc. DR147. Based on the location of the linked microsatellite markers, the resistance gene was located on the telomeric region of chromosome 2AL in wheat. Temporarily, the resistance gene was designated as PmDR147. The relation of this gene and Pm4 was discussed. Key words: resistance gene; microsatellite; molecular tagging; powdery mildew; durum wheat Powdery mildew, caused by Erysiphe graminis f. sp. tritici, is one of the most important diseases of wheat (Triticum aestivum) and causes serious yield loss in wheat growing regions with temperate climates. Breeding of resistant cultivars is the economical and environmentally safe way by reducing the application of fungicides to control this disease. Up to now, thirty-one gene loci for resistance to powdery mildew (Pm1–Pm31) have been identified and assigned to specific chromosomes of wheat (Xie et al., 2001; Liu et al., 2002; Zeller et al., 2002; McIntosh et al., 2003). A few of these have been utilized in commercial breeding. Because E. graminis f. sp. tritici has high genetic variability, new virulent pathogen mutants can overcome individual race-specific resistance genes in a relatively short period (Duan et al., 1998). It is necessary to search for new sources of resistance to guarantee the progress of resistance breeding. Durum wheat (Triticum durum) (2n = 4x = 28; genome AABB) is a valuable source of genes for diversifying pest resistance in common wheat. Durum wheat has been reported to carry resistance to leaf rust, stem rust, stripe rust, loose smut, Fusarium head blight, Septoria nodorum blotch, Karnal bunt, powdery mildew, and Hessian fly (McIntosh et al., 1967; Gupta et al., 1991; Cao et al., 2001; Singh et al., 2001; Mishhra et al., 2001). It also contributes towards superior quality characteristics. Molecular markers have been widely used for gene tagging, gene mapping, and other genetics research. These DNA markers are not influenced by environmental conditions and are detectable at all plant growth stages. Molecular markers tightly linked to the trait of interest can be used in marker-assisted selection (MAS) to improve the efficiency of conventional plant breeding (Gupta et al., 1999). Among various molecular markers available, microsatellites, or simple sequence repeats have been shown to be superior to other DNA markers with respect to their higher level of polymorphism and informativeness in hexaploid wheat (Plaschke et al., 1995; Bryan et al., 1997). Moreover, microsatellite markers are codominant, PCR-based, and functional across the wild relatives of wheat (Rafalski and Tingey, 1993; Röder et al., 1998). To date, several microsatellite maps of wheat have been constructed, with the microsatellite loci evenly distributed along the chromosome lengths to provide excellent coverage of the wheat genome (Röder et al., 1998; Stephenson et al., 1998; Pestsova et al., 2000; Gupta et al., 2002). Some microsatellite markers from hexaploid wheat also have been integrated into genetic linkage maps of durum wheat (Korzun et al., Received 7 Jul. 2003 Accepted 16 Dec. 2003 Supported by the State Key Basic Research and Development Plan of China (G1998010200) and the Hi-Tech Research and Development (863) Program of China (2001AA211031). * Author for correspondence. E-mail: <[email protected]>. 868 1999; Nachit et al., 2001). Closely linked microsatellite markers to many economically important trait genes including a few powdery mildew resistance genes in wheat have been identified (Zhu and Jia, 2003). The present study reports the identification and microsatellite markers of a powdery mildew resistance gene in common wheat introgressed from durum wheat. 1 Materials and Methods 1.1 Plant materials Triticum durum Desf. accession DR147, Aegilops caudata L. acc. Ae14, recurrent parent wheat (Triticum aestivum L.) cv. Laizhou 953, cv. Chinese Spring, the nullitetrasomic (NT) stocks N2AT2B, N2BT2A and N2DT2A of Chinese Spring were from the author’s institute. A cross was made between the amphidiploid of T. durum acc. DR147/Ae. caudata acc. Ae14 and T. aestivum cv. Laizhou 953, an agronomically superior wheat cultivar (Kong et al., 1999). The F1 was backcrossed to Laizhou 953 for three generations. Then a number of powdery mildew resistant BC3 progenies were selfed. A BC3F2 population and derived BC3F3 families were used in the study. 1.2 Powdery mildew evaluation Erysiphe graminis f. sp. tritici (Egt) isolate E09 was provided by Drs. DUAN Xia-Yu and ZHOU Yi-Lin, Institute of Plant Protection, The Chinese Academy of Agricultural Sciences. Resistance to powdery mildew of BC3F2 population was identified in the field by inoculating Egt isolate E09. The method of inoculation, conditions of incubation and disease assessment were according to Sheng (1991). T. durum acc. DR147, Ae. caudata acc. Ae14, recurrent parent wheat cv. Laizhou 953 and BC3F2-derived F3 families were evaluated for resistance to Egt isolate E09 at the seedling stage in a greenhouse. The method of inoculation, conditions of incubation and disease assessment were according to Duan et al. (2001). Egt isolate E09 is a prevalent virulence type in Beijing area with a virulence formula Pm1, Pm3a, Pm3c, Pm5, Pm7, Pm8, Pm17, Pm19. Chi-squared tests for goodness of fit were used for deviation of observed data from theoretically expected segregation ratios. Six infection types of hosts were distinguished: 0=immune, 0; = near immune, 1= high resistant, 2 = moderately resistant, 3 = moderately susceptible, and 4 = high susceptible (Sheng, 1991; Duan et al., 2001). 1.3 PCR amplification and product analysis Genomic DNA was extracted from leaf tissue samples as described by Sharp et al. (1988). Wheat microsatellite markers employed were described in Röder et al. (1998). Each PCR reaction contained 80 ng genomic DNA, 0.25 µmol/L Acta Botanica Sinica 植物学报 Vol.46 No.7 2004 of each primer, 1 U Taq DNA polymerase, 2 µL of 10 × PCR buffer containing 15 mmol/L MgCl2, 0.2 mmol/L of dNTPs in a total volume of 25 µL. PCR was performed in an MJ Research thermocycler, at 94 ℃ for 5 min, followed by 35 cycles of 94 ℃ for 1 min, 50, 55, or 60 ℃ (based on primer annealing temperature) for 1 min, and 72 ℃ for 1 min, with final incubation at 72 ℃ for 5 min before cooling to 4 ℃. Each PCR product was denatured by adding 8 µL loading buffer ( 98% formamide, 10 mmol/L EDTA, pH 8.0, 0.25% bromo-phenol blue, and 0.25% xylene cyanol) and denatured for 10 min at 95 ℃ and chilled on ice. Eight µL of each sample was loaded on 6% polyacyamide (19 : 1 acrylamide:Bis), 8 mol/L urea and 1× TBE (90 mmol/L Trisborate, pH 8.3, and 2 mmol/L EDTA) gels (40 cm length, 20 cm wide, 0.2 cm thick). Samples were then run at 2 500 V, 30 mA, 100 W for approximate 1 h, and the products were visualized by silver staining (Tixier et al., 1997). 1.4 Microsatellite marker analysis The identification of microsatellite markers linked to the resistance gene was accomplished by bulked segregation analysis (BSA) as described by Michelmore et al. (1991). The resistant bulk and the susceptible bulk were made by separately pooling equal amount of DNA from 10 resistant and 10 susceptible plants from the BC3F2 segregating population. The markers generating polymorphic microsatellite fragments between the bulks were further checked for their linkage to the resistance gene, using 108 plants of the BC3F2 population. 1.5 Linkage analysis Recombination frequencies (RF) or linkage relationships between microsatellite markers and the powdery mildew resistance gene were calculated using Mapmaker 3.0b (Lander et al., 1987) and converted to cM using the Kosambi mapping function (Kosambi, 1944). The decimal logarithm of likelihood ratio (LOD) was used as a test measure of reliability of linkage. The linkage is reliable if the markers were placed with an LOD threshold of 3.0. 2 Results 2.1 Powdery mildew resistance T. durum acc. DR147 and Ae. caudata acc. Ae14 were resistant to Egt isolate E09, recurrent parent wheat cv. Laizhou 953 was susceptible to Egt isolate E09. A total of 132 BC3F2 individuals were identified for resistance to powdery mildew in the field. The observed segregation of 100 resistant and 32 susceptible individuals fitted a 3:1 segregation ratio (Table 1). A total of 132 BC3F2-derived F3 families were tested with Egt isolate E09 in seedling to determine genotype of BC3F2 individuals. Thirty families were ZHU Zhen-Dong et al.: Identification and Microsatellite Markers of a Resistance Gene to Powdery Mildew in Common Wheat Introgressed from Triticum durum 869 Table 1 Segregation analysis for the resistance gene and microsatellite markers linked to the gene Number of Observed number BC3F2 D 132 100 108 81 108 82 or heterozygous; LL, Laizhou 953. χ2 3:1=3.84, P=0.05. Gene or marker PmDR147 Xgwm311 Xgwm382 D , DR147 homozygous resistance, 75 families were heterozygous resistant and 27 families were homozygous susceptible. The observed segregation fitted a 1:2:1 segregation ratio (χ 2 = 2.59). Both ratios supported a segregation of a single dominant locus. These results showed that the powdery mildew resistance of the BC3F1 derived from the amphidiploid of T. durum acc. DR147-Ae. caudata acc. Ae14 is controlled by single dominant gene. 2.2 Microsatellite markers linked to the resistance gene Based on the microsatellite map of wheat (Röder et al., 1998), 136 microsatellite markers were screened to identify polymorphic microsatellite markers between the resistant and susceptible DNA bulks. Two primer pairs WMS311 and WMS382 generated polymorphic DNA fragments between the bulks with size about 170 bp and 130 bp, respectively. Markers Xgwm311 and Xgwm382 were in the resistant pool, but absent in susceptible pool. These results indicated the two microsatellite markers could be linked to the resistance gene. Further, the two markers were used to check their linkage to the resistance gene using 108 plants of segregating BC3F2 population. The data is in Table 1, illustrated in Figs.1 and 2. Both markers Xgwm311 and Xgwm382 showed a 3:1segregation ratio, and were closely linked to the resistance gene with a map distance of 5.9 cM and 4.9 cM, respectively (Fig.3). 2.3 Origin and chromosomal location of the resistance gene Parents of the amphidiploid, T. durum acc. DR147 and Ae. caudata acc. Ae14, were resistant to powdery mildew. To determine the origin of the resistance gene in the BC3F2 population, we amplified DNA from T. durum acc. DR147, Fig.1. PCR bands amplified from the DNA of selected BC3F2 plants using primer pair WMS311. L, pBR322 DNA/MspⅠ marker; R, resistance plant; S, susceptible plant; →, specific bands linked to powdery mildew resistance gene from DR147. LL 32 27 26 χ 2 3:1 P 0.04 0.01 0.01 0.99–0.95 >0.99 >0.99 Fig.2. PCR bands amplified from the DNA of selected BC3F2 plants using primer pair WMS382. L, pBR322 DNA/Msp Ⅰ marker; R, resistance plant; S, susceptible plant; →, specific bands linked to powdery mildew resistance gene from DR147. Fig.3. Genetic linkage map of powdery mildew resistance gene PmDR147 and the linked microsatellite markers on wheat chromosome 2A. The genetic distances between gene Pm4 and Xgwm311 were based on published data by Röder et al. (1998). Ae. caudata acc. Ae14 and recurrent parent wheat cv. Laizhou 953 with primer pairs WMS311 and WMS382. The markers Xgwm311 and Xgwm382 related to resistance gene were present only in T. durum acc. DR147. The results indicated the resistance gene was from T. durum acc. DR147. Temporarily, the powdery mildew resistance gene was designated as PmDR147. According to microsatellite map of wheat (Röder et al., 1998), marker Xgwm311 was on wheat chromosome 2AL and 2DL, while marker Xgwm382 was on wheat chromosome 2AL, 2BL and 2DL. To verify the chromosome location of markers Xgwm311 and Xgwm382, the DNA from the NT stocks N2AT2B, N2BT2A and N2DT2A of Chinese 870 Spring wheat was amplified with primer pairs WMS311 and WMS382. Both markers were located on wheat chromosomes 2A and 2D. Figure 4 shows the location of Xgwm311. On a molecular linkage map for an intraspecific recombinant inbred population of durum wheat, two nearby microsatellite Xgwm382 loci are located on chromosome 2AL of durum wheat (Nachit et al., 2001). Since the resistance gene PmDR147 was from T. durum (genome AABB) and closely linked to the markers Xgwm311 and Xgwm382, it should be on the long arm of wheat chromosome 2A. Fig.4. Microsatellite DNA products amplified using primer pairs WMS311 in DR147 (lane 2), Laizhou 953 (lane 3), Ae14 (lane 4), resistant bulk (lane 5), susceptible bulk (lane 6), Chinese Spring (lane 7), N2AT2B, N2BT2A and N2DT2A of Chinese Spring (lanes 8-10), selected BC3F2 resistant plants (lanes 11- 13), selected BC3F2 susceptible plants (lanes 14-15). Lane 1 was pBR322 DNA/MspⅠ marker. Arrows indicate absence of Xgwm311 in N2AT2B or N2DT2A. 3 Discussion 3.1 Inheritance of microsatellite markers linked to the resistance gene The majority of documented microsatellite markers is inherited in a codominant manner (Röder et al., 1998). However, the microsatellite markers Xgwm311 and Xgwm382, linked to the powdery mildew resistance gene in this study, were inherited in a dominant manner, because they detected only resistance-related bands and segregation of the presence or absence of resistance band in tested segregating population fitted a 3 : 1 ratio. So markers Xgwm311 and Xgwm382 are “resistance-dominant” markers. Liu et al. (2001) identified microsatellite markers linked to six Russian wheat aphid resistance genes that were inherited in a dominant manner. The possible explanation for the dominance of these microsatellite markers with null alleles is most likely due to nucleotide-sequence alterations within the binding site for a DNA primer and results due to a primer site to close to the microsatellite. This may either inhibit primer binding, giving a faint band or may completely prevent this binding, leading to the loss of PCR product (Gupta and Varshney, 2000; Liu et al., 2001). Acta Botanica Sinica 植物学报 Vol.46 No.7 2004 3.2 Relationship between PmDR147 and Pm4 Classical genetic and molecular data show the genes for disease resistance are not randomly distributed all over the genome but rather frequently occur in clusters in particular chromosomes. For example, among the documented 28 powdery mildew resistance gene loci, Pm1, Pm3, Pm4, or Pm5 is a complex locus, and composed of 4, 10, 2, and 5 alleles, respectively (Hsam et al., 1998; Zeller et al., 1998; Huang et al., 2003). In our study, we located the gene PmDR147 on the distal region of long arm of chromosome 2A. Powdery mildew resistance genes Pm4a and Pm4b are also located on chromosome 2AL of wheat. Ma et al. (1994) identified RFLP markers linked to Pm4a using near-isogenic lines. Two markers Xcdo1231-2A (2) and Xcdo678-2A were found to be cosegregant with Pm4a. On genetic map of wheat, RFLP marker Xcdo678 is above microsatellite marker Xgwm382 on the wheat chromosome 2AL, with a map distance of 8.3 cM (Röder et al., 1998). The gene PmDR147 is below marker Xgwm382 with 4.9 cM. An estimative genetic distance between PmDR147 and Pm4a is 13.2 cM (Fig.3). Pm4a and Pm4b originated in T. dicoccum and T. carthlicum, respectively, while PmDR147 derived from T. durum. Therefore, we assume either the PmDR147 and Pm4a or Pm4b may be different genes present in a complex region on chromosome 2AL, or PmDR147 may be an allele of the complex Pm4 locus. 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