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QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli
Journal of Integrative Agriculture
May 2013
2013, 12(5): 756-764
RESEARCH ARTICLE
QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli Asad Muhammad Azeem1, BAI Bin1, LAN Cai-xia1, YAN Jun2, XIA Xian-chun1, ZHANG Yong1 and HE Zhong-hu1, 3 National Wheat Improvement Center/National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, P.R.China 2 Cotton Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Anyang 455000, P.R.China 3 International Maize and Wheat Improvement Center (CIMMYT) China Office, Beijing 100081, P.R.China 1
Abstract The Italian wheat cv. Strampelli displays high resistance to powdery mildew caused by Blumeria graminis f. sp. tritici. The objective of this study was to map quantitative trait loci (QTLs) for resistance to powdery mildew in a population of 249 F2:3 lines from Strampelli/Huixianhong. Adult plant powdery mildew tests were conducted over 2 yr in Beijing and 1 yr in Anyang and simple sequence repeat (SSR) markers were used for genotyping. QTLs Qpm.caas-3BS, Qpm.caas-5BL.1, and Qpm.caas-7DS were consistent across environments whereas, Qpm.caas-2BS.1 found in two environments, explained 0.4-1.6, 5.5-6.9, 27.1-34.5, and 1.0-3.5% of the phenotypic variation respectively. Qpm.caas-7DS corresponded to the genomic location of Pm38/Lr34/Yr18. Qpm.caas-4BL was identified in Anyang 2010 and Beijing 2011, accounting for 1.9-3.5% of phenotypic variation. Qpm.caas-2BS.1 and Qpm.caas-5BL.1 contributed by Strampelli and Qpm.caas-3BS by Huixianhong, seem to be new QTL for powdery mildew resistance. Qpm.caas-4BL, Qpm.caas-5BL.3, and Qpm.caas-7DS contributed by Strampelli appeared to be in the same genomic regions as those mapped previously for stripe rust resistance in the same population, indicating that these loci conferred resistance to both stripe rust and powdery mildew. Strampelli could be a valuable genetic resource for improving durable resistance to both powdery mildew and stripe rust in wheat. Key words: QTL analysis, SSR markers, Blumeria graminis, durable resistance, Triticum aestivum L.
INTRODUCTION Wheat is the most widely used food crop for humankind. Powdery mildew caused by Blumeria graminis f. sp. tritici is a major wheat disease worldwide and causes significant yield losses every year in many wheat growing regions in China (Zeng et al. 2010). Host plant resistance, being cost effective and environmentally friendly compared to the use of fungicides, is the best Received 19 July, 2012
option to control the disease (Gurung et al. 2009). Qualitative and quantitative forms of resistance to powdery mildew have been reported in wheat and its wild relatives. Qualitative resistance is usually highly protective throughout the growth cycle of the host, but when exploited in agriculture usually becomes ineffective within a relatively short period of adoption. This lack of durability is the consequence of increased frequencies of previously rare races of the pathogen, mutation from avirulence to virulence, or in some cases, if
Accepted 18 September, 2012
Asad Muhammad Azeem, E-mail: [email protected]; Correspondence HE Zhong-hu, Tel: +86-10-82108547, E-mail: [email protected]
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60297-X
QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli
resistance is based on combinations of such genes, by genetic recombination in the pathogen. Blumeria graminis f. sp. tritici is a haploid pathogen and although it usually propagates asexually by means of conidiospores it can also undergo sexual recombination, particularly at the beginning of the epidemic cycle. Most powdery mildew resistance genes commonly present in wheat are not effective in China (Zhou et al. 2002). The contrasting type of resistance, known as quantitative resistance, tends to confer partial resistance at post seedling growth stages, and although less effective, is considered to be durable. It is also called as adult plant resistance (APR). Although individual adult plant resistance (APR) genes or QTLs often confer inadequate resistance, combinations of a few such genes usually result in “near-immunity” or high levels of resistance (Gustafson and Shaner 1982; Liu et al. 2001). Studies of adult plant partial resistance to powdery mildew in wheat have generally found that it is an oligogenic trait and individual genes were mapped on many different chromosomes (Keller et al. 1999; Bougot et al. 2006; Lillemo et al. 2008; Lan et al. 2009, 2010b; Muranty et al. 2010). Generally, the highest levels of resistance occur in lines with the most resistance genes/ QTLs. The best characterized gene of this type is Pm38 (also known as Lr34 and Yr18) which is involved in multi-pathogen durable resistance. This gene has been cloned and characterized as a putative ABC transporter (Krattinger et al. 2009). Whether multiple disease protection is a general characteristic of other partial resistance genes is yet to be established. The Italian common wheat cv. Strampelli, introduced to Gansu Province of China in 1973 has adult plant resistance to stripe rust controlled by three QTLs, including Yr18 (Lu et al. 2009). It has also shown good resistance to powdery mildew in the field. The objective of this study was to determine the genetic basis of durable powdery mildew resistance in Strampelli.
RESULTS Field evaluation of mapping population Strampelli and Huixianhong expressed the maximum disease severities of 3.5 and 15%, 2.33 and 16.7%, and 4.0 and 33.3%, respectively, whereas the susceptible
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control Jingshuang 16 displayed 80-100, 60-90, and 90-100% in Anyang 2010, Beijing 2010, and Beijing 2011, respectively. Strampelli and Huixianhong expressed mean maximum disease severities (MDS) of less than 5 and 15-25%, respectively, over three environments. Powdery mildew MDS across the three environments were significantly correlated (r=0.67 to 0.69). Frequency distributions of MDS and area under disease progress curve (AUDPC) showed continuous variation in all environments, suggesting a quantitative basis of resistance to powdery mildew (Fig. 1). Broadsense heritabilities of MDS and AUDPC were 0.78 and 0.73, respectively. Analyses of variance showed significant variation among F2:3 lines for both MDS and AUDPC (Table 1).
QTLs for adult plant resistance (APR) to powdery mildew Seven QTLs based on MDS, five QTLs in averaged MDS over three environments, and three QTLs in averaged AUDPC over 2 yr in Beijing were mapped on chromosomes 2BS, 3BS, 4BL, 5BL, and 7DS (Fig. 2, Table 2). QTLs on 3BS and 7DS were stable across all environments, average MDS in three environments, and averaged AUDPC in Beijing over 2 yr. Except in Beijing 2011, QTL on 2BS was consistently identified in other environments, averaged MDS of three environments, and averaged AUDPC in Beijing over two years, whereas Qpm.caas-5BL.1 was detected in almost all environments except averaged AUDPC in Beijing over 2 yr. Qpm.caas-4BL, Qpm.caas-5BL.2 and Qpm.caas-5BL.3 were detected in two, three and one environments, respectively. QTLs on 2BS, 4BL, 5BL, and 7DS, and one on 3BS were contributed by Strampelli and Huixianhong, respectively. Qpm.caas-2BS mapped in the marker interval Xwmc25.1-Xwmc25.2, explained 1.0-3.5% of the phenotypic variances and was stable across two environments, in average MDS and average AUDPC in Beijing. A consistently detected QTL, Qpm.caas-3BS, was identified on the short arm of chromosome 3B between Xwmc366.1 and Xwmc366.2 across all environments. Qpm.caas-4BL explaining 1.9-3.5% of the phenotypic variance was located in the marker interval Xgwm165-Xgwm149 in two environments. Qpm.
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Fig. 1 Frequency distributions of the maximum powdery mildew severities (MDS) and area under the disease progress curve (AUDPC) values in F 2:3 lines derived from Strampelli/ Huixianhong. A, MDS in Anyang in 2010. B, MDS in Beijing in 2010. C, MDS in Beijing in 2011. D, average MDS across three environments. E, average AUDPC in Beijing over 2 yr. Mean values for the parents, Strampelli and Huixianhong, are indicated by arrows.
Table 1 Analysis of variance of the maximum powdery mildew severities (MDS) on penultimate leaves and area under the disease progress curve (AUDPC) for powdery mildew responses on F2:3 lines derived from Strampelli/Huixianhong Parameter
Source of variation
MDS
Replicates Environments Lines Lines × Environments Error Replicates Environments Lines Lines × Environments Error
AUDPC
**
df 2 2 248 494 1 379 2 1 248 248 944
Sum of squares 224 36 232 109 050 47 076 53 782 30 477 216 566 2 393 427 638 137 1 406 507
Mean square
F-value
112 18 116 439 95 39 15 238 216 566 9 650 2 573 1 414
2.88 464.51 ** 11.27 ** 2.44 ** 10.77 ** 153.05 ** 6.82 ** 1.82 **
, P<0.0001.
caas-5BL.1, identified in the marker interval Xgwm335Xbarc331 across all the environments, accounted for 5.5-6.9% of the phenotypic variance with additive effects of 1.42-3.39. Qpm.caas-5BL.2 in marker interval Xbarc331-Xwmc537 explained phenotypic variation 1.8-4.8% whereas Qpm.caas-5BL.3, in marker interval Xwmc415-Xwmc405 on the same chromosome explained 4.2% of the phenotypic variance in one environment. Use of STS marker csLV34 indicated the major stable QTL explaining 12.6-34.5% of phenotypic variances
across all environments was Pm38/Yr18/Lr34. Seven QTLs together accounted for 43.8-48.5% of the total phenotypic variance across three environments.
DISCUSSION In this mapping population, MDS and AUDPC showed continuous variation. The apparent lack of full susceptibility may be because the parents are homozygous at
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QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli
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Fig. 2 Logarithm of odds (LOD) contours obtained by inclusive composite interval mapping (ICIM) of QTLs for adult plant powdery mildew resistance in F2:3 lines of Strampelli/Huixianhong. A2010, maximum disease severities (MDS) in Anyang 2010; B2010 and B2011, maximum disease severities in Beijing 2010 and 2011, respectively; mean, mean MDS across three environments; AUDPC, average of area under the disease progress curve in Beijing over 2 yr. Logarithm of odds (LOD) threshold, 2.0.
common loci conferring partial resistance, which could result in decreased variance and absence of transgressive segregation. Disease severity range of F2:3 lines was quite below the susceptibility levels of cv. Jingshuang 16 suggesting that both parents possess some resistance loci in common that did not segregate in the cross, and there may be more minor resistance loci that could not be detected because of less number of simple sequence repeat markers used in this study. However, molecular mapping of the Strampelli/ Huixianhong population indicated the presence of seven QTLs for powdery mildew resistance on chromosomes 2BS, 4BL, 5BL, and 7DS in Strampelli and one on 3BS in Huixianhong. Previously, QTLs, QPm.crag-2BS (Mingeot et al. 2002) and QPm.caas-2BS (Lan et al. 2010b) were detected on chromosome 2BS in Festin and Lumai 21,
respectively. These genes are estimated to be about 22 and 43.9 cM, respectively, from QPm.caas-2BS.1 based on wheat consensus maps (Somers et al. 2004; http:// wheat.pw.usda.gov/GG2/index.shtml). The Pm26 (Rong et al. 2000) and Pm42 (Hua et al. 2009) resistance genes derived from wild emmer (Triticum turgidum var. dicoccoides) were estimated to be 33 and 22 cM from the QPm.caas-2BS.1 peak. This less effective QTL detected in two environments, average MDS in three environments, and averaged AUDPC in Beijing over 2 yr, seems to be a new locus for powdery mildew resistance on chromosome 2BS. A stable QTL, QPm.caas-3BS, was detected across all environments on chromosome 3BS. Chen et al. (2009) mapped a QTL on chromosome 3BS in hard winter wheat line 2174. It was centered on Xwms533 and 56 cM from QPm.caas-3BS. Pm13 derived from
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Table 2 Quantitative trait loci (QTLs) for adult-plant resistance (APR) to powdery mildew in F2:3 lines derived from Strampelli/ Huixianhong Location/Year MDS Anyang 2010
Beijing 2010
Beijing 2011
Averaged MDS in three environments
AUDPC Averaged AUDPC in Beijing
QTL1)
Marker interval
LOD 2)
AE 3)
R2 (%)4)
Total R2 (%)
QPm.caas-2BS.1 QPm.caas-3BS QPm.caas-4BL.1 QPm.caas-5BL.1 QPm.caas-5BL.2 QPm.caas-7DS QPm.caas-2BS.1 QPm.caas-3BS QPm.caas-5BL.1 QPm.caas-5BL.2 QPm.caas-5BL.3 QPm.caas-7DS QPm.caas-3BS QPm.caas-4BL.1 QPm.caas-5BL.1 QPm.caas-7DS QPm.caas-2BS.1 QPm.caas-3BS QPm.caas-5BL.1 QPm.caas-5BL.2 QPm.caas-7DS
Xwmc25.1-Xwmc25.2 Xwmc366.1-Xwmc366.2 Xgwm165-Xgwm149 Xgwm335-Xbarc331 Xbarc331-Xwmc537 csLV34-Xgwm295 Xwmc25.1-Xwmc25.2 Xwmc366.1-Xwmc366.2 Xgwm335-Xbarc331 Xbarc331-Xwmc537 Xwmc415-Xwmc405 csLV34-Xgwm295 Xwmc366.1-Xwmc366.2 Xgwm165-Xgwm149 Xgwm335-Xbarc331 csLV34-Xgwm295 Xwmc25.1-Xwmc25.2 Xwmc366.1-Xwmc366.2 Xgwm335-Xbarc331 Xbarc331-Xwmc537 csLV34-Xgwm295
2.7 4.0 2.7 3.8 3.4 14.5 2.3 3.4 4.2 3.1 3.0 14.9 3.5 1.9 4.6 22.0 2.1 3.3 4.9 2.1 16.6
1.36 0.56 0.74 1.42 1.262 3.34 1.00 0.01 1.30 1.06 1.32 4.74 0.97 1.09 3.39 10.00 0.72 0.06 2.36 0.38 5.90
3.5 1.6 3.5 5.5 4.8 27.2 1.7 0.4 6.9 4.0 4.2 27.1 0.5 1.9 5.9 34.5 1.0 0.1 6.1 1.8 25.9
46.1
QPm.caas-2BS.1 QPm.caas-3BS QPm.caas-7DS
Xwmc25.1-Xwmc25.2 Xwmc366.1-Xwmc366.2 csLV34-Xgwm295
1.8 3.3 6.8
4.74 0.24 19.3
0.8 0.1 12.6
13.5
48.5
43.8
34.9
QTLs that extend across single one-log support confidence intervals were assigned the same symbol. Logarithm of odds (LOD) score. 3) Additive effects. 4) Proportion of phenotypic variance explained by the QTL. 1) 2)
Ae. longissima is also located on 3BS (Donini et al. 1995). QPm.caas-3BS appears to be a new gene conferring adult plant resistance to powdery mildew. QPm.caas-4BL.1 was flanked by SSR markers Xgwm149 and Xgwm495. QPm.sfr-4B and QPm.ipk4B were mapped in cv. Forno (Keller et al. 1999) and the ITMI (Opata 85/W7984) population (Börner et al. 2002), flanked by markers Xpsr593b and Xpsr1112, and Xcdo795 and Xbcd1262, respectively, a similar position to QPm.caas-4BL.1. Liang et al. (2006) detected QPm.caas-4BL flanked by Xgwm375 and Xgwm251 in cv. Oligoculm in a similar position based on data from one test location. QPm.nuls-4BL flanked by XwPt1505 and Xgwm149, was mapped on the same chromosomal region of cv. Avocet in both Beijing and Norway (Lillemo et al. 2008). QYr.caas-4BL detected at one site and explaining 4.5% of the phenotypic variance in stripe rust response was mapped to a similar position (Lu et al. 2009). William et al. (2006) and Zwart et al. (2010) mapped stripe rust resistance QTL in crosses Avocet S/Pavon 76 and synthetic hexaploid CPI133872/
Janz and these also coincided with the position of QPm. caas-4BL.1. Thus results from a number of studies located QTLs for powdery mildew resistance in similar positions to those for stripe rust resistance. Three QTLs, QPm.ttu-5B, QPm.inra-5B.2, and QPm.nuls-5B were previously mapped on chromosome 5BS in cv. Tahti, Courtot and Saar, respectively (Bougot et al. 2006; Jakobson et al. 2006; Lillemo et al. 2008). Keller et al. (1999) mapped QPm.sfr-5B flanked by RFLP markers Xpsr580b-Xpsr143 on chromosome 5B. Our study has not determined a relationship of QPm. sfr-5B with QPm.caas-5BL.1 or QPm.caas-5BL.2 or QPm.caas-5BL.3 due to the different types of markers being used, and the positions of these markers are not comparable. QTLs, QPm.caas-5BL.1 and QPm.caas5BL.2 flanked by Xgwm335-Xbarc331 and Xbarc331Xwmc537 were located nearby with less than 20 cM distance between the resistance genes. We considered them same QTL as QPm.caas-5BL.1. QTL, QPm.caas5BL.3 flanked by Xwmc405 and Xwmc415, identified at distance more than 20 cM away from the peak of QPm.
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QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli
caas-5BL.2, considered as independent QTL. QPm. caas-5BL.1 conferred stable and consistent resistance across all environments, explaining 5.5-6.9% of the phenotypic variance. QYr.inra-5BL.1 accounting for 25% of the phenotypic variation in stripe rust response was flanked by Xgwm499 and Xgwm639 in the same region as QPm.caas-5BL.3 (Mallard et al. 2005). Lu et al. (2009) reported stripe rust APR QTLs on chromosome 5BL in cultivars Libellula and Strampelli located at a similar position to QPm.caas-5BL.3, indicating a common locus might confer resistance to both stripe rust and powdery mildew. QPm.caas-5BL.1 identified in the present study showed a stronger and more consistent effect across environments than QPm.caas5BL.3 which explained only 4.2% of the phenotypic variance in one environment. QPm.caas-5BL.1 appears to be a new locus for APR to powdery mildew. The most effective QTL was QPm.caas-7DS, inherited from Strampelli and explaining 27.1-34.5% of phenotypic variance across different environments. QTLs for slow mildewing in cv. W7984, FukuhoKomugi and Saar were previously mapped in the same genomic region as QPm.caas-7DS on chromosome 7DS (Börner et al. 2002; Liang et al. 2006; Lillemo et al. 2008). Lu et al. (2009) detected QYr.caas-7DS for stripe rust resistance in a similar position to QPm.caas7DS for powdery mildew resistance in Strampelli. The only presumed powdery mildew resistance gene in Strampelli was Pm38. The Pm38/Yr18/Lr34 gene is currently a prime target for durable resistance to multiple diseases because it is amenable to accurate molecular selection (Morgounov et al. 2012). Genes for resistance to powdery mildew in Strampelli seem to be more consistent and stable across environments than previously identified in Chinese wheat cultivars such as Yumai 2, Xiaoyang 6, Yumai 47, Lumai 21, and Bainong 64 (He et al. 2011, and personal communication). So far, Pm38/ Yr18/Lr34 has been identified in powdery mildew studies of Mexican (W7984 and Saar) and Japanese (Fukuho-Komugi) cultivars, and its identification in Strampelli, well adapted in Chinese environment seems to play a large role and should be useful for developing cultivars with potentially durable powdery mildew, stripe rust and leaf rust resistance simultaneously. During the past years, Strampelli was crossed with several
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widely grown Chinese wheat cultivars Zhongmai 175, Lunxuan 987, Jimai 22, and Zhoumai 18 in our breeding program. The molecular markers csLV34, wmc537, gwm149, gwm165, and barc331 closely linked to Pm38/Yr18/Lr34 and other QTLs (Lu et al. 2009) were used for marker-assisted selection in BC 1 or BC 2 progenies, and over 20 lines with good yield potential and resistance to powdery mildew, stripe rust and leaf rust were developed. This will provide a good example for marker-assisted selection targeting for durable resistance to powdery mildew, stripe rust and leaf rust in Chinese wheat breeding program.
MATERIALS AND METHODS Plant materials A total of 249 F 2:3 lines developed from Strampelli/ Huixianhong were available for QTL mapping. Strampelli has shown powdery mildew resistance at the adult plant stage in our nurseries over many years. Huixianhong, a landrace from Henan, is very susceptible at the seedling stage and moderately resistant at the adult stage. The F2:3 lines, derived from individual F2 plants are maintained as perpetual bulks of more than 50 plants.
Powdery mildew disease assessment F2:3 lines were sown in randomized complete blocks with three replications over 2 yr (2009-2010 and 2010-2011) at the CAAS Experimental Station, Beijing, and CAAS Cotton Research Institute, Anyang, Henan, (herein referred to as Beijing 2010, Beijing 2011, Anyang 2010). Fifty seeds of each line were sown in circular plots (8 cm diameter), and plots were spaced 30 cm apart to facilitate disease evaluation. Jingshuang 16 was planted at each tenth plot and around the experiment as a susceptible check and spreader to ensure the maximum inoculum for disease development. In Beijing, plots were inoculated prior to stem elongation with Blumeria graminis f. sp. tritici (Bgt) isolate E20. All entries were scored for the first time 5 wk after inoculation based on the actual percentage of leaf area covered by powdery mildew on five randomly selected penultimate (F-1) leaves and again 1 wk later when disease was at its maximum level around May 20. In Anyang, powdery mildew severities resulting from natural infection were evaluated once when infection levels on Jingshuang 16 had reached the maximum levels during the 3rd wk of May. Phenotypic data from Anyang in 2011 could not be used because the disease did not develop sufficiently due to dry spring conditions.
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gression (Wang et al. 2011). The PVE or R2 was calculated by the formula:
Statistical analysis AUDPC was calculated for each line using the formula: (Bjarko and Line 1988), where Xi is the severity value on date i, T i the number of days after inoculation on scoring date i, and n is the number of times of disease scoring. The correlation coefficients (r) and frequency distributions of powdery mildew response based on MDS in different environments were calculated with Microsoft Excel 2007. The statistical analyses of MDS and AUDPC were performed using the statistical package SAS ver. 9.1 by PROC GLM (SAS Institute 1997). Broad-sense heritability (h2) was calculated using the formula: h2=Var(G)/Var(P), where, Var(G) and Var(P) are genotypic and phenotypic variances, respectively (Allard 1960).
Simple sequence repeat (SSR) analysis Fresh young leaf tissue of the parents and each F 2:3 line (30-40 plants per F 2:3 line as a bulk) was used to extract genomic DNA using the procedure described by Sharp et al. (1988). A total of 943 sequences of available SSR markers (http://www.wheat.pw.usda.gov) and one STS marker csLV34 closely linked with the locus Lr34/Yr18 (Lagudah et al. 2006) were used. Based on MDS, resistant and susceptible bulks were constructed by mixing equal amounts of DNA from the five most resistant and five most susceptible F2:3 lines, respectively (Michelmore et al. 1991; Lan et al. 2010a). SSRs showing polymorphisms between the two parents and between the two bulks were used to genotype all 249 lines to determine their associations with powdery mildew responses. PCR reaction protocols, electrophoresis of PCR products and gel staining were conducted as described by Bryan et al. (1997) and Bassam et al. (1991). Polymorphic markers were used to genotype the population for linkage and QTL analysis.
Quantitative trait loci (QTL) analysis Linkage groups were established with the software Map Manager QTXb20 (Manly et al. 2001) and recombination values were converted to centiMorgans using the Kosambi mapping function (Kosambi 1944). Each linkage group was assigned to a chromosome based on previously published wheat genome maps (http://wheat.pw.usda.gov, Somers et al. 2004). QTL detection was performed using inclusive composite interval mapping (ICIM) with the software IciMapping v3.2 (Wang et al. 2011). A LOD threshold value of 2.0 was used for declaration of a QTL. The phenotypic variance explained (PVE or R2) by individual QTL and additive effects were obtained through stepwise re-
Where, VG is the genotypic variance of a QTL, and VP is the phenotypic variance of the trait in QTL mapping (Wang et al. 2011). QTLs overlapping within 20 cM intervals on the same chromosome across different environments were considered to be the same.
Acknowledgements We are grateful to the critical review of this manuscript by Prof. R. A. McIntosh, Plant Breeding Institute, University of Sydney. This study was supported by the National Basic Research Program of China (2013CB127700), the International Collaboration Project, Ministry of Agriculture, China (2011-G3), the National Natural Science Foundation of China (31261140370), and the China Agriculture Research System (CARS-3-1-3).
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QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli
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Blumeria graminis f. sp tritici using nested PCR. Journal of Phytopathology, 158, 227-235. Zhou Y L, Duan X Y, Chen G, Sheng B Q, Zhang Y. 2002. Analyses of resistance genes of 40 wheat cultivars or lines to wheat powdery mildew. Acta Phytopathology Sinica, 32, 301-305. Zwart R S, Thompson J P, Milgate A W, Bansal U K, Williamson P M, Raman H, Bariana H S. 2010. QTL mapping of multiple foliar disease and root-lesion nematode resistances in wheat. Molecular Breeding, 26, 107-124. (Managing editor WANG Ning)
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May 2013
2013, 12(5): 756-764
RESEARCH ARTICLE
QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli Asad Muhammad Azeem1, BAI Bin1, LAN Cai-xia1, YAN Jun2, XIA Xian-chun1, ZHANG Yong1 and HE Zhong-hu1, 3 National Wheat Improvement Center/National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Sciences, Chinese Academy of Agricultural Sciences (CAAS), Beijing 100081, P.R.China 2 Cotton Research Institute, Chinese Academy of Agricultural Sciences (CAAS), Anyang 455000, P.R.China 3 International Maize and Wheat Improvement Center (CIMMYT) China Office, Beijing 100081, P.R.China 1
Abstract The Italian wheat cv. Strampelli displays high resistance to powdery mildew caused by Blumeria graminis f. sp. tritici. The objective of this study was to map quantitative trait loci (QTLs) for resistance to powdery mildew in a population of 249 F2:3 lines from Strampelli/Huixianhong. Adult plant powdery mildew tests were conducted over 2 yr in Beijing and 1 yr in Anyang and simple sequence repeat (SSR) markers were used for genotyping. QTLs Qpm.caas-3BS, Qpm.caas-5BL.1, and Qpm.caas-7DS were consistent across environments whereas, Qpm.caas-2BS.1 found in two environments, explained 0.4-1.6, 5.5-6.9, 27.1-34.5, and 1.0-3.5% of the phenotypic variation respectively. Qpm.caas-7DS corresponded to the genomic location of Pm38/Lr34/Yr18. Qpm.caas-4BL was identified in Anyang 2010 and Beijing 2011, accounting for 1.9-3.5% of phenotypic variation. Qpm.caas-2BS.1 and Qpm.caas-5BL.1 contributed by Strampelli and Qpm.caas-3BS by Huixianhong, seem to be new QTL for powdery mildew resistance. Qpm.caas-4BL, Qpm.caas-5BL.3, and Qpm.caas-7DS contributed by Strampelli appeared to be in the same genomic regions as those mapped previously for stripe rust resistance in the same population, indicating that these loci conferred resistance to both stripe rust and powdery mildew. Strampelli could be a valuable genetic resource for improving durable resistance to both powdery mildew and stripe rust in wheat. Key words: QTL analysis, SSR markers, Blumeria graminis, durable resistance, Triticum aestivum L.
INTRODUCTION Wheat is the most widely used food crop for humankind. Powdery mildew caused by Blumeria graminis f. sp. tritici is a major wheat disease worldwide and causes significant yield losses every year in many wheat growing regions in China (Zeng et al. 2010). Host plant resistance, being cost effective and environmentally friendly compared to the use of fungicides, is the best Received 19 July, 2012
option to control the disease (Gurung et al. 2009). Qualitative and quantitative forms of resistance to powdery mildew have been reported in wheat and its wild relatives. Qualitative resistance is usually highly protective throughout the growth cycle of the host, but when exploited in agriculture usually becomes ineffective within a relatively short period of adoption. This lack of durability is the consequence of increased frequencies of previously rare races of the pathogen, mutation from avirulence to virulence, or in some cases, if
Accepted 18 September, 2012
Asad Muhammad Azeem, E-mail: [email protected]; Correspondence HE Zhong-hu, Tel: +86-10-82108547, E-mail: [email protected]
© 2013, CAAS. All rights reserved. Published by Elsevier Ltd. doi:10.1016/S2095-3119(13)60297-X
QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli
resistance is based on combinations of such genes, by genetic recombination in the pathogen. Blumeria graminis f. sp. tritici is a haploid pathogen and although it usually propagates asexually by means of conidiospores it can also undergo sexual recombination, particularly at the beginning of the epidemic cycle. Most powdery mildew resistance genes commonly present in wheat are not effective in China (Zhou et al. 2002). The contrasting type of resistance, known as quantitative resistance, tends to confer partial resistance at post seedling growth stages, and although less effective, is considered to be durable. It is also called as adult plant resistance (APR). Although individual adult plant resistance (APR) genes or QTLs often confer inadequate resistance, combinations of a few such genes usually result in “near-immunity” or high levels of resistance (Gustafson and Shaner 1982; Liu et al. 2001). Studies of adult plant partial resistance to powdery mildew in wheat have generally found that it is an oligogenic trait and individual genes were mapped on many different chromosomes (Keller et al. 1999; Bougot et al. 2006; Lillemo et al. 2008; Lan et al. 2009, 2010b; Muranty et al. 2010). Generally, the highest levels of resistance occur in lines with the most resistance genes/ QTLs. The best characterized gene of this type is Pm38 (also known as Lr34 and Yr18) which is involved in multi-pathogen durable resistance. This gene has been cloned and characterized as a putative ABC transporter (Krattinger et al. 2009). Whether multiple disease protection is a general characteristic of other partial resistance genes is yet to be established. The Italian common wheat cv. Strampelli, introduced to Gansu Province of China in 1973 has adult plant resistance to stripe rust controlled by three QTLs, including Yr18 (Lu et al. 2009). It has also shown good resistance to powdery mildew in the field. The objective of this study was to determine the genetic basis of durable powdery mildew resistance in Strampelli.
RESULTS Field evaluation of mapping population Strampelli and Huixianhong expressed the maximum disease severities of 3.5 and 15%, 2.33 and 16.7%, and 4.0 and 33.3%, respectively, whereas the susceptible
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control Jingshuang 16 displayed 80-100, 60-90, and 90-100% in Anyang 2010, Beijing 2010, and Beijing 2011, respectively. Strampelli and Huixianhong expressed mean maximum disease severities (MDS) of less than 5 and 15-25%, respectively, over three environments. Powdery mildew MDS across the three environments were significantly correlated (r=0.67 to 0.69). Frequency distributions of MDS and area under disease progress curve (AUDPC) showed continuous variation in all environments, suggesting a quantitative basis of resistance to powdery mildew (Fig. 1). Broadsense heritabilities of MDS and AUDPC were 0.78 and 0.73, respectively. Analyses of variance showed significant variation among F2:3 lines for both MDS and AUDPC (Table 1).
QTLs for adult plant resistance (APR) to powdery mildew Seven QTLs based on MDS, five QTLs in averaged MDS over three environments, and three QTLs in averaged AUDPC over 2 yr in Beijing were mapped on chromosomes 2BS, 3BS, 4BL, 5BL, and 7DS (Fig. 2, Table 2). QTLs on 3BS and 7DS were stable across all environments, average MDS in three environments, and averaged AUDPC in Beijing over 2 yr. Except in Beijing 2011, QTL on 2BS was consistently identified in other environments, averaged MDS of three environments, and averaged AUDPC in Beijing over two years, whereas Qpm.caas-5BL.1 was detected in almost all environments except averaged AUDPC in Beijing over 2 yr. Qpm.caas-4BL, Qpm.caas-5BL.2 and Qpm.caas-5BL.3 were detected in two, three and one environments, respectively. QTLs on 2BS, 4BL, 5BL, and 7DS, and one on 3BS were contributed by Strampelli and Huixianhong, respectively. Qpm.caas-2BS mapped in the marker interval Xwmc25.1-Xwmc25.2, explained 1.0-3.5% of the phenotypic variances and was stable across two environments, in average MDS and average AUDPC in Beijing. A consistently detected QTL, Qpm.caas-3BS, was identified on the short arm of chromosome 3B between Xwmc366.1 and Xwmc366.2 across all environments. Qpm.caas-4BL explaining 1.9-3.5% of the phenotypic variance was located in the marker interval Xgwm165-Xgwm149 in two environments. Qpm.
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Fig. 1 Frequency distributions of the maximum powdery mildew severities (MDS) and area under the disease progress curve (AUDPC) values in F 2:3 lines derived from Strampelli/ Huixianhong. A, MDS in Anyang in 2010. B, MDS in Beijing in 2010. C, MDS in Beijing in 2011. D, average MDS across three environments. E, average AUDPC in Beijing over 2 yr. Mean values for the parents, Strampelli and Huixianhong, are indicated by arrows.
Table 1 Analysis of variance of the maximum powdery mildew severities (MDS) on penultimate leaves and area under the disease progress curve (AUDPC) for powdery mildew responses on F2:3 lines derived from Strampelli/Huixianhong Parameter
Source of variation
MDS
Replicates Environments Lines Lines × Environments Error Replicates Environments Lines Lines × Environments Error
AUDPC
**
df 2 2 248 494 1 379 2 1 248 248 944
Sum of squares 224 36 232 109 050 47 076 53 782 30 477 216 566 2 393 427 638 137 1 406 507
Mean square
F-value
112 18 116 439 95 39 15 238 216 566 9 650 2 573 1 414
2.88 464.51 ** 11.27 ** 2.44 ** 10.77 ** 153.05 ** 6.82 ** 1.82 **
, P<0.0001.
caas-5BL.1, identified in the marker interval Xgwm335Xbarc331 across all the environments, accounted for 5.5-6.9% of the phenotypic variance with additive effects of 1.42-3.39. Qpm.caas-5BL.2 in marker interval Xbarc331-Xwmc537 explained phenotypic variation 1.8-4.8% whereas Qpm.caas-5BL.3, in marker interval Xwmc415-Xwmc405 on the same chromosome explained 4.2% of the phenotypic variance in one environment. Use of STS marker csLV34 indicated the major stable QTL explaining 12.6-34.5% of phenotypic variances
across all environments was Pm38/Yr18/Lr34. Seven QTLs together accounted for 43.8-48.5% of the total phenotypic variance across three environments.
DISCUSSION In this mapping population, MDS and AUDPC showed continuous variation. The apparent lack of full susceptibility may be because the parents are homozygous at
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QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli
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Fig. 2 Logarithm of odds (LOD) contours obtained by inclusive composite interval mapping (ICIM) of QTLs for adult plant powdery mildew resistance in F2:3 lines of Strampelli/Huixianhong. A2010, maximum disease severities (MDS) in Anyang 2010; B2010 and B2011, maximum disease severities in Beijing 2010 and 2011, respectively; mean, mean MDS across three environments; AUDPC, average of area under the disease progress curve in Beijing over 2 yr. Logarithm of odds (LOD) threshold, 2.0.
common loci conferring partial resistance, which could result in decreased variance and absence of transgressive segregation. Disease severity range of F2:3 lines was quite below the susceptibility levels of cv. Jingshuang 16 suggesting that both parents possess some resistance loci in common that did not segregate in the cross, and there may be more minor resistance loci that could not be detected because of less number of simple sequence repeat markers used in this study. However, molecular mapping of the Strampelli/ Huixianhong population indicated the presence of seven QTLs for powdery mildew resistance on chromosomes 2BS, 4BL, 5BL, and 7DS in Strampelli and one on 3BS in Huixianhong. Previously, QTLs, QPm.crag-2BS (Mingeot et al. 2002) and QPm.caas-2BS (Lan et al. 2010b) were detected on chromosome 2BS in Festin and Lumai 21,
respectively. These genes are estimated to be about 22 and 43.9 cM, respectively, from QPm.caas-2BS.1 based on wheat consensus maps (Somers et al. 2004; http:// wheat.pw.usda.gov/GG2/index.shtml). The Pm26 (Rong et al. 2000) and Pm42 (Hua et al. 2009) resistance genes derived from wild emmer (Triticum turgidum var. dicoccoides) were estimated to be 33 and 22 cM from the QPm.caas-2BS.1 peak. This less effective QTL detected in two environments, average MDS in three environments, and averaged AUDPC in Beijing over 2 yr, seems to be a new locus for powdery mildew resistance on chromosome 2BS. A stable QTL, QPm.caas-3BS, was detected across all environments on chromosome 3BS. Chen et al. (2009) mapped a QTL on chromosome 3BS in hard winter wheat line 2174. It was centered on Xwms533 and 56 cM from QPm.caas-3BS. Pm13 derived from
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Table 2 Quantitative trait loci (QTLs) for adult-plant resistance (APR) to powdery mildew in F2:3 lines derived from Strampelli/ Huixianhong Location/Year MDS Anyang 2010
Beijing 2010
Beijing 2011
Averaged MDS in three environments
AUDPC Averaged AUDPC in Beijing
QTL1)
Marker interval
LOD 2)
AE 3)
R2 (%)4)
Total R2 (%)
QPm.caas-2BS.1 QPm.caas-3BS QPm.caas-4BL.1 QPm.caas-5BL.1 QPm.caas-5BL.2 QPm.caas-7DS QPm.caas-2BS.1 QPm.caas-3BS QPm.caas-5BL.1 QPm.caas-5BL.2 QPm.caas-5BL.3 QPm.caas-7DS QPm.caas-3BS QPm.caas-4BL.1 QPm.caas-5BL.1 QPm.caas-7DS QPm.caas-2BS.1 QPm.caas-3BS QPm.caas-5BL.1 QPm.caas-5BL.2 QPm.caas-7DS
Xwmc25.1-Xwmc25.2 Xwmc366.1-Xwmc366.2 Xgwm165-Xgwm149 Xgwm335-Xbarc331 Xbarc331-Xwmc537 csLV34-Xgwm295 Xwmc25.1-Xwmc25.2 Xwmc366.1-Xwmc366.2 Xgwm335-Xbarc331 Xbarc331-Xwmc537 Xwmc415-Xwmc405 csLV34-Xgwm295 Xwmc366.1-Xwmc366.2 Xgwm165-Xgwm149 Xgwm335-Xbarc331 csLV34-Xgwm295 Xwmc25.1-Xwmc25.2 Xwmc366.1-Xwmc366.2 Xgwm335-Xbarc331 Xbarc331-Xwmc537 csLV34-Xgwm295
2.7 4.0 2.7 3.8 3.4 14.5 2.3 3.4 4.2 3.1 3.0 14.9 3.5 1.9 4.6 22.0 2.1 3.3 4.9 2.1 16.6
1.36 0.56 0.74 1.42 1.262 3.34 1.00 0.01 1.30 1.06 1.32 4.74 0.97 1.09 3.39 10.00 0.72 0.06 2.36 0.38 5.90
3.5 1.6 3.5 5.5 4.8 27.2 1.7 0.4 6.9 4.0 4.2 27.1 0.5 1.9 5.9 34.5 1.0 0.1 6.1 1.8 25.9
46.1
QPm.caas-2BS.1 QPm.caas-3BS QPm.caas-7DS
Xwmc25.1-Xwmc25.2 Xwmc366.1-Xwmc366.2 csLV34-Xgwm295
1.8 3.3 6.8
4.74 0.24 19.3
0.8 0.1 12.6
13.5
48.5
43.8
34.9
QTLs that extend across single one-log support confidence intervals were assigned the same symbol. Logarithm of odds (LOD) score. 3) Additive effects. 4) Proportion of phenotypic variance explained by the QTL. 1) 2)
Ae. longissima is also located on 3BS (Donini et al. 1995). QPm.caas-3BS appears to be a new gene conferring adult plant resistance to powdery mildew. QPm.caas-4BL.1 was flanked by SSR markers Xgwm149 and Xgwm495. QPm.sfr-4B and QPm.ipk4B were mapped in cv. Forno (Keller et al. 1999) and the ITMI (Opata 85/W7984) population (Börner et al. 2002), flanked by markers Xpsr593b and Xpsr1112, and Xcdo795 and Xbcd1262, respectively, a similar position to QPm.caas-4BL.1. Liang et al. (2006) detected QPm.caas-4BL flanked by Xgwm375 and Xgwm251 in cv. Oligoculm in a similar position based on data from one test location. QPm.nuls-4BL flanked by XwPt1505 and Xgwm149, was mapped on the same chromosomal region of cv. Avocet in both Beijing and Norway (Lillemo et al. 2008). QYr.caas-4BL detected at one site and explaining 4.5% of the phenotypic variance in stripe rust response was mapped to a similar position (Lu et al. 2009). William et al. (2006) and Zwart et al. (2010) mapped stripe rust resistance QTL in crosses Avocet S/Pavon 76 and synthetic hexaploid CPI133872/
Janz and these also coincided with the position of QPm. caas-4BL.1. Thus results from a number of studies located QTLs for powdery mildew resistance in similar positions to those for stripe rust resistance. Three QTLs, QPm.ttu-5B, QPm.inra-5B.2, and QPm.nuls-5B were previously mapped on chromosome 5BS in cv. Tahti, Courtot and Saar, respectively (Bougot et al. 2006; Jakobson et al. 2006; Lillemo et al. 2008). Keller et al. (1999) mapped QPm.sfr-5B flanked by RFLP markers Xpsr580b-Xpsr143 on chromosome 5B. Our study has not determined a relationship of QPm. sfr-5B with QPm.caas-5BL.1 or QPm.caas-5BL.2 or QPm.caas-5BL.3 due to the different types of markers being used, and the positions of these markers are not comparable. QTLs, QPm.caas-5BL.1 and QPm.caas5BL.2 flanked by Xgwm335-Xbarc331 and Xbarc331Xwmc537 were located nearby with less than 20 cM distance between the resistance genes. We considered them same QTL as QPm.caas-5BL.1. QTL, QPm.caas5BL.3 flanked by Xwmc405 and Xwmc415, identified at distance more than 20 cM away from the peak of QPm.
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QTL Mapping for Adult Plant Resistance to Powdery Mildew in Italian Wheat cv. Strampelli
caas-5BL.2, considered as independent QTL. QPm. caas-5BL.1 conferred stable and consistent resistance across all environments, explaining 5.5-6.9% of the phenotypic variance. QYr.inra-5BL.1 accounting for 25% of the phenotypic variation in stripe rust response was flanked by Xgwm499 and Xgwm639 in the same region as QPm.caas-5BL.3 (Mallard et al. 2005). Lu et al. (2009) reported stripe rust APR QTLs on chromosome 5BL in cultivars Libellula and Strampelli located at a similar position to QPm.caas-5BL.3, indicating a common locus might confer resistance to both stripe rust and powdery mildew. QPm.caas-5BL.1 identified in the present study showed a stronger and more consistent effect across environments than QPm.caas5BL.3 which explained only 4.2% of the phenotypic variance in one environment. QPm.caas-5BL.1 appears to be a new locus for APR to powdery mildew. The most effective QTL was QPm.caas-7DS, inherited from Strampelli and explaining 27.1-34.5% of phenotypic variance across different environments. QTLs for slow mildewing in cv. W7984, FukuhoKomugi and Saar were previously mapped in the same genomic region as QPm.caas-7DS on chromosome 7DS (Börner et al. 2002; Liang et al. 2006; Lillemo et al. 2008). Lu et al. (2009) detected QYr.caas-7DS for stripe rust resistance in a similar position to QPm.caas7DS for powdery mildew resistance in Strampelli. The only presumed powdery mildew resistance gene in Strampelli was Pm38. The Pm38/Yr18/Lr34 gene is currently a prime target for durable resistance to multiple diseases because it is amenable to accurate molecular selection (Morgounov et al. 2012). Genes for resistance to powdery mildew in Strampelli seem to be more consistent and stable across environments than previously identified in Chinese wheat cultivars such as Yumai 2, Xiaoyang 6, Yumai 47, Lumai 21, and Bainong 64 (He et al. 2011, and personal communication). So far, Pm38/ Yr18/Lr34 has been identified in powdery mildew studies of Mexican (W7984 and Saar) and Japanese (Fukuho-Komugi) cultivars, and its identification in Strampelli, well adapted in Chinese environment seems to play a large role and should be useful for developing cultivars with potentially durable powdery mildew, stripe rust and leaf rust resistance simultaneously. During the past years, Strampelli was crossed with several
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widely grown Chinese wheat cultivars Zhongmai 175, Lunxuan 987, Jimai 22, and Zhoumai 18 in our breeding program. The molecular markers csLV34, wmc537, gwm149, gwm165, and barc331 closely linked to Pm38/Yr18/Lr34 and other QTLs (Lu et al. 2009) were used for marker-assisted selection in BC 1 or BC 2 progenies, and over 20 lines with good yield potential and resistance to powdery mildew, stripe rust and leaf rust were developed. This will provide a good example for marker-assisted selection targeting for durable resistance to powdery mildew, stripe rust and leaf rust in Chinese wheat breeding program.
MATERIALS AND METHODS Plant materials A total of 249 F 2:3 lines developed from Strampelli/ Huixianhong were available for QTL mapping. Strampelli has shown powdery mildew resistance at the adult plant stage in our nurseries over many years. Huixianhong, a landrace from Henan, is very susceptible at the seedling stage and moderately resistant at the adult stage. The F2:3 lines, derived from individual F2 plants are maintained as perpetual bulks of more than 50 plants.
Powdery mildew disease assessment F2:3 lines were sown in randomized complete blocks with three replications over 2 yr (2009-2010 and 2010-2011) at the CAAS Experimental Station, Beijing, and CAAS Cotton Research Institute, Anyang, Henan, (herein referred to as Beijing 2010, Beijing 2011, Anyang 2010). Fifty seeds of each line were sown in circular plots (8 cm diameter), and plots were spaced 30 cm apart to facilitate disease evaluation. Jingshuang 16 was planted at each tenth plot and around the experiment as a susceptible check and spreader to ensure the maximum inoculum for disease development. In Beijing, plots were inoculated prior to stem elongation with Blumeria graminis f. sp. tritici (Bgt) isolate E20. All entries were scored for the first time 5 wk after inoculation based on the actual percentage of leaf area covered by powdery mildew on five randomly selected penultimate (F-1) leaves and again 1 wk later when disease was at its maximum level around May 20. In Anyang, powdery mildew severities resulting from natural infection were evaluated once when infection levels on Jingshuang 16 had reached the maximum levels during the 3rd wk of May. Phenotypic data from Anyang in 2011 could not be used because the disease did not develop sufficiently due to dry spring conditions.
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gression (Wang et al. 2011). The PVE or R2 was calculated by the formula:
Statistical analysis AUDPC was calculated for each line using the formula: (Bjarko and Line 1988), where Xi is the severity value on date i, T i the number of days after inoculation on scoring date i, and n is the number of times of disease scoring. The correlation coefficients (r) and frequency distributions of powdery mildew response based on MDS in different environments were calculated with Microsoft Excel 2007. The statistical analyses of MDS and AUDPC were performed using the statistical package SAS ver. 9.1 by PROC GLM (SAS Institute 1997). Broad-sense heritability (h2) was calculated using the formula: h2=Var(G)/Var(P), where, Var(G) and Var(P) are genotypic and phenotypic variances, respectively (Allard 1960).
Simple sequence repeat (SSR) analysis Fresh young leaf tissue of the parents and each F 2:3 line (30-40 plants per F 2:3 line as a bulk) was used to extract genomic DNA using the procedure described by Sharp et al. (1988). A total of 943 sequences of available SSR markers (http://www.wheat.pw.usda.gov) and one STS marker csLV34 closely linked with the locus Lr34/Yr18 (Lagudah et al. 2006) were used. Based on MDS, resistant and susceptible bulks were constructed by mixing equal amounts of DNA from the five most resistant and five most susceptible F2:3 lines, respectively (Michelmore et al. 1991; Lan et al. 2010a). SSRs showing polymorphisms between the two parents and between the two bulks were used to genotype all 249 lines to determine their associations with powdery mildew responses. PCR reaction protocols, electrophoresis of PCR products and gel staining were conducted as described by Bryan et al. (1997) and Bassam et al. (1991). Polymorphic markers were used to genotype the population for linkage and QTL analysis.
Quantitative trait loci (QTL) analysis Linkage groups were established with the software Map Manager QTXb20 (Manly et al. 2001) and recombination values were converted to centiMorgans using the Kosambi mapping function (Kosambi 1944). Each linkage group was assigned to a chromosome based on previously published wheat genome maps (http://wheat.pw.usda.gov, Somers et al. 2004). QTL detection was performed using inclusive composite interval mapping (ICIM) with the software IciMapping v3.2 (Wang et al. 2011). A LOD threshold value of 2.0 was used for declaration of a QTL. The phenotypic variance explained (PVE or R2) by individual QTL and additive effects were obtained through stepwise re-
Where, VG is the genotypic variance of a QTL, and VP is the phenotypic variance of the trait in QTL mapping (Wang et al. 2011). QTLs overlapping within 20 cM intervals on the same chromosome across different environments were considered to be the same.
Acknowledgements We are grateful to the critical review of this manuscript by Prof. R. A. McIntosh, Plant Breeding Institute, University of Sydney. This study was supported by the National Basic Research Program of China (2013CB127700), the International Collaboration Project, Ministry of Agriculture, China (2011-G3), the National Natural Science Foundation of China (31261140370), and the China Agriculture Research System (CARS-3-1-3).
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