Chand R

Joshi AK

Kumar U

Kumar, S

Röder MS

English

Sydney eScholarship

 1QTLs for resistance to spot blotch of wheat caused by Bipolaris sorokiniana  Kumar U1, Joshi AK1, Kumar S2, Chand R3 and Röder MS4 1Department of Genetics and Plant Breeding, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005 India; 2Department of Biotechnology, Sardar Vallabh Bhai Patel University of Agriculture and Technology, Meerut 250110, India; 3Department of Mycology and Plant Pathology, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221005 India; 4Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Gatersleben, Germany  INTRODUCTION  Spot blotch disease of wheat caused by Bipolaris sorokiniana is a disease causing substantial damage to wheat (Triticum aestivum L. em. Thell) in South Asia (Saari, 1998; Joshi et al., 2007a). Some traits viz., erect leaf posture (Joshi and Chand 2002), leaf tip necrosis (Joshi et al. 2004a) and stay green trait (Joshi et al. 2007b) have been demonstrated to have positive effects on resistance to spot blotch. However, reports on suitable molecular markers are not available. Thus the objective of the present study was to identify QTLs associated with spot blotch resistance in spring wheat.  MATERIALS AND METHODS  One hundred and thirty nine single seed descent (SSD) derived Recombinant Inbred Lines (RILs, F8) of the cross between two wheat genotypes Yangmai 6 (Resistant) and Sonalika (Susceptible) were investigated. The F6, F7 and F7:8 generations of RILs of Yangmai 6×Sonalika were evaluated in for spot blotch resistance in RCBD during the crop seasons 2003-04, 2004-05 and 2005-06 at the Agricultural research farm of Banaras Hindu University, Varanasi, India. Spreader rows of A-9-30-1 were also planted to induce disease development. Agronomic practices recommended for normal fertility (120 Kg N: 60 Kg P2O5: 40 Kg K2O) were followed.   A pure culture of the most aggressive isolate of B. sorokiniana (isolate No. ICMP 13584, Auckland, New Zealand) (Chaurasia et al., 2000) was used for artificial epiphytotic. Disease severity (%) of each row was recorded at three different growth stages (GS) viz., GS 63 (beginning of anthesis to half complete); GS 69 (anthesis complete) and GS 77 (late milking) on Zadoks scale (Zadoks et al., 1974). Area under disease progress curve (AUDPC) based on disease severity was estimated.  Leaves were harvested from 15 days old seedlings of the RILs (F8). Genomic DNA was isolated using the CTAB method described by Doyle and Doyle (1990). PCR reactions of gwm (gatersleben wheat microsatellite) and barc (Beltsville Agriculture Research Center) microsatellite markers were performed as described by Röder et al (1998) and Somers et al (2004). Microsatellite fragments were detected on an automated laser fluorescence ALF express sequencer (Amersham Biosciences Europe GmbH, Freiburg, Germany). An external standard with four fragments (73, 122, 196 and 231bp) was loaded in one lane. The order and distribution of the microsatellite markers were based on the ITMI (International Triticeae Mapping Initiative) population of wheat. Single marker regression analysis was performed using the software Qgene developed by Nelson (1997). When a marker was observed significant (P < 0.001), all RILs were genotyped with the respective marker and QTL Cartographer version 2.5 (Wang et al. 2005) was used to confirm the QTLs by IM (interval mapping) and CIM (composite interval mapping).  Analysis of variance (ANOVA) for mean AUDPC values of each year was performed using SAS software (version 6.03; SAS Institute Inc., Cary, NC 1997). Heritability (h2) was estimated following Singh and Ceccarelli (1996). Analysis of variance was performed to test the significance of marker trait association for each marker in all RILs. Phenotypic correlation coefficients of spot blotch disease severity and AUDPC values were calculated using Qgene software (Nelson 1997).  Mapmaker v 2.0 (Lander et al., 1987) was used for all polymorphic markers to create a framework map with 129 markers. Later on some more markers were added to enrich the neighbouring regions. Finally, Mapmaker was used to create linkage map of significant markers. The linkage map was constructed using the LOD (Likelihood of odd ratio) of >3 and recombination fraction of <0.4. QTL analysis was performed with the software QTL Cartographer version 2.5. QTLs were verified by LOD scores compared to as empirical genome-wide significant threshold calculated from 1000 permutations for P < 0.01 to control Type–I error. QTLxQTL and QTLxEnvironment interactions were calculated using QTL Network V 1.60 and PlabQTL V 1.2 (http://www.uni-hohenheim.de/~ipspwww/soft.html) respectively. The names of the QTLs were assigned following International Rules of Genetic Nomenclature (http://wheat.pw.usda.gov/ggpages/wgc/98/Intro.htm): QSb.bhu as QTL for resistance to spot blotch disease detected at Banaras Hindu University.    2 RESULTS  The distribution of spot blotch AUDPC and the test of normality using Shapiro-Wilk test (W = 0.9892, P = 0.3463) revealed that the RILs data fit a normal distribution. The analysis of variance for AUDPC revealed significant variation for genotypes and genotype x year interaction. However, the difference among the three groups of genotypes and Group x Year interaction was non-significant. Broad sense heritability for AUDPC was moderate to high.  High Pearson correlation coefficients were observed between disease severity and AUDPC within years with a range from 0.82 to 0.90 (P < 0.0001). Moderate correlations were observed between years ranging from 0.39 to 0.78 for AUDPC (P < 0.001 or P < 0.0001). The correlation coefficient between days to heading and AUDPC was non-significant (0.087).  Four QTLs were detected for spot blotch AUDPC (Table 1). Individual QTLs explained between 8.04% and 41.10% of phenotypic variance in composite interval mapping. The two most consistent QTLs mapped on the short arm of chromosome 2B and the long arm of chromosome of 5B (Fig. 1), detected in all three years, while other QTLs present at least in two years were located on the long arm of chromosome 2A and the long arm of chromosome 6D. The QTL on 5BL explained the largest part of phenotypic variance in the third year (41.10%). In the second year, maximum phenotypic variation (14.89%) was controlled by the QTL located on chromosome 2BS. QTLs mapped on 2AL, 2BS, 5BL and 6DL (Table 1) accounted for 14.80%, 20.50%, 38.6% and 15.6% of phenotypic variation based on mean over years, respectively  The QTL, QSb.bhu-2A was located on the long arm of chromosome 2A between the markers interval Xbarc353-Xgwm445 (37.4 cM). The main QTL QSb.bhu-5B was located on the long arm of chromosome 5B between Xgwm067 and Xgwm371 (13.2 cM). The other QTLs, QSb.bhu-2B and QSb.bhu-6D were mapped between Xgwm148-Xgwm374 (15.0 cM) and Xgwm732-Xgwm1103 (3.2 cM), respectively.   DISCUSSION  The distribution of 139 RILs suggested that spot blotch resistance is polygenic in the ‘Yangmai 6’ × ‘Sonalika’ cross. Earlier studies (Joshi et al. 2004b) also suggest a polygenic control for spot blotch resistance.   Four QTLs were identified for spot blotch resistance on 2A, 2B, 5B and 6D. We found a major QTL in the marker interval Xgwm067-Xgwm371 (13.2 cM) on chromosome 5B. In the deletion maps, the microsatellite locus, Xgwm445 flanking QSb.bhu-2A was assigned to the 2AL1-0.78 deletions bin. The loci Xgwm148 and Xgwm374 flanking the QTL QSb.bhu-2B have been assigned to the 2BS1-0.53-0.75  and the C-2BS1-0.53 deletion bins, respectively, while the loci Xgwm067 and Xgwm371 flanking QSb.bhu-5B were assigned to the C-5BL6-0.29 and 5BL1-0.55-0.75 bins, respectively (http://wheat.pw.usda.gov/GG2/index.shtml). In the public database 'GrainGenes' no physical mapping data were available for markers Xgwm732 and Xgwm1103 flanking the QTL QSb.bhu-6D.   ACKNOWLEDGEMENTS  Part of this research was supported by a DAAD-Leibniz (German Academic Exchange Service) fellowship to U. Kumar. Authors are thankful to Ram Awadh, Anette Heber and Sonja Allner for excellent technical assistance. The help rendered by Dr. Rajender Parsad, Indian Agricultural Statistical Research Institute, N. Delhi in the analysis of data is gratefully acknowledged.  REFERENCES Chaurasia S, Chand R, Joshi AK (2000) Relative dominance of Alternaria triticina Pras. et Prab and Bipolaris sorokiniana (Sacc.) Shoemaker, in different growth stages of wheat (T. aestivum L). J Plant Dis Protec 107:176-181 Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:12-15. Joshi AK, Chand R, Kumar S, Singh RP (2004a) Leaf tip necrosis: A phenotypic marker associated with resistance to spot blotch disease in wheat. Crop Sci 44:792-796 Joshi AK, Kumar S, Chand R, Ortiz-Ferrara G (2004b) Inheritance of resistance to spot blotch caused by Bipolaris sorokiniana in spring wheat. Plant Breed 123:213-219 Joshi AK, Ortiz-Ferrara G, Crossa J, Singh G, Alvarado G, Bhatta MR, Duveiller E, Sharma RC, Pandit DB, Siddique AB, Das SY, Sharma RN, Chand R (2007a) Associations of environments in South Asia based on spot blotch disease of wheat caused by Cochliobolus sativus. Crop Sci. 47:1071-1081 Joshi AK, Kumari M, Singh VP, Reddy CM, Kumar S, Rane J, Chand R (2007b) Stay green trait: Variation, inheritance and its association with spot blotch resistance in spring wheat (Triticum aestivum L.). Euphytica 153: 59-71 Nelson J (1997) Qgene: software for marker-based genomic analysis and breeding. Mol. Breed. 3:239-245 Röder MS, Korzun V, Wendehake K, Plaschke J, Tixier M-H, Leroy P, Ganal MW (1998) A microsatellite map of wheat. Genetics 149:2007-2023 Saari EE (1998) Leaf blight diseases and associated soil borne fungal pathogens of wheat in north and south East Asia. In: Helminthosporium Blights of wheat: Spot Blotch and Tan Spot (eds.) by Duveiller E, Dubin HJ, Reeves J and Mc Nab A, CIMMYT, Mexico, D.F. pp37-51 Singh M, Ceccarelli S (1996) Estimation of heritability of Crop Traits from Variety Trial Data. Technical Manual No. 21, ICARDA, Syria. Zadoks JC, Chang TT, Konjak CF (1974) A decimal code for the growth stages of cereals. Weed Res 14:415-421 3       Fig. 1. LOD curves obtained by composite interval mapping for four quantitative trait loci mapped on chromosome 2BS and 5BL indicated by arrow that reduce spot blotch severity in ‘Yangmai 6’ x ‘Sonalika’ recombinant inbred population. The vertical line indicate the threshold LOD value (2.2) determining significant QTLs (LR = LOD x 4.6). Short arms are toward the top and open triangles indicate the probable position of centromeres.          Table 1. Effects of quantitative loci (QTLs) that reduce spot blotch  Severity in ‘Yangmai 6 x Sonalika’ recombinant inbred (RI)  population detected by composite interval mapping (CIM).  The marker intervals cited are those flanking the peak of the  LOD scan. R2 represents the percentage of phenotypic variance  explained for each QTL   QTLs Marker Interval Interval Size (cM) Chrom R2  mean across years Q x E LOD R2 QSb.bhu-2A Xbarc353 – Xgwm445 37.4 2AL 3.5 14.8 * QSb.bhu-2B Xgwm148 – Xgwm374 15.0 2BS 5.8 20.5 ** QSb.bhu-5B Xgwm067 - Xgwm371 13.2 5BL 12.9 44.6 ** QSb.bhu-6DXgwm732 - Xgwm1103 3.0 6DL 4.1 15.6 * R2    19.4 60.2  QTLxQTL      2A x 2B  - -  Ns 2B x 5B  - -  Ns 5B x 6D  - -  Ns 

QTLs for resistance to spot blotch of wheat caused by Bipolaris sorokiniana

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QTLs for resistance to spot blotch of wheat caused by Bipolaris sorokiniana

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