Fusarium head blight (FHB or scab), commonly caused by Fusarium graminearum Schwabe and Fusarium culmorum (W.G. Smith) is one of the most destructive diseases of wheat, Triticum aestivum L., in humid and semi-humid areas worldwide. The development of cultivars with high FHB resistance is the most economical and effective method to control the disease. Although diverse sources of resistance to FHB are available in wheat, breeding for FHB resistance with classical selection methods is costly and time-consuming (Mardi et al., 2004). Mapping of quantitative trait loci (QTL) associated with FHB resistance and application of marker-assisted selection (MAS) can be used to accelerate the selection and development of FHB resistant cultivars (Anderson et al., 2007). DNA markers linked to FHB resistance have been identified in several studies using different mapping populations (Anderson et al., 2007; Mardi et al., 2006; Mardi et al., 2005; Lin et al., 2004; Buerstmayr et al., 2003).
Recent advances in molecular characterization of plant resistance genes (R-genes) have led to development of direct markers known as resistance gene analog polymorphism (RGAP) markers. RGAP markers are based on designing primers from the conserved domains including nucleotide-binding site (NBS), leucine-rich repeat (LRR) and protein kinase of resistance genes (Chen et al., 1998). Several RGAP markers have been used successfully to develop molecular markers linked to resistance genes in wheat (Chen et al., 2006; Xie et al., 2004; Yan et al., 2003), barley (Yan et al., 2006) and maize (Wenkai et al., 2006). The objective of this study was to identify RGAP markers that co-localize with the major QTL for FHB resistance, Qfhs.ndsu-3BS, in wheat using a ‘Wangshuibai’ derived population. The ultimate goal was to find out the possibility of identifying FHB candidate resistance genes that to be investigated in future studies.
Hundred and seventy one F3 individual plants, one from each F2 individual, and their derived F3:5 obtained from a cross between ‘Wangshuibai’, a highly resistant Chinese landrace, and ‘Seri82’, a susceptible Mexican spring cultivar, were used in this study. The pedigree of ‘Seri82’ is ‘Kavkaz’/’Buho-sib’//’Kalyansona’/’Bluebird’. The 171 F3 plants and their derived F3:5 were assessed for disease by evaluating the area under the disease progress curve (AUDPC) in field experiments carried out by Mardi et al. (2005). Total genomic DNA was isolated using the cetyltrimethyl-ammonium bromide (CTAB) method (Saghai-Maroof et al., 1984) with minor modifications. DNA quantity and quality were measured with a DU- 530® UV-Photometer (Beckman, USA). Fifty degenerate primers based on the sequence of known R-genes were used (Table 1). PCR analysis was performed as described by Chen et al. (1998). Amplification was performed in a thermocycler (BioRad Laboratories Inc., USA) programmed for 5 min at 94ºC for initial denaturation and 45 cycles each consisting of 1 min at 94ºC, 1 min at 45ºC and 2 min at 72ºC, followed by a final 7 min extension at 72ºC. The PCR products were separated on 6% (w/v) denaturing polyacrylamide gel followed by silver-staining as described by Bassam et al. (1991). The RGAP markers were integrated into linkage maps constructed with 421 simple sequence repeat (SSR) and amplified fragment length polymorphism (AFLP) markers covering a genetic distance of 2554 cM (Mardi et al., 2005), considering a minimum logarithm of the odds ratio (LOD) of 5 and a maximum genetic distance of 30 cM. The Kosambi mapping function (Kosambi, 1994) was used to convert recombination frequencies into genetic distances. QTL analysis was performed based on composite interval mapping using the PLABQTL program (Utz and Melchinger, 1996).Cloning of the PCR product was performed by using the pGEM-T Easy vector cloning kit (Promega, USA). Five positive clones of each amplified fragment were sequenced using the ABI Prism 3130xL Genetic Analyzer. The Basic Local Alignment Search Tool (BLAST) algorithm (Altschul et al., 1997) was used for similarity search of the sequenced fragment with known resistance gene sequences.
Eight out of the 50 primer combinations showed polymorphism between parental lines (Table 2) and produced 16 RGAP markers (Fig. 1). Two markers were integrated into the chromosal region containing the major QTL for FHB resistance, Qfhs.ndsu-3BS (Fig. 2). Composite interval mapping detected two QTLs in a genomic region that were coincident with Qfhs.ndsu-3BS (Fig. 2). A QTL in the map interval of Xrga1-Xgwm533 with 4.2 cM was detected and accounted for up to 12.5% of the phenotypic variation (Table 3). The second QTL was detected in the map interval Xrga9-Xgwm493 with 9.6 cM. This QTL explained up to 8.5% of the phenotypic variation (Table 3). Both QTL alleles conferring resistance were contributed by ‘Wangshuibai’ with additive effects and were tagged with flanking SSR markers. In this study, we found two RGAP markers significantly associated with FHB resistance and co-localizing with the major QTL for FHB resistance on the 3BS chromosome. The major QTL, Qfhs.ndsu-3BS, from Sumai3 and its derivatives were consistently observed to have a major effect on FHB resistance in various genetic backgrounds (Anderson et al., 2007; Buerstmayr et al., 2003). Since Xrga1 and Xrga9 loci were coincident with Qfhs.ndsu-3BS, they could be converted into sequence-characterized amplified region markers, i.e. single band present/absent markers which may be easily used as a low-cost, high-throughput alternative to conventional phenotypic screening in wheat-breeding programs to improve FHB resistant lines. Guo et al. (2006) converted one RGA associated with FHB resistance in wheat to a sequence tagged site (STS) marker and mapped it onto chromosome 1AL.
Of 87 BLAST hits, with up to 97% homology to Xrga1, 70 had similarity to Pto and Pto-like genes in Lycopersicon spp. and Capsicum spp., 3 to the N gene in Nicotiana spp. and 1 to the putative resistance protein in Triticum monococcum. There are some indications that Qfhs.ndsu-3BS may be in a gene-rich region (Liu et al., 2003) where protein kinase genes have been found (Feuillet et al., 1997). The Pto interacts with proteins that bind to the cis-element of pathogenesis-related genes in order to confer resistance in tomato bacterial speck disease (Zhou et al., 1997). However the role of Pto and Pto-like genes in enhancing FHB resistance in wheat remains to be determined. The Xrga9 sequence was highly similar to the gypsy-type retrotransposon reverse transcriptase and CBF transcription factors at the frost tolerance locus Fr-Am2 (AY951944) in Triticum monococcum. However, Xrga9 sequence did not have homology with any known resistance genes. Previous studies have reported that many RGAP cloned fragments do not have significant sequence homology to known resistance genes because of rearrangement of genome and transposon elements (Shi et al., 2001; Leister et al., 1996; Yu et al., 1996).
As reported by Shi et al. (2001); Yan et al. (2003) and Xie et al. (2004), our results also showed that the RGAP markers could be efficient tools for mapping of resistance genes in wheat. Identifying RGAP markers can reduce linkage drag associated with the QTL to obtain improved FHB resistant lines in wheat breeding programs. Fine resolution of the major QTL for FHB resistance, Qfhs.ndsu-3BS, may further be improved by subsequent study of this small interval and the QTL thus be assigned to intervals sufficiently small for physical mapping and map-based cloning. The present study provides that known R-genes, namely Pto and Pto-like genes, may be considered as FHBcandidate resistance genes underlying Qfhs.ndsu-3BS and may be used in future studies.
This research was supported by the Agricultural Biotechnology Research Institute of Iran (ABRII).