Resistance Gene Analog Polymorphism (RGAP) Markers Co-Localize with the Major QTL of Fusarium Head Blight (FHB) Resistance, Qfhs.ndsu-3BS in Wheat

Document Type: Brief Report

Authors

1 Department of Genomics, Agricultural Biotechnology Research Institute of Iran (ABRII), Mahdasht Road, P.O. Box 31535-1897, Karaj, I.R. Iran

2 Department of Agronomy and Plant Breeding, Faculty of Agricultural, University of Tehran, P.O. Box 31587-11167, Tehran, I.R. Iran

Abstract

Resistance gene analog polymorphism (RGAP) markers linked to Fusarium head blight resistance (FHB) and co-localize with Qfhs.ndsu-3BS were identified using F3 plants and F3:5 lines derived from a ‘Wangshuibai’ (resistant) / ‘Seri82’ (susceptible) cross. The mapping populations were genotyped using 50 degenerate primers designed based on the known R genes. Out of the 50 designed primer combinations, eight showed polymorphism and produced 16 RGAP markers. Out of the 16 RGAP markers, two were integrated into the major QTL for FHB resistance, Qfhs.ndsu-3BS. Composite interval mapping (CIM) analysis detected two QTLs in a genomic region that were coincident with Qfhs.ndsu-3BS, thus explaining up to 12.5% of the phenotypic variations. The nucleotide sequence analysis of the positive subjected RGAP markers showed that known R-genes, namely Pto and Pto-like genes, may be considered as FHB candidate resistance genes underlying Qfhs.ndsu-3BS and may be used in future studies.

Keywords


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.


Acknowledgments

This research was supported by the Agricultural Biotechnology Research Institute of Iran (ABRII).

Altschul SF, Madden TI, Schaffer AA, Zhang JH, Zhang Z, Miller W, Lipman DJ (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-3402.
Anderson JA, Chao S, Liu S (2007). Molecular breeding using a major QTL for Fusarium head blight resistance in wheat. Crop Sci. 47: 112-119.
Bassam BJ, Caetano-Anolles G, Gresshoff PM (1991). Fast and sensitive silver staining of DNA in acrylamide gels. Anal Biochem. 196: 80-83.
Buerstmayr H, Steiner B, Hartl L, Griesser M, Angerer N, Lengauer D, Miedaner T, Schneider B, Lemmens M (2003). Molecular mapping of QTLs for Fusarium head blit resistance in spring wheat. II. Resistance to fungal penetration and spread. Theor Appl Genet. 107: 503-508.
Chen XM, Line RF, Leung H (1998). Genome scanning for resistance-gene analogs in rice, barley, and wheat by high-resolution electrophoresis. Theor Appl Genet. 97: 345-355.
Chen YP, Wang HZ, Cao AZ, Wang CM, Chen PD (2006). Cloning of a resistance gene analog from wheat and development of a codominant PCR marker for Pm21. JIPB. 48: 715-721.
Feuillet C, Schachermayr G, Keller B (1997). Molecular cloning of a new receptor-like kinase gene encoded at the Lr10 disease resistance locus of wheat. Plant J. 11: 45-52.
Guo PG, Bai GH, Li RH, Shaner G, Baum M (2006). Resistance gene analogs associated with Fusarium head blight resistance in wheat. Euphytica 151: 251-261.
Kosambi DD (1994). The estimation of map distances from recombination values. Ann Eugen. 12: 172-175.
Leister D, Ballvora A, Salamini F, Gebhardt C (1996). A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide application in plant. Nature Genet. 14: 421-429.
Lin F, Kong ZX, Zhu HL, Xue SL, Wu JZ, Tian DG, Wei JB, Zhang CQ, ZQ MA (2004). Mapping QTL associated with resistance to Fusarium head blight in the Nanda2419 × Wangshuibai population. I. Type II resistance. Theor  Appl Genet. 109: 1504-1511.
Liu S, Anderson A (2003). Targeted molecular mapping of a major wheat QTL for Fusarium head blight resistance using wheat ESTs and synteny with rice. Genome 46: 817-823.
Mardi M, Ghareyazie B, Buerstmayr H, Lemmens M, Moshrefzadeh N, Ruckenbauer P (2004). Combining ability analysis of resistance to head blight caused by Fusarium graminearum in spring wheat. Euphytica 139: 45-50.
Mardi M, Buerstmayr H, Ghareyazie B, Lemmens M, Mohammadi SA, Nolz R, Ruckenbauer P (2005). QTL analysis of resistance to Fusarium head blight in wheat using a ‘Wangshuibai’ derived population. Plant Breed. 124: 329-333.
Mardi M, Pazouki L, Delavar H, Kazemi MB, Ghareyazie B, Steiner B, Nolz R, Lemmens M, Buerstmayr H (2006). QTL analysis of resistance to Fusarium head blight in wheat using a ‘Frontana’-derived population. Plant Breed. 125: 313-317.
Saghai-Maroof MA, Soliman K, Jorgensen RA, Allard RW (1984). Ribosomal DNA spacer-length polymorphism in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA. 81: 8014-8018.
Shi ZX, Chen XM, Line RF, Leung H, Wellings CR (2001). Development of resistance gene analog polymorphism markers for the Yr9 gene resistance to wheat stripe rust. Genome 44: 509-516.
Utz HF, Melchinger AE (1996). PLABQTL: a program for composite interval mapping of QTL. J Agric Genomics. URL: http://www.ncgr.org/research/jag/papers96/paper196/indexp196.html.
Wang SP, Liu KD, Wang J, Zhang QF (1998). Identifying candidate disease resistance genes in rice by sequence homology and chromosomal locations. Acta Bot Sin. 40: 42-50.
Wenkai X, Mingliang X, Jiuren Zh, Fengge W, Jiansheng L, Jingrui D (2006). Genome-wide isolation of resistance gene analogs in maize ( Zea mays L.). Theor Appl Genet. 113: 63-72.
Xie C, Sun Q, Ni Z, Yang T, Nevo E, Fahima T (2004). Identification of resistance gene analogue markers closely linked to wheat powdery mildew resistance gene Pm31-short communication. Plant Breed. 124: 198-200.
Yan GP, Chen XM (2006). Molecular mapping of a recessive gene for resistance to stripe rust in barley. Theor Appl Gene. 113: 529-537.
Yan GP, Chen XM, Line RF, Wellings CR (2003). Resistance gene-analog polymorphism markers co-segregating with the YR5 gene for resistance to wheat stripe rust. Theor Appl Genet. 106: 636-643.
Yu YG, Buss GR, Maroof MA (1996). Isolation of a super family of candidate disease resistance genes in soybean based on a conserved nucleotide-binding site. Proc Natl Acad Sci USA. 93: 11751-11756.
Zhou J, Tang X, Martin GB (1997). The Pto kinase conferring resistance to tomato bacterial speck disease interacts with proteins that bind a cis- element of pathogenesis-related genes. EMBO J. 16: 3207-3218.