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a IFFIVE-CICVA, INTA. Camino 60 Cuadras Km 5 1/2 X5020ICA, Córdoba, Argentina
b ADVANTA SEMILLAS SAIC Ruta 226 Km 60,5 (7620) Balcarce, Buenos Aires, Argentina
* Corresponding author (slenard{at}infovia.com.ar)
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Abbreviations: CM, chlorotic mottling • DAS-ELISA, double antibody sandwich-enzyme linked inmunosorbent assay • ICP, isolated chlorotic pinpoint • MM, mild mosaic • SCM, severe chlorotic mottling • SCP, scarce chlorotic pinpoint • SuCMoV, Sunflower chlorotic mottle virus
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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SuCMoV infected plants usually present individual, brilliant yellow blotches, which later coalesce, reducing and distorting leaf morphology and producing plant stunting (Dujovny et al., 1998). Yield parameters such as plant height, stem and capitulum diameter, seed yield, and weight of 1000 seeds were significantly reduced by SuCMoV infections occurring at various ontogenetic stages (Lenardon et al., 2001).
Breeding for virus resistance is a simple way to manage natural virus infestations since it does not require specific cultural practices, which is important in a sustainable agricultural system. To our knowledge, no virus resistance source(s) for SuCMoV has been detected so far.
The objectives of this study were (i) to detect sources of resistance to SuCMoV by the screening of sunflower inbreeds and (ii) to map SuCMoV resistance genes. Molecular markers closely linked to this gene may be useful for selection in breeding programs (Young, 1996).
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Eighteen public sunflower inbred lines (DM2, HA61, HA89, HA124, HA335, HA342, HA372, HA383, HA821, HA850, HAR2, RHA265, RHA271, RHA274, RHA276, RHA295, RHA340, and RHA856) and 214 proprietary ones, representing an ample genetic base within cultivated H. annuus germplasm, were mechanically inoculated with SuCMoV. Seeds of all the lines were provided by Advanta Semillas, S.A.I.C. The three most resistant lines (L33, L74, L52) and a susceptible control were reevaluated on July 1999, to confirm prior results.
Artificial Inoculation and Phenotypic Scoring
A SuCMoV isolate maintained on sunflower plants in the greenhouse was used as the inoculum source. Infected leaves were ground in 0.01 M Na phosphate buffer, pH 7, containing 0.1% (w/v) Na2 SO3, and silicon carbide 600 mesh added as abrasive (0.25 g/10 mL slurry). Inoculum was applied on the vegetative stage V 1-2 of expanding sunflower leaves (Schneiter and Miller, 1981) with a high-pressure airbrush apparatus.
The plant virus infection(s) were evaluated 15 d after inoculation. Plant symptoms were classified as severe chlorotic mottling (SCM); chlorotic mottling (CM); mild mosaic (MM); isolated chlorotic pinpoint (ICP); very scarce, isolated, chlorotic pinpoint (SCP), and without any visible symptoms (R).
Virus Quantification by DAS-ELISA
Crude extracts from infected or healthy sunflower leaves were prepared by grinding tissues 1:5 (w/v) in the presence of extraction buffer [PBS + 0.05% (v/v) Tween 20 + 2% (w/v) polyvinylpyrolidone 40000]. The homogenate was centrifuged at 5000 g for 5 min and the supernatant was used for DAS-ELISA.
Wells in ELISA microtiter plates (Nunc Inter Med., Denmark) were coated with 100 µL of a 1:2000 dilution of SuCMoV specific rabbit IgG in carbonate buffer, pH 9.6. The secondary rabbit antibody was alkaline phosphatase-conjugated (1 mg/mL, Sigma, St. Louis MO) at a 1:2000 dilution.
DAS-ELISA was performed as described by Clark and Adams (1977), and the reactions were recorded at 405 nm with a DYNEX MRXII spectrophotometer (Dynateck, UK).
Effect of Viral Inoculation on Morphological Characteristics
The resistant line L33 and the susceptible line L2 were tested in hybrid combinations with the susceptible lines L1 and L3.
A trial with a randomized block design, three replications and two factors: (four hybrids) and treatments (inoculated vs. non inoculated) was performed in the 2000-2001 crop season. Each replication consisted of three rows with 20 plants in each one. Middle row plants were inoculated with the virus, or not inoculated according to the treatment. At the reproductive stage R6 (Schneiter and Miller, 1981), all plants in the central row were evaluated for virus symptoms development. Plant height, capitulum diameter, and width of the totally expanded leaves from the upper part were recorded in five plants per row. Data were analyzed by a two-way analysis of variance.
Mapping Population Development
The line with the highest SuCMoV resistance, detected in the previous experiments (L33), and a very susceptible one (L2) were crossed to obtain an F1 hybrid, and then self pollinated to produce an F2 population.
The parental lines, F1 and F2 plants were tested in two trials, one in the greenhouse, and the other one in the field using artificial inoculations.
Greenhouse Trial
A total of 186 F2 plants of the biparental cross (L2 x L33), and nine plants of each parental line and the F1 were sown in pots (three plants/pot) under the described conditions.
Field Trial
Two rows of each parental line, one row of F1 plants and 16 rows of F2 population (160 plants) were sown at Balcarce (Buenos Aires Province, Argentine) in the 1999-2000 crop season. Each row included 10 plants.
Molecular Markers and Linkage Analysis
DNA Extraction
Leaf tissues were collected from the young leaves of individual SuCMoV-inoculated plants. The DNA was isolated by a modified CTAB miniprep method described by Haymes (1996).
Briefly, the leaf samples were mechanically ground with a homogenizer in the presence of the extraction buffer [100 mM Tris-HCl pH 8; 1.4 M NaCl; 20 mM EDTA pH 8; 2% (w/v) hexadecyl-trimethyl-ammonium bromide (CTAB); 0.4% (v/v) β-mercaptoethanol. The samples were incubated for 30 min at 65°C in a water bath. Chloroform/octanol (24:1) was added, mixed by hand, and centrifuged at 14000 g for 15 min to separate the organic phase. The aqueous phase was recovered and transferred to a new tube that contained ethanol/sodium acetate [0.12 M NaAc, 96% (v/v) ethanol, pH 5.2]. The mixture was then incubated at room temperature for 15 min to allow DNA precipitation. Finally, the DNA was recovered by centrifugation and washed with 70% (v/v) ethanol, air dried, and resuspended in the TE buffer (Tris-HCl 10 mM, EDTA 1 mM, pH = 8).
Molecular Markers Analysis
Public and proprietary markers were screened for polymorphism detection. The MS markers correspond with Advanta proprietary microsatellites and the ORS markers correspond with public ones (Tang et al., 2002).
PCR reactions were performed in 20 µL of reaction mixture containing 2 µL 10x PCR buffer, 2.5 mM Mg2+, 0.2 mM each of dNTPs, 7.5 pmol of each primer, 0.7 units of Platinum Taq polymerase (Invitrogene Life Technologies, Carlsbad, CA, USA), and 20 ng of genomic DNA. The microsatellite procedure was described in Tang et al. (2002). PCR reactions were then performed in a PTC200 thermocycler (MJ Research, Waltham, MA, USA). The PCR products were separated by electrophoresis in agarose gels [1.5% (w/v) in TBE pH 8.3] or denaturing polyacrylamide gels [6% (w/v) acrylamide/bisacrylamide, 20:1, 8 M urea in TBE, pH 8.3]. Gels were stained with a SYBR Gold nucleic acid gel stain (Molecular Probes, Eugene, OR, USA) and visualized in a Fluor-S multimager (Biorad, Hercules, CA, USA).
One hundred eighty microsatellites and 40 proprietary indel markers were screened comparing the parental lines and progeny individuals to find polymorphic markers. Thirty-six microsatellite markers and four indel markers distributed on the 17 linkage groups of sunflower genome (Tang et al., 2002) were polymorphic between the parental lines.
The markers were scored in 186 F2 individuals from the cross L2 x L33, previously scored for SuCMoV reaction(s) in the greenhouse. The most probable order of the markers in the linkage map, and the distances between the resistance gene and the molecular markers, were determined by multipoint maximum likelihood estimations with the computer program Carthagene (Chabrier et al., 2000), using default parameters of LOD = 3 and a maximum Kosambi distance of 50 cM and the default algorithm. Permuting all possible orders and comparing likelihood confirmed the order of the markers.
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RESULTS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The partial resistance of L33, L52, and L74 was confirmed in a second experiment including visual evaluations and DAS-ELISA. The disease symptoms clearly differentiated the three tolerant lines from the susceptible (SCM) controls. Lines L52 and L74 were moderately susceptible and similar to each other, but less resistant than line L33 (ICP). The kinetics of viral particles accumulation in L33 was different from that of the susceptible line (Fig. 1) .
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All the F1 individual plants showed ICP scores similar to those of the resistant line suggesting a dominant behavior. For the F2 populations a 3:1 ratio was determined from the phenotypic analysis (75.4% resistant; 24.6% susceptible) considering plants with R, and ICP scores as resistant and those that presented CM and SCM scores as susceptible. These results suggest that a single dominant gene controls virus resistance in this population.
There was a significantly positive association between disease score and the DAS-ELISA absorbance values (Fig. 2) (Spearman's rank correlation rho = 0.6, p value < 2.2 10–16).
Marker Polymorphism and Linkage Analysis
Linkage analysis including molecular markers scores and phenotypic data (using the resistant and susceptible classification described above) belonging to F2 plants from the cross L33 x L2 detected a single locus for resistance located in Linkage Group 14, flanked by markers MS0022 (5 cM) and ORS307 (4 cM) (Fig. 3)
. We propose the name Rcmo-1 (Resistance to chlorotic mottle) for this locus.
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DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A single resistance gene with dominant effects was detected in L33 line. Dominant resistance is the most frequent type of resistance to plant viruses, with nearly 80% of the host–virus combinations analyzed displaying such disease-resistance combination (Provvidenti and Hampton, 1992; Khetarpal et al., 1998).
Resistance genes to downy mildew (caused by Plasmopara halstedii (Farl.) Berl. & De Toni in Sacc.) (Vear et al., 2000) and black rust (caused by Puccinia helianthi Schwein.) (Lawson et al., 1996, 1998) have been characterized and mapped in sunflower. Resistance gene analogs corresponding to TIR-NBS-LRR and non-TIR-NBS-LRR resistance genes (Gedil et al., 2001; Radwan et al., 2003) have been mapped in the same chromosome regions in Groups 8 and 13 where the downy mildew and black rust resistance genes were located. Resistance gene analogs linked to potyvirus resistance genes have been described in other crops like soybean (Gore et al., 2002). However, until now, no resistance gene analogs have been reported in the Rcmo-1 gene region. The markers detected in the present study provide an initial step to develop high resolution mapping populations that can be used to clone Rcmo-1 by a map-based approach.
In sunflower plants, leaves in the upper third intercept more than 90% of the total incident light (Aguirrezábal et al., 1996). A reduction in leaf area during the critical period for yield (flowering and post flowering period) will affect both the number and the weight of achenes and consequently might be related with yield reduction (Andrade and Sadras, 2000). When the L33 resistant line was tested in hybrid combinations with two susceptible lines, plant height and leaf width were not significantly reduced, while susceptible hybrids were severely affected. The detected level of resistance could be useful for agronomic purposes, and may contribute to reducing yield losses caused by this disease. Studies are currently being performed to determine the effect of the L33 resistance gene on yield.
The selection for this trait can be done by inoculation and direct phenotypic selection or by indirect selection with the flanking molecular markers, as was done for improving resistance to the potyviruses in other crops (Miklas et al., 2000).
Mapping of other resistance genes as those potentially present in Lines L52 and L74 could be an important next step toward a better SuCMoV control through genetic pyramiding. Pyramiding resistance genes can be accelerated with the availability of molecular markers and can be used to breed for a more durable resistance.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Received for publication June 9, 2004.
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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