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Published in Crop Sci 39:1456-1463 (1999)
© 1999 Crop Science Society of America
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Crop Science 39:1456-1463 (1999)
© 1999 Crop Science Society of America

CELL BIOLOGY & MOLECULAR GENETICS

Genetic Variability as Identified by AP-PCR and Reaction to Mid-Stem Infection of Sclerotinia sclerotiorum among Interspecific Sunflower (Helianthus annuus L.) Hybrid Progenies

H. Köhlera and W. Friedta

a Institute of Crop Science and Plant Breeding I, Faculty of Agriculture and Environment Preservation, Justus-Liebig-University, Ludwigstr. 23, D-35390 Giessen, Germany

wolfgang.friedt{at}agrar.uni-giessen.de


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 Materials and methods
 Results
 Discussion
 REFERENCES
 
Various diseases limit productivity in the majority of sunflower (Helianthus annuus L.) growing areas. Sunflower cultivars lack acceptable levels of resistance to Sclerotinia sclerotiorum (Lib.) de Bary, which is a major pathogen in sunflower production. Several wild Helianthus species are potential sources of genes for disease resistance and can be used in interspecific crosses to increase the genetic variability of cultivated sunflower. Progenies of interspecific hybrids between cultivated sunflower lines and Helianthus mollis Lam., H. decapetalus L., H. maximiliani Schrader, H. giganteus L., H. tuberosus L., and H. pauciflorus Nutt were tested for their mid-stem Sclerotinia reaction by an artificial infection method. Compared with the susceptible commercial hybrid check cv. Frankasol, interspecific hybrid progenies were selected that showed reduced Sclerotinia infection. It could be demonstrated by results of AP-PCR fingerprinting that interspecific hybrids can be a valuable tool for broadening genetic variability in sunflower. In total, 20 AP-PCR primers were used for the characterization of interspecific hybrid progenies. It was possible to discriminate between the H. annuus parents and their progenies, where an increased genetic variability resulting from the interspecific hybridizations was detectable. Results of an UPGMA cluster analysis showed that interspecific hybrid progenies which originate in the same cross combination share a common cluster. These lines exhibited a large genetic distance from the parental sunflower inbred lines and form their own distinct genetic pool.

Abbreviations: bp, base pair • kb, kilobases • AP-PCR, arbitrarily primed polymerase chain reaction • QTL, quantitative trait locus • RFLP, restriction fragment length polymorphism • SSR, simple sequence repeat • RAPD, random amplified polymorphic DNA • UPGMA, unweighted pair group method of arithmetic means


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
Materials and methods
 Results
 Discussion
 REFERENCES
 
THE GENUS Helianthus consists of 49 species, i.e., 13 annual and 36 perennials with different ploidy levels (Schilling and Heiser, 1981). In agriculture, only two species have been used as crop plants so far: Helianthus tuberosus L. (Jerusalem artichoke) and H. annuus L., the commercial sunflower, which can be used as an oilseed, confectionery, or an ornamental crop. Sunflower is one of the most important annual oil crops of the world. It's oil can be used for both human consumption, and as a raw material for oleochemistry. As in other cultivated crops, hybrid breeding in sunflower utilizes a comparatively narrow genetic base and the wild Helianthus germplasm seems to be useful for broadening the genetic variation (e.g., Korell et al., 1996a,b).

In temperate climates like central Europe, successful sunflower production is endangered by many fungal pathogens. Sclerotinia sclerotiorum (Lib.) de Bary is a major problem in sunflower production in central Europe's temperate and humid climate and persists for many years in the soil, with a very wide host range of about 225 plant genera (Gulya et al., 1997). On sunflower, it causes a basal stalk rot and wilt, head rot, midstem rotting, and breaking; the infection starts from the leaf axils or originates from a leaf infection (Sackston, 1992; Masirevic and Gulya, 1992). Losses may be severe, with nearly 100% loss in parts of affected fields or even entire fields under extreme circumstances. Cultivated sunflower lacks an acceptable level of resistance to Sclerotinia. However, several wild Helianthus species are potential sources of genes for disease resistance and can be used in interspecific crosses to increase the genetic variability of cultivated sunflower (Seiler, 1992; Skoric and Rajcan, 1992; Skoric et al., 1995; Köhler, 1997; Köhler et al., 1997). The conservation and exploitation of these resources is a permanent challenge for sunflower researchers. An embryo rescue technique has been used successfully to overcome incompatibilities between H. annuus and several wild species allowing researchers to produce a large number of interspecific hybrids (Dahlhoff, 1994; Friedt, 1992; Korell et al., 1996a,b; Kräuter et al., 1991).

Knowledge of genetic relationships in novel plant material derived from interspecific crosses is essential for breeding. Molecular techniques recently developed are highly specific tools to differentiate and classify genotypes. These modern tools complement the more classical methods which are based mainly on morphological, biochemical, and physiological traits and allow the determination of the degree of genetic similarity within populations of a species and between species and related genera. Additionally, the success of interspecific crosses can be verified by molecular methods (e.g., Kräuter et al., 1991). DNA fingerprinting techniques such as RFLPs, microsatellites (simple sequence repeats, SSR), RAPDs (random amplified polymorphic DNA), or AP-PCRs (Arbitrarily Primed PCR) have been shown to be useful tools for genotype characterization in numerous crop species including sunflower (e.g., Berry et al., 1994; 1995; Gentzbittel et al., 1992; Hongtrakul et al., 1997; Mösges and Friedt, 1994).

The objective of the present study was to characterize interspecific hybrids which were developed by an embryo rescue technique described elsewhere (Kräuter et al., 1991; Dahlhoff, 1994). Reaction to mid-stem S. sclerotiorum infection was tested by an artificial infection technique and AP-PCR markers were used as a molecular tool to characterize the interspecific hybrid progenies.


   Materials and methods
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
Results
 Discussion
 REFERENCES
 
Genetic Material
Inbred lines derived from interspecific hybrids that had been repeatedly self-pollinated and/or backcrossed were used to analyze the genetic variation and the Sclerotinia reaction of H. annuus genotypes (hybrids) and Helianthus wild species. Sclerotinia tests were carried out with three commercial sunflower hybrids and 29 interspecific progenies (Table 1) . The interspecific hybrids were produced in a previous work by means of the embryo rescue technique (Kräuter et al., 1991).


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  		Table 1 Sunflower material used for mid-stem Sclerotinia sclerotiorum testing

 
In addition, the perennial, wild species H. mollis (MOL Rh, MOL 1873), H. giganteus (GIG 1897), H. maximiliani (MAX 40), H. pauciflorus (PAUC), H. decapetalus (DEC Dijon), H. tuberosus (TUB 1705), and two sterile inbred lines of sunflower, HA89 and Baso, were used for molecular analyses.

Tests for Mid-Stem Sclerotinia sclerotiorum Reaction
Tests for Sclerotinia reaction were carried out by an artificial inoculation method which was done with slight modifications according to the stem test of Thuault and Tourvieille (1988) and Castano et al. (1993). For that purpose, sclerotia originating from the field station of Gross-Gerau (near Frankfurt/Main, Germany) were harvested from sunflower capitula after natural head infection. Inoculum was produced by placing one sterilized sclerotium in a Petri dish containing a potato-dextrose agar medium (PDA) for 6 d in a growth chamber at 15°C (12h/12h light/dark). One mycelial disk (1cm) was taken from the periphery of the colony and used for plant infection. The stems of sunflower plants were infected on an internode of the upper third of the stem approximately 3 wk before flowering. Three days after infection the size at the lesion was measured and continued for 5 wk with one measurement per week. The test was performed twice (I, II) in 1995 with 24 plants of each genotype (in one replication) under high humidity in a vinyl house.

Statistical Analysis of the Mid-Stem Sclerotinia Data
An analysis of variance was performed with the software program Plabstat (Utz, 1991).

DNA Extraction
Arbitrarily Primed-PCR analysis was carried out with genomic DNA extracted by a method according to Doyle and Doyle (1990) with some modifications. Sunflower leaf tissue was ground to a fine powder in liquid nitrogen. The frozen powder (2.5 g) was transferred to 15 mL hot hexadecyltrimethylammonium bromide (CTAB) extraction buffer [2% (w/v) CTAB, 100 mM Tris HCl (pH 8.0), 20 mM EDTA, 1% Na2S2O5 (w/v), 0.2% (v/v) ß-mercaptoethanol] and incubated at 65°C for 30 min with occasional shaking. An equal volume of chloroform:isoamylalcohol (24:1 v/v) was added and mixed by inversion, then centrifuged at 1600 g for 10 min. The aqueous phase was transferred to a fresh tube and reextracted with an equal volume of chloroform:isoamylalcohol and centrifuging at 2000 g for 10 min. The aqueous phase was removed and transferred to a fresh tube again and precipitated in 1.0 mL ammonium acetate (10 M), 1.0 mL sodium acetate (3 M, pH 5.5), and 2/3 VT 2-propanol (4°C). At last, the precipitated DNA was dried and resuspended in TE buffer. After treatment with RNase, the DNA concentration was measured with a fluorometer (Model TKO 100, Hoefer Scientific Instruments, Serva, Germany).

DNA Amplification
After an initial screen of 160 primers, i.e., short RAPD primers (10 bp) and longer AP-PCR primers (17–20 bp), 20 AP-PCR primers were used for the amplification of random DNA sequences (Table 2) . Primer selection was based on the information content, clarity, and reproducibility of banding patterns. Amplifications were carried out in a 25-µL volume containing 0.2 units Taq DNA polymerase Goldstar (Eurogentec Bel AS, Seraing, Belgium); 1x reaction buffer; 6 mM MgCl2; 0.1 mM each of dATP, dCTP, dGTP, and dTTP; 25 ng of template DNA and 0.2 µM of primer. The amplifications were performed using a PE 9600 DNA thermal cycler (Perkin Elmer Cetus, Norwalk, CT). The annealing temperature of the primers was divided into groups of similar melting-point temperatures, while temperatures were set at 10 to 12°C lower than the average melting point temperature (Itakura et al., 1984) (Table 2). The thermal cycler was programmed for a first denaturation step of 1 min at 94°C followed by 45 cycles of 1 min at 94°C, 1 min at primer specific temperature (Table 2), and 2 min at 72°C. Amplification products were resolved by gel electrophoresis in 2% (w/v) agarose in 0.5x TBE (89 mM Tris pH 8.0, 89 mM boric acid, 0.5 M EDTA). Molecular sizes of the amplification products were estimated by means of a 100-bp DNA ladder (Gibco BRL Life Technologies, Inc., Burlington, ON).


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  		Table 2 List of the primers used to characterize interspecific progenies in Helianthus

 
Analysis of Amplification Profiles
Amplification profiles of Helianthus genotypes were compared with each other and bands of DNA fragments were scored as present (1) or absent (0). The computer program RFLPscan (Scanalytics, Billerica, MA) was used to estimate the data for all the 20 primers. The similarity was then analyzed on the basis of the number of shared amplification products according to Nei and Li (1979). A dendrogram based on similarity coefficients was generated with the program NTSys (Version 1.80, Exeter Software, Setauket, NY) by means of the unweighted pair group method of arithmetic means (UPGMA).


   Results
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
Discussion
 REFERENCES
 
Genotypic Reaction to Mid-Stem Sclerotinia Infection
The interspecific hybrid progenies were tested by artificial stem infection induced by Sclerotinia mycelium cultured in vitro which caused mid-stem rotting. The infections resulted in lesions of variable length on the sunflower stems. A large extension of the lesions was interpreted as a susceptible reaction and a small extension was considered a resistant reaction. The lesion lengths on the stem were statistically evaluated by an analysis of variance. The measurement of the stem lesions from the first to the third week after infection gave no significant differences between the tested genotypes. After 4 wk, there were significant genotypic differences for Sclerotinia mid-stem infection (data not shown) and at the last scoring date (5 wk after artificial infection), there were significant genotypic differences for lesion extension on the stem (Table 3) . Some interspecific hybrid progenies exhibited significantly reduced mid-stem Sclerotinia infection, compared with the commercial hybrid Frankasol (#14) which was used as a susceptible check. The size of the lesions at the last scoring date, averaged over two experimental times, is given in Fig. 1 . Interspecific genotypes, represented by black bars, showed a significantly better level of resistance to Sclerotinia than Frankasol. The interspecific hybrid progenies #4 and #6 derived from crosses to the wild species H. mollis and the lines #24, #32, and #36, derived from the crosses to H. tuberosus, showed a degree of partial resistance to mid-stem Sclerotinia infection.


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  		Table 3 Analysis of variance for the size of lesions caused by artificial mid-stem infection of Sclerotinia sclerotiorum in 29 interspecific hybrid progenies and three commercial hybrids (two replications, 1995 I, II)

 


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  		Fig. 1 Genotypic reaction of sunflower interspecific progenies to Sclerotinia after artificial mid-stem inoculation. Data shown as the extension of stem lesions (cm) determined the last scoring date. Interspecific genotypes that showed a significantly lower average level of Sclerotinia infection than Frankasol (# 14) are represented by black bars. For description of the genotypes see Table 1

 
Molecular Analysis of Interspecific Hybrid Progenies
The molecular characterization of the novel sunflower germplasm derived from interspecific crosses was carried out by AP-PCR. The AP-PCR primers generated a higher degree of polymorphism in sunflower than did RAPD primers (data not shown) and resulted in distinct and clear patterns. In total, 20 AP-PCR primers were used for fingerprinting and their patterns resulted in a molecular range between 0.2 and 3.6 kb. Altogether 620 markers were scored with the 20 primers, of which AP07, AP09, AP12, AP17, and AP19 revealed most of the polymorphisms (Fig. 2) .



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  		Fig. 2 AP-PCR fingerprint of interspecific progenies (H. annuus x H. tuberosus); PCR carried out with the primer AP17. Lanes 2, 3: parents of the interspecific cross (HA89cms, H. tuberosus1705). Lanes 4 through 17: interspecific progenies that were backcrossed to H. annuus twice and self-pollinated three times (BC2S3). Lanes 1 and 18: 100-bp ladder (Gibco BRL, Life Science Technology)

 
The similarity matrix obtained after multivariate analysis by means of Nei and Li's coefficient is presented in Table 4 . These similarity coefficients were used to generate a dendrogram (Fig. 3) by UPGMA analysis to determine the grouping of the genotypes. In general, the degree of similarity ranged from 31.7% (H. tuberosus vs. H. decapetalus) to 96% (lines #3 vs. #8, both are progenies from a H. mollis cross). The wild species showed the lowest similarity with the other genotypes. Consequently, these species are easily distinguishable in the dendrogram because they do not cluster with either the interspecific hybrids or the H. annuus lines. The commercial sunflower hybrids Alphasol (#7), Frankasol (#14), and Sunking-256 (#1) also proved to be quite distinct from the interspecific hybrids and the wild species. As expected, the H. annuus sterile inbred lines `HA89' and `Baso' showed a closer similarity to the interspecific hybrids because they were used as female parents for the interspecific hybridizations.


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  		Table 4 Similarity matrix for Nei and Li's coefficient of seven Helianthus wild species, two sunflower inbred lines, three commercial sunflower hybrids and 29 interspecific progenies (pedigrees of these genotypes are given in Table 1)

 


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  		Fig. 3 Dendrogram generated from AP-PCR data determined by the unweighted pair group method of arithmetic means (UPGMA). Data of seven wild Helianthus species, two sunflower inbred lines, three sunflower commercial hybrids, and 29 interspecific progenies (#2–6, 8–13 and 15–36; the wild species that were used for the initial crosses are noted in parenthesis) were analyzed. The scale is based on Nei and Li's coefficient of similarity

 
The interspecific hybrid progenies (#2-6, 8-13, and 15-36) were also clearly distinguishable from each other. Lines which originate from the same interspecific cross form specific clusters, i.e., related lines, could be grouped into distinct genetic pools; this was especially true of the progenies of the hybrid H. annuus x H. tuberosus which are different from the rest of the interspecific hybrids. In the same way, the different genotypes derived from the interspecific crosses H. annuus x H. mollis and H. annuus x H. giganteus form separate groups. In spite of the fact that some of these lines were backcrossed twice with H. annuus and self-pollinated several times (Table 1), proportions of the alien genome(s) are still detectable and are highly specific for the respective wild species used for the interspecific hybridization.


   Discussion
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
 Discussion
REFERENCES
 
As in other cultivated crops, trait incorporation in elite sunflower germplasm has been restricted through breeding bottle-necks, included are (i) oilseed traits, (ii) self-compatibility and self-pollination, and (iii) hybrid seed production traits (Hongtrakul et al., 1997; Korell et al., 1992). Moreover, in commercial hybrid seed production, female lines with the male sterility inducing cytoplasm derived from the wild species H. petiolaris (Leclercq, 1969) are used exclusively. For these reasons, interspecific hybridization is an important tool for the creation of novel genetic and cytoplasmic variability (see, Kräuter et al., 1991; Korell et al., 1996a,b; Whelan, 1980, 1981).

Several Helianthus wild species are also potential sources of disease resistance genes. Consequently, interspecific hybridization presents the possibility of transferring these genes into sunflower (Seiler, 1992; Skoric and Rajcan, 1992; Skoric et al., 1995). Complete resistance to Sclerotinia has not been observed in sunflower so far. Gulya (1985) tested about 750 accessions including open-pollinated cultivars from 35 countries, several inbred lines and commercial hybrids, and wild H. annuus. None of the genotypes were immune to Sclerotinia wilt and no commercial hybrid was completely resistant, but some inbred lines and wild H. annuus collections have high levels of resistance. While the occurrence of wilt resistance in H. annuus may be rare, these results show that a certain level of resistance can be identified if enough diverse germplasm is evaluated. Partial resistance to S. sclerotiorum has been reported in several wild species. Skoric and Rajcan (1992) mentioned that H. maximiliani populations probably possess a degree of resistance to Sclerotinia white rot and Skoric et al. (1995) were able to find a moderate resistance in populations of H. mollis, H. maximiliani, and H. tuberosus.

Our results obtained by artificial mid-stem Sclerotinia inoculation confirm the existence of resistance genes in wild sunflower species and interspecific hybrids. Complete resistance to Sclerotinia stem infection was not observed, but several interspecific hybrid progenies derived from the wild parents H. mollis and H. tuberosus had higher resistance levels than the susceptible check cultivar. In accordance with this result, Skoric and Rajcan (1992) and Skoric et al. (1995) described some partial resistance to Sclerotinia infection in H. mollis and H. tuberosus. In addition, H. tuberosus was reported to be resistant to Sclerotinia head rot (Seiler, 1992). Later, Hammann et al. (1994, 1995) found varying degrees of partial Sclerotinia resistance in progenies of interspecific hybrids of cultivated sunflower and H. mollis, H. giganteus, H. tuberosus, and H. pauciflorus which were compared with European commercial hybrids of early, medium, and late maturity. According to our investigations and on the basis of related materials, the latter authors demonstrated that interspecific hybrids represent an extended genetic resource and are valuable sources for disease resistance breeding in sunflower.

By AP-PCR fingerprinting, we demonstrated in the present work that interspecific hybridizations are in general a very useful tool for the transfer of genomic portions from the wild species into the cultivated sunflower. In contrast to the use of RAPD primers (9–10 bp), AP-PCR was carried out with longer primers (>10 bp) (Welsh and McClelland, 1990). As described by Welsh et al. (1991) for maize (Zea mays L.), the AP-PCR is also a highly useful tool for genetic fingerprinting in sunflower. On the basis of our AP-PCR fingerprint results, it was possible to discriminate between the H. annuus parents and their progenies, where an increased genetic variability resulting from the interspecific hybridization was detectable (Fig. 3). The interspecific progenies which originated in the same cross combination each form a cluster. Interspecific lines showed a large genetic distance from the inbred sunflower lines and commercial hybrids and form a distinct genetic pool.

The low similarity of H. tuberosus with other genotypes could be due to the fact that it is a hexaploid species , in contrast to the diploid species H. annuus, H. mollis, H. giganteus, and H. maximiliani . The chromosome number of H. decapetalus may either be , and a hexaploid chromosome number of has been described for the wild H. pauciflorus (Rogers et al., 1982).

Biochemical and molecular markers have already been employed for germplasm characterization in sunflower. Isozyme markers were successfully used for the characterization of interspecific sunflower hybrids (Friedt et al., 1991; Dahlhoff et al., 1992; Köhler et al., 1992, 1994) and for the identification of anther culture-derived genotypes in sunflower (Nurhidayah et al., 1994, 1996; Friedt et al., 1996). Arias and Rieseberg (1995) used random amplified polymorphic DNA (RAPDs), while Berry et al. (1995) and Gentzbittel et al. (1992; 1995) applied restriction fragment length polymorphisms (RFLP) to check the relationships between sunflower genotypes. AFLPs were used for DNA fingerprinting of sunflower germplasm to evaluate the genetic diversity among oilseed inbred lines (Hongtrakul et al., 1997). Furthermore, different microsatellite motifs have been applied for analyzing genetic relationships in cultivated sunflower. For example, Dehmer and Friedt (1998) were able to clearly separate German sunflower materials and USDA lines by using different simple sequence repeats (SSRs).

Knowledge of genetic relationships of the basic breeding material is essential for sunflower breeders in planning parental line and hybrid development. Crosses between genetically divergent parents are expected to yield a higher degree of heterosis in hybrids and a larger genetic variance among progenies in subsequent selfing generations than crosses of closely related parents (Messmer et al., 1993). Interspecific hybrids represent a very valuable basic material for broadening the genetic variation in sunflower and molecular markers can help the breeder to characterize this novel material. As described above, the fingerprint data of the interspecific hybrid progenies are very helpful for the estimation of the genetic relationships, since most of the respective wild genome portion can be lost during backcrossing with sunflower lines. After interspecific hybridization, techniques such as AP-PCR markers can be used for monitoring the introgression of wild genome portions into sunflower material during the backcross procedure. By a direct comparison of the genetic similarity between the interspecific progenies and their recurrent sunflower parent, a selection of divergent pools with a high level of genetic distinctness should be possible.

For quantitative traits like Sclerotinia reaction, the use of molecular tools will be essential in the transfer of (a) defined (set of) gene(s) into the genome of the cultivated sunflower without transferring the undesirable traits of the wild genome. However, we could not identify (data not shown) a significant correlation between the portion of the wild species' genome in the interspecific hybrid progenies, which was calculated on the basis of AP-PCR fingerprint data, and their Sclerotinia reaction. However, it is possible that by the application of genetic markers for S. sclerotiorum resistance quantitative trait loci (QTL), the number of Sclerotinia resistance tests can be reduced. In cultivated sunflower, molecular markers for Sclerotinia resistance QTL (leaf resistance and capitulum resistance loci) have been developed (Mestries et al., 1998). These markers will be a very helpful in Sclerotinia resistance breeding in sunflower if they can be used successfully in other populations.


   ACKNOWLEDGMENTS
 
We thank C. Löwer, S. Schreiner, A. Runkel, K. Striedelmeyer, T. Hain, and M. Tolksdorf for excellent technical assistance and Drs. L. Brahm and M. Korrell for their help with the Sclerotinia test. We are also grateful to the German Association of Private Plant Breeders (GFP), Bonn, for financial support of the research.

Received for publication September 11, 1998.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 Materials and methods
 Results
 Discussion
 REFERENCES
 




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