The Journal of Heredity 2001:92(1)
© 2001 The American Genetic Association 92:38-42
Identification of Wild and Cultivated Sunflower for Breeding Purposes by AFLP Markers
From the Dipartimento di Produzione; Vegetale e Tecnologie Agrarie, Università di Udine, Via delle Scienze 208, 33100 Udine, Itally (Quagliaro, Vischi, and Oliveri) and Institut of Genetics and Plant Breeding, University Agricultural, Lublin, Poland (Turka).
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Abstract |
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Amplified fragment length polymorphisms (AFLPs) represent one of the most powerful polymerase chain reaction (PCR)-based markers which enables one to discriminate single plants by DNA analysis. To date this technique has only been applied in cultivated sunflower to detect genetic diversity among oilseed inbred lines. In this article we report the use of AFLP markers to investigate the level of diversity within and between populations of Helianthus argophyllus collected in the Maputo area, Mozambique, both for taxonomic and breeding purposes. Three primer combinations gave the best results with 92 polymorphic fragments and were able to discriminate these wild endemic populations from H. annuus and from one of its interspecific hybrids. Most of the variation (71%) observed was within population, and the dendrogram based on shared fragments did not divide the H. argophyllus genotypes into distinct groups resembling different populations. Moreover the hybrid genotypes formed distinguishable subgroups with the cultivated sunflower genotype, confirming the suitability of this technique for taxonomic and phylogenetic studies. From a breeding point of view, although the 12 populations of H. argophyllus represent a new valuable genetic resource, only two of them possessed most of the variation observed, suggesting that they can be the most promising material for crossing with cultivated sunflower.
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Introduction |
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The study of phylogenetic and taxonomic relationships requires a flexible and reliable marker system to detect high levels of polymorphism. Traditionally a combination of morphologic and agronomic traits has been used to measure genetic diversity, but most of the vegetative characteristics are influenced by environmental factors, and show continuous variation and have a high degree of plasticity. In an attempt to overcome these problems, biochemical and molecular techniques have been used to monitor sunflower (Helianthus annuus L.) genetic variability and to solve taxonomic and phylogenetic problems. Helianthinin electrophoresis was used for identification of sunflower lines and varieties (Anisimova et al. 1991). Molecular markers such as restriction fragment length polymorphisms (RFLPs) were used to investigate polymorphism levels between inbred lines of cultivated sunflower (Berry et al. 1994) and helped to identify restorer and maintainer germplasm pools in cultivated sunflower (Gentzbittel et al. 1994). The ancestral relationships between sunflower inbred lines, wild populations, and land races were studied using randomly amplified polymorphic DNAs (RAPDs) (Arias and Reiseberg 1995) and RFLPs (Berry et al. 1995; Gentzbittel et al. 1994). Microsatellite markers detected polymorphism in hybrid lines of H. annuus L. (Brunel 1994). Recently, amplified fragment length polymorphism (AFLP) was developed by Vos et al. (1995), and combines the features of RFLP and polymerase chain reaction (PCR), does not require prior sequence characterization of the target genome, and is readily applicable to a wide variety of crops. The number of polymorphisms detected and the level of fingerprint reproducibility per reaction is much higher than revealed by RFLPs and RAPDs because of the simultaneous coverage of many loci in a single assay.
To date, this technique has been used in cultivated sunflower to detect genetic diversity among oilseed inbreed lines (Hongtrakul et al. 1997). In this article we report the use of AFLP markers to investigate the level of diversity within and between populations of H. argophyllus collected in the Maputo area of Mozambique for both taxonomic and breeding purposes.
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Materials and Methods |
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Plant Material
Ten open-pollinated populations of H. argophyllus, two half-sib H. argophyllus families, one population of cultivated sunflower and selected selfed material from (H. annuus x H. argophyllus) x H. annuus population were used (Table 1). H. argophyllus populations were sampled in Maputo province (Mozambique): eight of them from the coast over a distance of about 4 km, and the others from inland areas around the city of Maputo; backcross material was obtained from a garden in Maputo. Four to eight individuals were randomly sampled from the above-mentioned populations, resulting in a total of 89 genotypes.
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DNA Extraction
The genomic DNA of the 89 samples was extracted from young leaves and stored at -80°C, according to the Doyle and Doyle (1990) protocol. The DNA concentration was determined by 1% agarose gel electrophoresis and the final concentration was adjusted to 50 ng/µl.
Double Digestion of the DNA Solution and Ligation with the Adapters
AFLP analysis was carried out with the Keygene N.V. protocol (Zabeau and Vos 1993) with slight modifications. Genomic DNA (500 ng) was digested overnight at 37°C using 5 U MseI, 5 U EcoRI, and 8 µl 5x restriction-ligation buffer (50 mM Tris-acetate, 50 mM magnesium-acetate, 250 mM potassium-acetate, 25 mM DDT, 50 ng/µl BSA) in a final volume of 40 µl. The DNA fragments were ligated to EcoRI (5 pmol) and MseI (50 pmol) adapters after 3-h incubation at 37°C in the following solution: 1 µl ATP 10 mM, 1 µl buffer One for All 10x, 1 U T4 DNA ligase, and sterile distilled water to a final volume of 50 µl. The primer adapter sequences used are listed in Table 2.
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Preselective Amplification Reaction
This reaction allowed for a 16-fold reduction of the initial number of fragments generated, because each of the two primers have one selective nucleotide. The primers used (Table 2) were E01 with M02 or E02 with M01. Five microliters 1:10 diluted, digested, and ligated DNA solution was mixed with 30 ng primer (each), 0.4 U Taq gold polymerase (Perkin Elmer), 2 µl buffer 10x (Perkin Elmer), 0.2 mM dNTPs, in a final volume of 20 µl. The PCR reaction was carried out in a Perkin Elmer 9600 thermal cycler (Perkin Elmer) starting at 95°C for 10 min. DNA was amplified according to the following temperature profile: one cycle at 94°C for 30 s, 65°C for 30 s, 72°C for 60 s, 12 cycles with a 0.7°C annealing temperature decrease each cycle, and 23 cycles at 94°C for 30 s, 56°C for 30 s, and 72°C for 60 s. Preamplified DNA was checked by 1.5 % agarose gel.
Selective Amplification Reaction
This step allowed a 256-fold reduction of the preselected fragment number because each primer has two more selective nucleotides (Table 2). EcoRI primers (5 ng) were labeled in the following solution: 0.1 µl 
-33P] ATP, 0.1 U T4 kinase (Biolabs), 0.05 µl buffer 10x (Biolabs), and 0.26 µl sterile distilled water. The solution was incubated for 35 min at 37°C and then at 70°C for 10 min. Selective restriction fragment amplification was performed with 5 µl 1:10 diluted preamplified solution, 0.5 µl labeled EcoRI primer (10 ng/µl), 0.6 µl unlabeled MseI primer (50 ng/µl), 2 µl buffer 10x (Perkin Elmer), 1.6 µl dNTPs (2.5 mM), 0.4 U Taq gold polymerase (Perkin Elmer) in a final volume of 20 µl. For E01/M02 preamplified fragments, the following combinations of primers were used: E40/M47, M48, M51, M55, M62 and E41/M47, M48, M51, M55, M62, whereas E47/M33, M34, M37, M39 combinations were used for E02/M01 (Table 2). The amplification conditions were the same as described in the preselective amplification reaction.
Separation of Amplified Fragments
The PCR products obtained in the selective reaction were added to 20 µl loading buffer (98% formamide, 10 mM ETDA, 0.025% xylene cyanol, and 0.025% bromophenol blue) and incubated at 95°C for 5 min. A volume of 5.5 µl for each sample was loaded into a denaturing polyacrylamide gel. The gels were run at 58 W for 2 h, vacuum dried, and exposed to X-ray film for 2–4 days.
Data Analysis
The resultant band patterns obtained were manually scored for the presence or absence of bands. Because AFLPs are dominant markers, we assumed that each band position corresponded to a single character with two alleles, presence and absence of the band, respectively. Phenotypic frequencies of AFLP bands were established within populations (fi) and for all sets of genotypes (f). Shannon's index (Chalmers et al. 1992; Zhu et al. 1998) of within-population phenotypic diversity (Hs) was computed using the following formula:
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The average intrapopulation diversity,
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The genetic similarities were calculated from the AFLP data using the Dice similarity index (Dice 1945). The matrix values estimated the number of AFLP fragments shared (or not shared) between two individuals. Cluster analyses were based on similarity matrices obtained from average pairwise similarities for individuals grouped in populations (Chalmers et al. 1992; Williams et al. 1994) and from individual pairwise comparisons with the unweighted pair group method using arithmetic averages (UPGMA; Rohlf 1990). Relationships between populations were visualized as dendrograms; the cophenetic coefficients between the similarity matrix and the matrix of cophenetic values for these dendrograms were computed using appropriate subroutines of the NTSYS-pc package. The significance of cophenetic correlation observed were tested using the Mantel matrix correspondence test (Mantel 1967).
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Results |
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AFLP fragment size ranged from approximately 35 to 400 bp. Polymorphic fragments were distributed across the entire size range with the major portion (60%) between 101 and 200 bp. The remaining polymorphic fragments were shared equally through the fingerprint. E41/M51 (Pr1), E41/M47 (Pr2), and E41/M62 (Pr3) primer combinations produced the best result with 92 scoreable fragments and 30, 32, and 30 polymorphic fragments, respectively (Table 3). No other combinations were considered in this study because of their low number of scoreable bands.
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The frequency of a single AFLP marker in all populations ranged from 0.08 to 0.84; no population-specific markers were identified. However, all populations were identified on the basis of the lack of specific bands. The highest numbers of polymorphic fragments and the largest within-population variability were observed in populations arg5 (85 bands, Hs = 8.7) and arg11 (83 l200bands, Hs = 8.2). These two populations were sufficient to represent all bands/markers observed in 12 populations of H. argophyllus. Estimates of within-population variability are given in Table 4. A relatively small number of polymorphic bands with a lower variation were found in half-sib population arg3A (55 bands, Hs = 5.5) and arg8 (60 bands, Hs = 5.9).
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Only 29% of the overall variation could be assigned to interpopulation diversity (Table 5); most of the variation (71%) was observed within populations. The highest between-population variability was detected with the third primer combination. To complement the analysis, based on phenotypic frequencies, the Dice index was used to generate a similarity matrix. These individual similarity coefficients were reduced to population similarities by averaging individuals within populations (Table 6). The similarity matrix highlighted high within-population variability in the analyzed material. Populations an and (H. annuus x H. argophyllus) x H. annuus exhibited low similarity values (range 0.31–0.44 and 0.28–0.36, respectively) compared to all other populations of H. argophyllus. This suggested that they were phylogenetically distinct.
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The UPGMA dendrograms representing relationships between individuals and populations are shown in Figures 1 and 2, respectively. The cophenetic correlation coefficient used as a measure of goodness-of-fit for cluster analysis was very high for dendrogram clustering populations and high for individuals (r = 0.954 and r = 0.827, respectively).
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The dendrogram based on shared fragments (Figure 1) did not divide the H. argophyllus genotypes into distinct groups resembling the different populations. Generally genotypes were evenly distributed along the dendrogram, confirming big intrapopulation diversities. Nevertheless, the arg3A (used as control), arg4, and arg8 populations formed distinct subgroups. Moreover, the hybrid genotypes formed distinguishable subgroups with the cultivated sunflower genotypes, confirming the suitability of this technique for taxonomic and phylogenetic studies.
The UPGMA method revealed no distinct clusters (Figure 2). However, the an and anxarg populations were most distanced from the rest of populations being analyzed thus confirming results obtained using evaluation based on unreduced similarity values. Moreover, the two half-sib populations, arg3A and arg3B, used as a control, were closely linked when the neighbor joining method of clustering was used (not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The usefulness of AFLP technology to investigate genetic relationships has already been established for many species, such as rice (Zhu et al. 1998), lettuce (Hill et al. 1996), and maize (Pejic et al. 1998). It was found to be useful in analyzing variability between and within several wild populations of H. argophyllus: 8 of the 92 generated markers were neither present in the hybrid population nor in the an population of cultivated sunflower. This suggests the possibility of further increasing the gene pool diversity of cultivated sunflowers. The various statistical approaches used confirmed high within-population diversity in the examined material. The analysis was based on the number of shared bands which represents an estimator of relatedness between genotypes and their populations. According to what was found in barley by Waugh et al. (1997), we assumed that fragments with the same electrophoretic mobility were amplified from the same locus (homology). Alternatively the possibility that two or more bands of the same size are derived from two or more distinct loci (homoplasy) was disregarded in this contest, as Waugh et al. (1997) reported that it can occur with higher probability when distant taxonomic units are considered.
The validity of AFLP markers, although obtained with only three pairs of primers, has been clearly demonstrated in wild and domesticated sunflower species. Selected material coming from a backcross generation (anxarg) showed the smallest mean value of genetic similarity between individuals representing this group, followed by the open-pollinated H. annuus population (Table 6). Both populations were clearly distinguishable from all other H. argophyllus populations, which could not be related to sampling locations. This could be explained by considering the small area (about 20 km x 10 km) sampled and the fact that the material would have the same origin, because it was probably introduced during the slave trade. In this context, even the sampling areas (coastal or inland) and the half-family progeny subsets would have had little importance given the outcrossing species, the large presence of insects, and the high salinity in the groundwater.
From a breeding point of view, although the 12 populations of H. argophyllus represent a new valuable genetic resource, two of them (arg5 and arg11) possessed in themselves most of the variation observed, suggesting that they can be the most promising material for crossing with cultivated sunflower.
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Footnotes |
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Address correspondence to Massimo Vischi at the address above.
Corresponding Editor: J. Perry Gustafson
Received July 13, 2000
Accepted October 31, 2000
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References |
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