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Instituto de Agricultura Sostenible (CSIC). Apartado 4084. E-14080 Córdoba, Spain
* Corresponding author (bperez{at}cica.es)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: HPLC, high-performance liquid chromatography • INDEL, insertion-deletion polymorphisms • SSR, simple sequence repeats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Conventional sunflower seeds mainly contain alpha-tocopherol, which accounts for more than 900 g kg–1 total tocopherols. Beta- and gamma-tocopherol can be present in sunflower seeds, usually in amounts below 20 g kg–1 of the total tocopherols (Demurin, 1993; Dolde et al., 1999). Sunflower germplasm with modified tocopherol profile has been developed. Demurin (1993) reported the line LG-15, with increased concentration of beta-tocopherol (500 g kg–1 tocopherols), and the line LG-17, with increased concentration of gamma-tocopherol (950 g kg–1 tocopherols), both of them developed from segregating accessions identified in the evaluation of a germplasm collection. Genetic characterization of both lines concluded that the increased levels of beta-tocopherol were produced by recessive alleles at the Tph1 locus, whereas the increased levels of gamma-tocopherol were the result of recessive alleles at the Tph2 locus (Demurin et al., 1996). Also through the evaluation of the natural variability existing in germplasm collections, Velasco et al. (2004a) developed the line T589, with a beta-tocopherol content above 300 g kg–1 total tocopherols, and the line T2100, with a gamma-tocopherol content above 850 g kg–1. Velasco and Fernández-Martínez (2003) reported the presence of recessive alleles at a single locus underlying each of the modified tocopherol profiles, i.e., the increased beta-tocopherol concentration in seeds of T589 and the high gamma-tocopherol content in seeds of T2100. Comparative genetic studies concluded that tph1 alleles were present in both LG-15 and T589 lines (Demurin et al., 2004; Vera-Ruiz et al., 2005), and tph2 alleles were present in both LG-17 and T2100 lines (Demurin et al., 2004).
Additional variation for gamma-tocopherol content was created in sunflower by using chemical mutagenesis (Velasco et al., 2004b). The authors isolated the lines IAST-1 and IAST-540, with gamma-tocopherol content above 850 g kg–1 total tocopherols. No comparative genetic studies have been conducted to determine whether the high gamma-tocopherol lines developed by mutagenesis are allelic to those developed through germplasm evaluation.
Recent advances in molecular marker technologies in sunflower, especially the development of public SSRs (microsatellites) (Tang et al., 2002), SNPs (single nucleotide polymorphisms) (Lai et al., 2005), and integrated genetic linkage maps (Gedil et al., 2001; Yu et al., 2003; Lai et al., 2005) have made possible the genetic mapping and dissection of quantitative and qualitative traits in this crop and the application of this technology to sunflower breeding. Genetic mapping of tocopherol biosynthesis genes and identification of molecular markers linked to them would provide important tools for increased selection efficiency and for investigating the function and organization of these genes. Currently, only the Tph1 gene, conferring increased beta-tocopherol content to sunflower seeds, has been mapped in the sunflower genetic map (Vera-Ruiz et al., 2006). This gene mapped to the upper end of linkage group 1 and cosegregated with the SSR markers ORS1093, ORS222, and ORS598.
The objectives of the present research were (i) to conduct a comparative genetic analysis of the four sources of high gamma-tocopherol developed so far in sunflower, (ii) to identify PCR-based molecular markers linked to the Tph2 gene controlling gamma-tocopherol accumulation in sunflower seeds, and (iii) to map Tph2 in the sunflower genetic map.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Genetic Study
Twenty-four half seeds of HA89, CAS-12, LG-17, T2100, IAST-1, and IAST-540 were nondestructively analyzed for tocopherol profile as described below, germinated and planted in pots in a field screenhouse in spring 2003. Plants of T2100, used in all cases as females, were crossed with plants of LG-17, IAST-1, and IAST-540. Plants of CAS-12, used as females, were crossed with plants of IAST-540. Crossing was done by emasculating florets of the female parent followed by pollination of their stigmas with pollen from the male parent. Half seeds of the parents as well as F1 half-seeds were analyzed for tocopherol profile. F1 and parent half seeds were sown in September 2003 and the corresponding plants were grown in the greenhouse. F1 plants were self-pollinated to obtain the F2 generation.
F2 half seeds from one (T2100 x LG17; CAS-12 x IAST-540) to four (rest of the crosses) F1 plants were analyzed for tocopherol profile. Forty-eight F2 half seeds per F1 plant were analyzed in crosses involving high gamma-tocopherol parents, whereas 294 F2 half seeds from a single F1 plant were analyzed in the cross CAS-12 x IAST-540. F2 half-seeds from the latter cross were germinated and the corresponding plants were transplanted to the field in spring 2004. Germination was low in this population, which resulted in a population of 145 F2 plants. F2 plants were selfed and ninety of them each produced more than 12 F3 seeds, which was the minimum number of seeds used for genotypic classification of the F2 individuals. Twelve to twenty-four F3 seeds from each of the 90 F2 plants were analyzed for tocopherol profile. F2 individuals were classified as Tph2Tph2 if their F3 seeds had a uniform low gamma-tocopherol content (<20 g kg–1 total tocopherols), Tph2tph2 if their F3 seeds segregated for low and high (>850 g kg–1 total tocopherols) gamma-tocopherol content, and tph2tph2 if their F3 seeds showed a uniform high gamma-tocopherol content.
Bulked Segregant Analysis
Two fully expanded leaves were cut from each of the 145 F2 plants from the mapping population CAS-12 x IAST-540 and frozen at –80°C. The leaf tissue was lyophilized and ground to a fine powder in a laboratory mill. DNA was isolated from ground leaf tissue from each F2 plant as described in Berry et al. (1995). DNA was also isolated from three plants of the CAS-12 and IAST-540 parents. For bulked segregant analysis (Michelmore et al., 1991), two bulks were constructed by pooling aliquots (20 µL) of DNA from two sets of individuals with contrasting genotypes. The low gamma-tocopherol bulk was made up from 12 F2 individuals classified as Tph2Tph2, and the high gamma-tocopherol bulk was constructed from 12 individuals classified as tph2tph2. Homozygosity of F2 individuals included in the bulks was verified through the analysis of their respective F3 seeds. Two replicate samples of each bulk and the parental lines were screened with a genome-wide framework of 95 sunflower SSRs (Tang et al., 2003). For SSRs analyses, PCRs were performed as described by Pérez-Vich et al. (2004), and the amplification products were resolved on 3% (w/v) Metaphor (BMA, Rockland, ME) agarose gels in 1x TBE buffer with ethidium bromide incorporated in the gel.
Linkage between Tph2 and the SSR markers polymorphic between the low gamma-tocopherol and the high gamma-tocopherol bulks was verified by genotyping these SSR markers on the 145 F2 individuals from CAS-12 x IAST-540. The significance of each marker's association with the gamma-tocopherol content was determined by one-way analysis of variance (ANOVA) using the statistical package SPSS v 12.0 (SPSS for Windows; SPSS Inc., Chicago, IL), with marker genotypes being classes. Additionally, linkage of these markers and Tph2 was also verified by running a preliminary linkage analysis with MAPMAKER/EXP v 3.0b (Whitehead Institute, Cambridge, MA; Lander et al., 1987) using segregation data from the markers and Tph2. The genotypes for the Tph2 gene were inferred from gamma-tocopherol phenotypes in F2 and F3 seeds. On the basis of the F3 analyses, F2 plants were classified as Tph2Tph2, Tph2tph2, or tph2tph2 as described above. F2 individuals not producing the minimum number of seeds for F3 analyses (55 of a total of 145), were classified as Tph2__ if they had a low F2 gamma-tocopherol content (<20 g kg–1) and tph2tph2 if they had a high F2 gamma-tocopherol content (>850 g kg–1). Linkage was considered significant if the LOD score was >8.0. For consideration of the positions of the SSR marker loci relative to the target locus Tph2, linkage distances were calculated as two-point data.
F2 SSR Genotyping, Map Construction, and Tph2 Mapping
Once the Tph2 linkage group location was identified, all SSR markers known to map to the same linkage group (Tang et al., 2002, 2003; identified by prefixes ORS and CRT), excluding those already used for BSA, were screened for polymorphisms between the parental lines CAS-12 and IAST-540 to construct a complete genetic map including the Tph2 gene. Additionally, INDEL (insertion-deletion polymorphisms) markers mapping to the same linkage group were also used, and they are identified by ZVG prefixes (Yu et al., 2003). Primer sequences from nonpublished markers were kindly provided by Dr. S.J. Knapp (Center for Applied Genetic Technologies, University of Georgia, Athens, Georgia) and Dr. A.J. Leon (Advanta Seeds, Buenos Aires, Argentina). SSR marker analyses were performed as described above. INDEL analyses were performed following Yu et al. (2003).
The SSR and INDEL polymorphic markers were then genotyped in the 145 F2 individuals from CAS-12 x IAST-540, and a linkage map including Tph2 was constructed with MAPMAKER. The genotypes for the Tph2 gene were deduced as described above, and mapped accordingly. Two-point analysis was used to group all SSR marker loci and Tph2 at a LOD score of 4 and a maximum recombination frequency of 0.35. Three-point and multi-point analyses were used to determine the order and interval distances between the markers. The map distances, expressed in centimorgans (cM), were calculated by means of the Kosambi mapping function. Linkage group maps were drawn by the MapChart software (Voorrips, 2002). Chi-square analyses were performed on each locus to detect deviations from the expected Mendelian ratios for codominant (1:2:1) or dominant (3:1) markers.
A regression interval mapping analysis by the PLABQTL 1.1 software (Utz and Melchinger, 1996) was performed using the genetic map constructed to assess the effect of Tph2 on the tocopherol content. For this analysis, a new genetic map was constructed removing Tph2 segregation data and calculating map distances using the Haldane mapping function. The phenotypic data consisted of trait values (gamma-tocopherol content) for each F2 half-seed. The genetic map was scanned for the presence of QTLs at a LOD threshold of 3.0 at every 2.0-cM interval. Gene action was tested by fitting QTLs to dominant and additive genetic models.
Analysis of Tocopherols by High-Performance Liquid Chromatography
The analysis of tocopherol profile followed the method of Goffman et al. (1999). Half seeds were placed into 10-mL tubes with 2 mL iso-octane. The half seeds were then crushed with a stainless steel rod as finely as possible. The samples were stirred and extracted overnight at room temperature in darkness (extraction time about 16 h). After extraction, the samples were stirred again, centrifuged, and filtered. Five microliters of the extract were analyzed by HPLC using a fluorescence detector at 295-nm excitation and 330-nm emission and iso-octane/tert-butylmethylether (94:6) as eluent at an isocratic flow rate of 0.8 mL min–1. Chromatographic separation of the tocopherols was performed on a LiChrospher 100 diol column (250- x 2-mm I.D.) with 5-µm spherical particles, connected to a silica guard column (LiChrospher Si 60, 5- x 4-mm I.D.). The peak areas of the individual tocopherols were corrected according to their previously calculated response factors, which follow: alpha-tocopherol = 1.0; beta-tocopherol = 1.80; gamma-tocopherol = 1.85; delta-tocopherol = 2.30. The relative content of individual tocopherols in a single half seed was calculated based on corrected peak areas and expressed as g kg–1 of the total tocopherols present in the half seed.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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F1 seeds from crosses of LG-17, IAST-1, and IAST-540 with T2100 had a uniformly high gamma-tocopherol content of 947 ± 32 g kg–1 (T2100 x LG-17), 944 ± 23 g kg–1 (T2100 x IAST-1), and 941 ± 21 g kg–1 (T2100 x IAST-540). These results were confirmed in the analysis of F2 seeds, which also showed high gamma-tocopherol contents of 960 ± 25 g kg–1 (T2100 x LG-17), 970 ± 34 g kg–1 (T2100 x IAST-1), and 980 ± 16 g kg–1 (T2100 x IAST-540). Demurin et al. (2004) evaluated the F1 from a cross between the line VK 175, a line with tph2tph2 genotype derived from LG-17, and T2100, concluding that T2100 was allelic to tph2. The results of the present research suggested that tph2 alleles are present in the four lines.
The line IAST-1 was selected from a population that exhibited a wide segregation for gamma-tocopherol content, from zero to 845 g kg–1 total tocopherols, including intermediate levels (Velasco et al., 2004b). This had not been observed in the other three high gamma-tocopherol lines, which were selected from populations that only showed segregation for low and high gamma-tocopherol content but not for intermediate values (Demurin, 1993; Velasco et al., 2004a, 2004b). The different segregation pattern in the population from which IAST-1 was selected initially suggested that the line might carry an allele different to tph2 (Velasco et al., 2004b). This view has been discarded in the present research. However, the occurrence of intermediate levels of gamma-tocopherol (between 50 and 850 g kg–1) in the original population from which IAST-1 was isolated cannot be completely explained taking into account only the presence of tph2 alleles, and its characterization will require additional specific research.
Molecular Mapping of the Tph2 Gene
Seeds of the line CAS-12 had a low gamma-tocopherol content of 5 ± 2 g kg–1. Seeds of the line IAST-540 had a high gamma-tocopherol content of 946 ± 32 g kg–1. F1 seeds from the CAS-12 x IAST-540 cross exhibited a low gamma-tocopherol phenotype of 12 ± 3 g kg–1, which is in agreement with previous reports on the recessive character of the trait (Demurin et al., 1996; Velasco and Fernández-Martínez, 2003). F2 seeds segregated following a bimodal distribution that was not significantly different (
2 = 2.40, p = 0.12) from a 3:1 (low: high gamma-tocopherol content) ratio (Fig. 1
), which indicated segregation of a single, recessive gene. The analysis of 90 F2:3 families allowed the classification of F2 genotypes into three classes, characterized by uniformly low gamma-tocopherol content (n = 23; genotype Tph2Tph2), segregating for gamma-tocopherol content (n = 53; Tph2tph2), and uniformly high gamma-tocopherol content (n = 14; tph2tph2). Such a distribution fit the expected 1:2:1 segregation ratio (2 = 4.60, P = 0.10) that confirms monogenic inheritance of gamma-tocopherol content in IAST-540. According to the allelic study described above, the altered locus in IAST-540 is the Tph2 locus reported by Demurin et al. (1996).
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All ORS-SSR, CRT-SSR, and ZVG-INDEL markers known to map to LG 8 (Tang et al., 2002, 2003; Yu et al., 2003), excluding those already used for BSA, were screened for polymorphisms between CAS-12 and IAST-540 to construct a complete linkage map of LG 8. Two codominant (ORS243, and CRT35) and four dominant (ORS830, ORS312, ORS599, and ZVG35) marker loci were then genotyped on the 145 F2 individuals from the CAS-12 x IAST-540 population. Linkage analysis was performed, including segregation data from Tph2. All markers were grouped together. LG 8 comprised 9 marker loci, including the Tph2 gene, and was 44.6 cM long (Fig. 3 ). The locus order for the SSR markers and the reference linkage maps (Tang et al., 2002, 2003) was identical, except for the ORS456 locus. The Tph2 gene mapped 26.9 cM downstream from the upper end of LG 8, between markers ORS312 and ORS599. The ORS312 and the ORS599 markers were 3.6 cM proximal and 1.9 cM distal, respectively, of the Tph2 locus (Fig. 3).
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Other genes controlling tocopherol biosynthesis have been mapped in sunflower. The Tph1 gene determining increased beta-tocopherol content in sunflower lines LG-15 and T589 (Demurin et al., 2004) was mapped to LG 1 of the sunflower genetic map (Vera-Ruiz et al., 2006). The results of the present research demonstrated that both the Tph1 and the Tph2 genes will segregate independently, since they are located in different linkage groups, and the identification of molecular markers linked to them provides an efficient system to select the tph1tph1tph2tph2 genotype. Recombination of tph1 and tph2 alleles produces novel tocopherol profiles of great potential value for sunflower oil quality. Thus, Demurin et al. (1996) reported the occurrence of 220 g kg–1 delta-tocopherol in segregants from the cross between the line LG-15 (tph1tph1) and the high gamma-tocopherol line LG-17 (tph2tph2), whereas Velasco et al. (2004b) reported levels of 700 g kg–1 beta-tocopherol and 580 g kg–1 delta-tocopherol, respectively, in two lines developed from the cross between the line T589 (tph1tph1) and the high gamma-tocopherol line IAST-1 (tph2tph2).
The development of different combinations of fatty acid and tocopherol profiles for specific end uses of the oil is now an interesting possibility in sunflower, since a wide range of fatty acid and tocopherol profiles are available (Fernández-Martínez et al., 2004). For that purpose, absence of linkage between genes controlling fatty acid and tocopherol profiles is desired. So far, only the Es3 gene associated with increased stearic acid levels in the CAS-14 mutant has been located on LG 8 of the sunflower genetic map (Pérez-Vich et al., 2006). This gene mapped between the ORS243 and the ORS1161 markers, and genetic distance between Es3 and Tph2 was estimated to be 11.5 cM. Even though this distance is large enough to obtain recombinants es3es3tph2tph2 expressing both an increased stearic acid and gamma-tocopherol content, breeding for this phenotype would be easier with other sources of high stearic acid content determined by genes located at different linkage groups, for example, CAS-3 with a major gene located at LG 1 (Pérez-Vich et al., 2002).
Alpha-tocopherol is synthesized from gamma-tocopherol by a methylation reaction mediated by the enzyme gamma-tocopherol methyl transferase, which also mediates the conversion of delta- to beta-tocopherol (DellaPenna, 2005). The gene encoding the enzyme has been cloned in Arabidopsis through a genomics-based approach and overexpressed in the Arabidopsis genome, which led to a drastic alteration in tocopherol profile, from 50 g kg–1 alpha-tocopherol and 950 g kg–1 gamma-tocopherol to 950 g kg–1 alpha-tocopherol and 50 g kg–1 gamma-tocopherol (Shintani and DellaPenna, 1998). Such an alteration of tocopherol profile is of similar magnitude but opposite direction to that occurring in the sunflower germplasm with high gamma-tocopherol content carrying tph2 alleles, which suggests that gamma-tocopherol methyl transferase activity might be altered in this germplasm. The location of the Tph2 gene in the sunflower genetic map and the identification of molecular markers associated with it opens up the possibility of testing this hypothesis through map-based cloning or candidate gene strategies.
In summary, the present research concluded that the four sources of high gamma-tocopherol content identified so far in sunflower are allelic to each other, with recessive alleles at the Tph2 locus determining the trait. The gene has been mapped to LG 8 of the sunflower genetic map and molecular markers flanking the gene have been identified, which will facilitate marker-assisted selection in breeding programs focused on introgressing the trait into elite germplasm. Additionally, the results of the present research provide a basis for determining the function of the Tph2 gene in the tocopherol biosynthesis pathway.
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ACKNOWLEDGMENTS |
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Received for publication October 25, 2005.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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kori
, and D. Karlovic. 1996. Genetic variability of tocopherol composition in sunflower seeds as a basis of breeding for improved oil quality. Plant Breed. 115:33–36.[CrossRef]
ipspwww/soft.html; verified 12 July 2006.
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