Journal of Heredity Advance Access originally published online on May 22, 2008
Journal of Heredity 2008 99(6):593-597; doi:10.1093/jhered/esn041
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Genetic and Linkage Analysis of Purple–blue Flower in Soybean
From the National Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518 Japan (Takahashi); and the University of Tsukuba, 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518 Japan (Takahashi, Matsumura, Oyoo, and Khan)
Address correspondence to R. Takahashi at the address above, or e-mail: masako{at}affrc.go.jp.
Flower color of soybean is primarily controlled by genes W1, W3, W4, Wm, and Wp. In addition, the soybean gene symbol W2, w2 produces purple–blue flower in combination with W1. This study was conducted to determine the genetic control of purple–blue flower of cultivar (cv). Nezumisaya. F1 plants derived from a cross between Nezumisaya and purple flower cv. Harosoy had purple flowers. Segregation of the F2 plants fitted a ratio of 3 purple:1 purple–blue. F3 lines derived from F2 plants with purple–blue flowers were fixed for purple–blue flowers, whereas those from F2 plants with purple flowers fitted a ratio of 1 fixed for purple flower:2 segregating for flower color. These results indicated that the flower color of Nezumisaya is controlled by a single gene whose recessive allele is responsible for purple–blue flower. Complementation analysis revealed that flower color of Nezumisaya is controlled by W2. Linkage mapping revealed that W2 is located in molecular linkage group B2. Sap obtained from banner petals of cvs. with purple flower had a pH value of 5.73–5.77, whereas that of cvs. with purple–blue flower had a value of 6.07–6.10. Our results suggested that W2 is responsible for vacuolar acidification of flower petals.
Flower color of soybean (Glycine max [L.] Merr.) is primarily controlled by 5 genes (W1, W3, W4, Wm, and Wp) (reviewed by Palmer et al. 2004). The W1 gene has a pleiotropic effect on flower and hypocotyl color: Soybean cultivars having purple/white flower color have purple/green hypocotyls (Takahashi and Fukuyama 1919). Chromatographic experiments suggested that W1 is responsible for the formation of flavonoids with 3', 4', 5' hydroxylation pattern (Buzzell and Buttery 1982; Buzzell et al. 1987) suggesting that W1 encodes a flavonoid 3'5'-hydroxylase (F3'5'H). Zabala and Vodkin (2007) cloned soybean F3'5'H gene and confirmed that W1 encodes F3'5'H.
W3 and W4 alleles have epistatic effects: Under W1 genotype, soybean genotype with W3W4 has dark purple, W3w4 has dilute purple or purple throat, w3W4 has purple, and w3w4 has near white flowers (Hartwig and Hinson 1962). Restriction fragment length polymorphism of dihydroflavonol 4-reductase (DFR) gene cosegregated with flower color variants in an F2 population segregating for the W3 locus (Fasoula et al. 1995). Low transcript levels or abnormal transcript products of DFR2 gene were associated with mutation of W4 gene (Palmer RG, personal communication, 2007). These results suggested that W3 and W4 might encode DFR.
A Harosoy mutant (T235) with magenta flower was found in Urbana, IL, in 1957 (Buzzell et al. 1977). Buttery and Buzzell (1976) revealed that magenta flower allele (wm) is associated with low amounts of flavonol glycosides in leaves. Takahashi et al. (2007) cloned cDNA of flavonol synthase and revealed that Wm encodes flavonol synthase. A pink flower mutant was found in 1989 (Stephens and Nickell 1991). Genetic analysis revealed that pink flower is controlled by a recessive allele at the Wp locus (Stephens and Nickell 1992). cDNA microarray suggested that Wp corresponds to flavanone 3-hydroxylase gene (Zabala and Vodkin 2005).
Nagai (1926) crossed a white flower cultivar (cv). Shakujou with a purple flower cv. Nakaide. The F2 plants segregated into 9 purple:3 purple–blue:4 white flowers. Based on these results, Matsuura (1933) assigned the gene symbol W2, w2 produces purple–blue flower in combination with W1. However, progeny tests to ascertain this genetic model have not been conducted. Subsequently, a review article defined W2 as a gene that produces intense-purple flower in combination with W1 (Weiss 1949). Takahashi (unpublished results, 2004) repeated the cross between cvs. Shakujou and Nakaide and observed that the 77 F2 plants segregated into 36 purple:20 purple–blue:21 white flower in agreement with the previous results (
2 = 3.54, 0.1 < P < 0.2) of Nagai (1926). A line fixed for blue–purple flower was developed from the cross and designated as w2-20. Takahashi further found that the Japanese landrace Nezumisaya has similar purple–blue flower color and that another Japanese landrace Yogetsu-1 segregated into plants with purple and purple–blue flowers. Purple–blue flowered plants of Yogetsu-1 were selfed and fixed for purple–blue flower. The line was designated as Yogetsu-1-blue.
Iwashina et al. (2007, 2008) analyzed the flavonoids in flower petals of soybean. The primary anthocyanins in purple flower cultivars were malvidin 3,5-di-O-glucoside, delphinidin 3,5-di-O-glucoside, petunidin 3,5-di-O-glucoside, and delphinidin 3-O-glucoside. Iwashina et al. (2007, 2008) also identified 8 flavonol glycosides, kaempferol 3-O-gentiobioside, kaempferol 3-O-rutinoside, kaempferol 3-O-glucoside, kaempferol 3-O-glycoside, kaempferol 3-O-rhamnosylgentiobioside, kaempferol 7-O-glucoside, kaempferol 7-O-diglucoside, and quercetin 3-O-gentiobioside and 1 dihydroflavonol, aromadendrin 3-O-glucoside in purple flowers. No anthocyanins were detected in Clark near-isogenic lines for flower color genes, Clark-w1 (white) and Clark-w4 (near white), whereas a trace amount was detected in Clark-W3w4 (dilute purple).
Blue flower color generally depends on the production of appropriate anthocyanin pigments like delphinidin-derived anthocyanins, self-association, intra- or intermolecular copigmentation, association with metal ions, and the vacuolar pH (Goto and Kondo 1991; Davies and Schwinn 1997). In cornflower, blue color is produced by a tetrametal complex consisted of 6 molecules each of cyanidin-type anthocyanin and flavone, with 1 ferric iron, 1 magnesium, and 2 calcium ions (Shiono et al. 2005). Iwashina et al. (2008) analyzed the flavonoids in purple–blue flowers of Nezumisaya, Yogetsu-1-blue, and w2-20. Flavonoids were largely similar to those obtained from the purple flowers of Clark. Similar to purple flowers, purple–blue flowers contained malvidin 3,5-di-O-glucoside, kaempferol 3-O-gentiobioside, and aromadendrin 3-O-glucoside as major anthocyanin, flavonol, and dihydroflavonol, respectively. The results indicated that purple–blue flower color was not caused by structural or quantitative differences in flavonoids. This study was conducted to determine the genetic control and physiological mechanism of purple–blue flower color of Nezumisaya.
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Materials and Methods |
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Plant Materials
A Japanese landrace Nezumisaya (W1W1 w3w3 W4W4 WmWm WpWp tt i-ii-i) with purple–blue flower, gray pubescence, and buff hilum was crossed with Harosoy with purple flower, gray pubescence, and yellow hilum (W1W1 w3w3 W4W4 WmWm WpWp tt II) in 2004. Hybridity of the F1 plants was ascertained by purple flower color. One hundred and twenty F2 seeds were planted on 13 June 2005, and 30 seeds each of 100 F3 lines were planted on 8 June 2007 at National Institute of Crop Science, Tsukuba, Japan (36°06'N, 140°05'E). N, P, and K were applied at 3.0, 4.4, and 8.3 g m–2, respectively. Flower color was scored for individual F2 and F3 plants. Hilum color of the F3 seeds was also recorded.
A purple–blue flowered line, w2-20 with tawny pubescence and brown hilum (W1W1 w2w2 w3w3 W4W4 WmWm WpWp TT i-ii-i) was developed from a cross between cultivars Shakujou and Nakaide following the report of Nagai (1926). For complementation analysis, Nezumisaya was crossed with w2-20 in 2006. Hybridity of the F1 plants was ascertained by tawny pubescence color. Eighty-three F2 plants were grown with similar levels of fertilizers in pots (24 cm diameter) in a glasshouse in 2007. For measurement of vacuolar pH of flower petals, cultivars with purple flower, Tachinagaha, and Bay and cultivars with purple–blue flower, Nezumisaya, and w2-20 were similarly grown in the above field in 2007.
SSR Analysis and Linkage Mapping
Total DNA of the parents and 92 F2 plants was extracted from trifoliolate leaves by cetyl trimethyl ammonium bromide (CTAB) method (Murray and Thompson 1980). Simple sequence repeat (SSR) primers developed by the USDA (Cregan et al. 1999; Song et al. 2004) were used. The polymerase chain reaction (PCR) mixture contained 15 ng of genomic DNA, 5 nmol of primer, 10 pmol of nucleotides, and 1 unit of ExTaq in 1 x ExTaq buffer supplied by the manufacturer (TAKARA BIO, Ohtsu, Japan) in a total volume of 5 µl. An initial 15 min denaturation at 95 °C was followed by 35 cycles of 1 min denaturation at 92 °C, 1 min annealing at 46 °C, and 1 min extension at 68 °C. PCR was performed in an Applied Biosystems 9700 thermal cycler (Applied Biosystems, Foster City, CA). The PCR products were separated on 8% acrylamide gels (39:1), and the fragments were visualized by ethidium bromide staining.
The observed segregation ratios of morphological and molecular markers were tested by chi-square analyses. Unstable or weak markers were eliminated. A linkage map was constructed using MapMaker/EXP. version 3.0 (Lander et al. 1987) with the threshold LOD score of 3.0.
Measurement of Vacuolar pH of Flower Petals
Banner petals (1.5 g) for each cultivar were collected at the date of opening in 3 replications from Tachinagaha, Bay, Nezumisaya, and w2-20. The flower petals were ground into a fine powder using mortar and pestle under liquid nitrogen. The powder was transferred with chilled spatula to a centrifugation filtration unit (Low-binding Durapore PVDF membrane, 0.22 µm mesh, Millipore, Bedford, MA) and centrifuged at 1780 x g for 5 min at 4 °C. One hundred and fifty microliters of the filtrated sap were immediately applied to the flat sensor of a B-212 twin pH meter (HORIBA, Ltd., Kyoto, Japan). The pH values from 3 replicated samples for each cultivar were subjected to analysis of variance to evaluate varietal differences using the Statistica software (StatSoft, Inc. Tulsa, OK).
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Genetic Analysis
The phenotypes of F1 plants, F2 population, and F3 lines obtained from the cross between Nezumisaya and Harosoy are presented in Table 1. The F1 plants had purple flowers indicating that purple flower is dominant to purple–blue flower. One hundred and fifteen F2 plants that grew normally segregated into 92 plants with purple flower and 23 plants with purple–blue flower. They fitted to a 3:1 ratio, suggesting the involvement of a single gene. All the 23 F3 lines derived from F2 plants with purple–blue flower had purple–blue flower. Seventy-seven F3 lines derived from F2 plants with purple flower segregated into 22 lines fixed for purple flower and 55 lines segregating for purple and purple–blue flower. The segregation fitted to a 1:2 ratio expected from a single-gene model. The above results indicated that the purple–blue flower of Nezumisaya is controlled by a single gene, and its recessive allele is responsible for purple–blue flower color.
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To ascertain its identity with the W2 gene proposed by Nagai (1926) and Matsuura (1933), Nezumisaya was crossed with w2-20. Three F1 plants had purple–blue flowers. Eighty-three F2 plants were grown in the glasshouse. All the F2 plants had purple–blue flower indicating that purple–blue flower of Nezumisaya is controlled by the W2 gene.
Linkage Mapping
Preliminary screening using 5–6 SSR primers polymorphic between the parents for each linkage group revealed a linkage between SSR markers in molecular linkage group (MLG) B2 and W2. A total of 21 SSR markers located in the MLG B2 were then used for screening of the parents and the F2 population. Among them, 9 SSR markers exhibited a distinct segregation in the F2 population and were used for linkage mapping. W2 was mapped in the MLG B2 flanked by Satt318 and Satt020 with a distance of 1.1 and 3.9 cM, respectively (Figure 1).
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Vacuolar pH
In plants, the vacuole occupies a large part (up to 90%) of the cell volume (reviewed by Taiz 1992). To investigate the physiological basis of flower color, we measured the pH value of sap from banner petals of cultivars with purple flowers, Tachinagaha, and Bay and cultivars with purple–blue flowers, w2-20, and Nezumisaya. The filtered sap had similar dark purple color in all cultivars. The purple flower cultivars had a pH value of 5.73–5.77, whereas the purple–blue flower cultivars had a pH value of 6.07–6.10 (Table 2). Varietal differences were significant at 1% level only among cultivars with different flower color, suggesting that increase in vacuolar pH may be responsible for purple–blue color. To confirm pH values of flower petals, absorption spectra of flower sap or solution containing major anthocyanins and copigments under various pH should be compared with spectra of intact flower petals. It may be possible to obtain more distinct varietal differences in vacuolar pH similar to that obtained for morning glory (Yoshida et al. 1995; Yamaguchi et al. 2001) if separation and pH measurement of epidermal cells where anthocyanins are deposited is possible in soybean. W2 may be responsible for the acidification of flower petals. Purple–blue flower color may have been caused by dysfunction of the W2 gene.
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In petunia, 7 genes, PH1 to PH7, have been identified that, when mutated, cause a bluer flower color and an increase in the pH of crude petal extracts, suggesting that these genes are required for acidification of the vacuole (de Vlaming et al. 1983; Chuck et al. 1993; van Houwelingen et al. 1998). Among them, PH4 and PH6 have been cloned and determined to encode transcription factors, an R2R3 MYB protein and a basic helix-loop-helix protein, respectively (Spelt et al. 2002; Quattrocchio et al. 2006). However, it is not clear how these transcription factors control vacuolar pH of flower petals (Spelt et al. 2002; Quattrocchio et al. 2006). Cloning the W2 gene may help determine how soybean flowers become blue.
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Funding |
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The Japanese Government (MEXT) Scholarship (062155 to M.E.O. and 030004 to N.A.K.).
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Acknowledgments |
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We are grateful to the National Institute of Agrobiological Sciences (NIAS) Genebank for providing the seeds of Nezumisaya and Yogetsu-1; Dr K. Takeda (Tokyo Gakugei University) for advice; Dr M. Ishimoto (National Agricultural Research Center for Hokkaido Region) and Society for Techno-innovation of Agriculture, Forestry, and Fisheries (STAFF) for parental screening of SSR markers; and Dr Joseph G. Dubouzet (NIAS) for critical reading of the manuscript.
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Footnotes |
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Corresponding Editor: Reid G. Palmer
Received February 14, 2008
Accepted April 21, 2008
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