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a National Institute of Crop Science, Kannondai 2-1-18, Tsukuba, Ibaraki, 305-8518 Japan
b Faculty of Agriculture, Utsunomiya University, 350 Mine-Machi, Utsunomiya, 321-8505 Japan
c National Agriculture Research Center for Hokkaido Region, Hitsujigaoka 1, Sapporo, 062-8555 Japan
d Tokachi Agricultural Experiment Station, Memuro, Hokkaido, 082-0071 Japan
* Corresponding author (masako{at}affrc.go.jp)
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: DAO, days after opening of individual flowers • ILD, incandescent long daylength • NILs, near-isogenic lines
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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In Japan, cultivars with brown and yellow hilum generally have tawny and gray pubescence, respectively. Soybean breeders in Hokkaido (northern Japan) have observed that chilling tolerance in terms of seed yield reduction is associated with pubescence color rather than hilum color based on the observation of chilling tolerance of populations segregating for both hilum and pubescence color. Takahashi and Asanuma (1996) evaluated chilling tolerance of a pair of NILs for pubescence color, To7B with tawny pubescence (T) and To7G with gray pubescence (t) and found that seed yield and nitrogen-fixation ability of To7B was higher than To7G only under chilling treatment. Gene T is involved in flavonoid biosynthesis and is presumed to encode a flavonoid 3'-hydroxylase that hydroxylates the 3' position of the B-ring in flavonoids (Buttery and Buzzell, 1973). The T gene also has a pleiotropic effect on seed coat color (Palmer et al., 2004). Toda et al. (2002) cloned and characterized the flavonoid 3'-hydroxylase gene from To7B and To7G. Sequence analysis revealed that a single-base deletion of C occurs in the coding region of To7G. The deletion generated a truncated polypeptide lacking the GGEK consensus sequence and the heme-binding domain resulting in nonfunctional protein.
To investigate the genetic basis of tolerance to seed coat pigmentation and cracking, Takahashi and Asanuma (1996) and Takahashi (1997) evaluated the roles of genes T and I (responsible for distribution of seed coat color) using NILs for the two loci. Independent of the genotypes at I locus, the dominant allele T completely suppressed the development of pigmentation around the hilum region and partly suppressed seed coat cracking. Dominant allele I also suppressed seed coat pigmentation and cracking under the genotype of tt, although its inhibitory effect was not as obvious as gene T. Flavonoids with 3', 4'-dihydroxy configuration possess a high antioxidant activity relative to those with a single hydroxyl group on the B-ring (Pratt, 1976). Most likely, the dominant allele T suppresses pigmentation by inhibiting the oxidation of phenolic compounds.
In addition to genes T and I, there is genetic variation in the tolerance to pigmentation among cultivars with yellow hilum (I) and gray pubescence (t). Genetic analysis revealed that a few major genes were involved in tolerance, and that one of the genes for tolerance was closely associated with a dominant gene for late maturity, the recessive allele of which was involved in floral induction under artificially induced long days by means of incandescent lamps (Takahashi and Abe, 1994).
Eight loci have been reported to control time to flowering and maturity in soybean: E1, E2 (Bernard, 1971), E3 (Buzzell, 1971), E4 (Buzzell and Voldeng, 1980), E5 (McBlain and Bernard, 1987), E6 (Bonato and Vello, 1999) for long juvenility, E7 (Cober and Voldeng, 2001), and J (Ray et al., 1995) for long juvenility. Of these, E3, E4, and E7 are involved in the response of flowering to long daylength. The e3 locus controls the insensitivity to fluorescent long daylength obtained by extending natural daylength to 20 h using cool white fluorescent lamps with a low FR/R ratio, whereas e4 combines with e3 to control the insensitivity to incandescent long daylength (ILD) by extending natural daylength to 20 h using incandescent lamps with a high FR/R ratio (Buzzell, 1971, Buzzell and Voldeng, 1980). Insensitivity to ILD is an adaptive trait at high latitude regions where a short growth period is required.
To evaluate the separate effects of five soybean maturity genes (E1 to E5) on the intensity of seed coat pigmentation and cracking, Takahashi and Abe (1999) treated soybean cv. Harosoy (e1e2E3E4e5E7) and its NILs for E1 to E5 loci with chilling temperatures. Intensity of pigmentation was not affected by e3, slightly reduced by E2 and e4, and profoundly reduced by E1 and E5. Degree of cracking was slightly increased by e3 and drastically reduced by e4, E1, and E5. Dominant alleles E1 and E5 were most effective in suppressing both pigmentation and cracking.
Benitez et al. (2004) found that E7 also had inhibitory effects on pigmentation and cracking. The linked E1 and E7, E2, E3 and E4 loci were presumed to locate in molecular linkage groups C2, O, L, and I, respectively (Cregan et al., 1999; Cober and Voldeng, 2001; Abe et al., 2003). It is unlikely that the genes responsible for tolerance to seed coat pigmentation and cracking are located proximal to the maturity genes found in various positions in the genome. Each maturity gene may have pleiotropic effects on the low temperature–induced seed coat pigmentation and cracking, although the underlying physiological mechanisms remain to be identified. Benitez et al. (2004) further investigated the combined effects of E3 and E4 using a double recessive NIL and found that Harosoy-e3e4 had an intermediate degree of pigmentation and cracking relative to Harosoy-e3 and Harosoy-e4, suggesting that the effects of E3 and E4 loci were additive. The first objective of this study was to investigate the combination effects between the E1 and E3E4 loci on seed coat pigmentation and cracking.
Investigation of the relationship between maturity genes and chilling tolerance in terms of seed yield reduction may also be important to efficiently develop chilling tolerant cultivars. However, late-maturing genotypes generally have longer growth period, larger plant mass, and higher productivity. It is therefore difficult to identify the direct effect of maturity genes on chilling tolerance, because quantitative and qualitative effects of maturity genes cannot be easily dissected. The second objective of this study was to investigate the effects of maturity genes on chilling tolerance in terms of seed yield reduction by exchanging a combination of maturity genes between E1e3e4 and e1E3E4 to avoid substantial differences in growth period. Effects of genotypes at T locus were also evaluated.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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The chilling treatment was done by transferring plants from the greenhouse to a phytotron set at 15° ± 0.5°C (day/night). Light was supplied at a photosynthetic photon flux density (400 to 700 nm) of 200 µmol m–2 s–1 using metal halide lamps [DR 400/T(L), Toshiba Co., Tokyo, Japan] at a 14/10 h light/dark regime. The mean minimum and maximum temperatures in the control greenhouse during the correspondent period of the chilling treatment in the phytotron were 20.1 and 27.4°C, respectively. Plants were exposed to chilling for 14 d beginning 8 d after flowering of individual plants. Because the flowering period in soybean spans over 2 wk, the chilling treatment of an individual plant leads to exposure of flowers at various stages of development. Accordingly, flowers were individually labeled with the date of opening and only the flowers that opened before the beginning of the chilling treatment (flower age of 0 to 8 d at the beginning of chilling treatment) were used for analysis. After 14 d of the chilling treatment, pots were returned to the greenhouse.
The number of days from planting to opening of the first flower (R1, Fehr et al., 1971), and from planting to maturity (R8), when 95% of pods had mature color, were recorded for individual plants. Means of days from planting to flowering and maturity among NILs were compared with Tukey's HSD test using the Statistica software (StatSoft, Inc., Tulsa, OK). The degree of pigmentation and cracking of each seed was scored using a pigmentation index (0 = not pigmented to 4 = severely pigmented) and a cracking index (0 = not cracked to 4 = severely cracked) as previously described (Takahashi, 1997; Takahashi and Abe, 1999). The effect of a 14-d chilling treatment initiated at different flower stages on pigmentation and cracking was evaluated by averaging the indices of seeds obtained from flowers which opened on the same day.
Each temperature treatment was conducted in one phytotron or greenhouse. Pots were distributed at random both in the phytotron and the greenhouse and repositioned twice a week in the greenhouse and daily in the phytotron. Pigmentation and cracking indices were subjected to two-way analysis of variance to evaluate effects of NILs, age of flowers, and their interactions. Each pot was considered as a replicate in the statistical analysis.
Yield Component Experiments
Experiments were conducted from May to October at the Natl. Agric. Research Center for Hokkaido Region (Sapporo, 43°03' N, 141°20' E) in 2002 and at the Tokachi Agric. Exp. Stn. (Memuro, 42°53' N, 143°05' E) in 2003 according to the conventional procedure of the respective laboratories to evaluate chilling tolerance. Although the latitudes are largely similar between the two locations, mean temperature of soybean growing season in Memuro is generally 2 to 3°C lower than Sapporo because of chilling wind from the Sea of Okhotsk.
In 2002, eight seeds of Harosoy, Harosoy-T, Harosoy-E1e3e4, and Harosoy-E1e3e4T were planted in pots (15.5 cm diameter) filled with 4.5 kg soil (low-humic andosols) supplemented with ammonium sulfate (2 g), monocalcium phosphate (4 g), fused magnesium phosphate (8 g) and potassium sulfate (2 g) on May 31. One week after emergence, seedlings were thinned to two per pot. Ten pots (20 plants) per line were grown in an unheated vinyl plastic greenhouse. The control plants were continuously grown in the greenhouse. The average temperature throughout the growth of the control plants was 17.1°C.
At anthesis (R1), chilling treatment was imposed by transferring five pots per genotype from the greenhouse to a phytotron set at 15 ± 0.5°C (day/night) with 75 ± 5% relative humidity. After 4 wk of chilling treatment, plants were returned to the greenhouse and grown until maturity. The mean photosynthetic photon flux density (400–700 nm) at canopy level was 
307 µmol m–2 s–1 in the phytotron and 407 µmol m–2 s–1 in the greenhouse at the time of treatment. The mean minimum and maximum temperatures in the greenhouse during the corresponding period of the chilling treatment in the phytotron were 16.5 and 23.5°C, respectively.
In 2003, 12 seeds of Harosoy, Harosoy-T, Harosoy-E1e3e4 and Harosoy-E1e3e4T were planted in pots (24 cm diameter) filled with 9 kg soil (dry andosols) supplemented with a synthetic fertilizer (24 N–200 P2O5–104 K2O kg ha–1) on May 31. One week after emergence, plants were thinned to two per pot and grown in an unheated vinyl plastic greenhouse. Twenty-four plants (12 pots) were grown for each NIL. At flowering (R1), chilling treatment was imposed by transferring six pots per genotype from the greenhouse to a phytotron set at 18 ± 0.5/13 ± 0.5°C (day/night) with approximately 55% shading of natural daylight using a plastic mesh net with evenly spaced reflective aluminum strips to mimic chilling weather under field conditions. The control plants were transferred to the phytotron set at 25 ± 0.5/20 ± 0.5°C (day/night) without shading during the period of chilling treatment, because chilling damage occasionally occurs at Memuro even without chilling treatments. After 4 weeks of treatments, all plants were returned to the plastic greenhouse and were grown until maturity. The mean photosynthetic photon flux density (400–700 nm) at canopy level in the control and the chilling treatment chamber of the phytotron were 368 and 166 µmol m–2 s–1, respectively. The average temperature throughout the growth of the control plants was 14.9°C.
Flowering date (R1), maturity date (R8), and characters related to productivity were recorded for individual plants. Each temperature treatment was conducted in one phytotron or greenhouse. Pots were distributed at random in the phytotron and in the greenhouse and repositioned twice a week. Effects of genotypes at maturity genes and pubescence color gene, and their interactions under control and chilling treatments were analyzed by two-way analysis of variance using the Statistica software. Each pot was considered as a replicate in the statistical analysis.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Harosoy-E1e3e4 showed an intermediate degree of pigmentation and cracking relative to Harosoy-E1 and Harosoy-e3e4 suggesting that the effects of E1 and E3E4 may be additive. This is consistent with the result of Benitez et al. (2004) who reported that the effects of E3 and E4 were additive. Their presumption that combination effects of maturity genes might roughly be estimated by the individual gene actions was applicable to triple gene action as well. E1e3e4 genotype may therefore be better than e1E3E4 genotype in terms of seed quality under chilling conditions.
Yield Component Experiments
Aside from the obvious environmental differences associated with each experimental location, the main procedural differences in these two experiments were: (i) the control plants were grown in a phytotron set at 25/20°C in the 2003 experiment at Memuro whereas the control plants in the 2002 experiment at Sapporo were left in the unheated plastic house during the chilling treatment and (ii) the chilling treatment in Sapporo was performed in a phytotron set at 15°C whereas the chilling treatment in Memuro was performed in a partially shaded phytotron set at 18/13°C. Despite these procedural limitations, statistically significant differences could be detected in the agronomic characters vis-à-vis chilling exposure and the genes for maturity and pubescence color.
Results of two-way analysis of variance and mean value of the characters related to productivity are presented in Tables 4 and 5. Under control conditions, the average temperature throughout soybean culture period at Memuro was 2.2°C lower than at Sapporo. The low temperature conditions at Memuro may be responsible for lower plant height and lower weight of seed per plant in 2003. Light intensity under chilling treatment was lower than control conditions in both years. Generally, light intensity is low when chilling temperatures prevail under field conditions. Although the effects of chilling temperatures could not be separated from low light intensity in this study, the results obtained may be applicable to chilling response of soybean under field conditions.
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Under control conditions, the allelic combination E1e3e4 showed reduced plant height and decreased number of nodes in 2002, whereas the same genotype had a slightly increased plant height and increased number of nodes in 2003. The allelic combination of E1e3e4 increased the number of branches by 112% (control) and 61% (chilling) in 2002, and by 61% (control) and 73% (chilling) in 2003. Number of pods per plant was not different among NILs for maturity genotypes under control conditions in both years, whereas NILs with E1e3e4 produced 39% (in 2002) and 89% (in 2003) more pods compared to NILs with e1E3E4 under chilling treatment.
In 2002, weight of seed per plant in NILs with e1E3E4 was slightly higher than NILs with E1e3e4 under control conditions whereas NILs with E1e3e4 produced 42% higher amount of seed compared to NILs with e1E3E4 under chilling treatments. In 2003, weight of seed per plant of NILs with E1e3e4 was 20% higher than NILs with e1E3E4 under control conditions. Under chilling treatments, weight of seed per plant of NILs with E1e3e4 was double that of NILs with e1E3E4. The NIL differences in weight of seed per plant under control conditions in 2003 were probably due to low temperature conditions at Memuro.
Insensitivity of flowering to long daylength is an essential trait in adaptation of soybean to high latitudes with short growing seasons and long daylengths. The double recessive genotype e3e4 is insensitive to incandescent long daylength and flowers under long daylength conditions (Saindon et al., 1989). NILs with E1e3e4 matured significantly earlier than NILs with e1E3E4 in 2002 and similar in 2003 with chilling treatment. Weight of seed per plant in NILs with E1e3e4 under chilling treatment was higher than NILs with e1E3E4 in both years, suggesting that E1e3e4 genotype may be preferable to e1E3E4 to increase seed yield under chilling conditions.
Higher productivity of NILs with E1e3e4 under chilling treatment was not ascribable to plant height, node number, seed size, nor length of growth period. Higher branch number of NILs with E1e3e4 is possibly related to chilling tolerance. The differences in seed yield reduction under chilling conditions were associated with number of pods on main stems. The number of pods on main stems in NILs with e1E3E4 was 87% higher than NILs with E1e3e4 under control conditions, whereas NILs with e1E3E4 produced 15% lower number of pods in contrast to NILs with E1e3e4 in 2002 under chilling treatment (Tables 4 and 5).
Chilling treatment not only reduces pod number presumably by causing flower or pod abortion, it also decreases fertility within pod as indicated by the drastically lower number of seeds per pod. In the present study, number of seeds per pod was not significantly different under control conditions, whereas the allelic combination of E1e3e4 increased number of seeds per pod by 19% (in 2002) and 31% (in 2003) compared to NILs with e1E3E4 under chilling treatment (Tables 4 and 5). Assimilation ability and flowering-pod set dynamics in connection with plant morphology under chilling conditions should be investigated further to determine the mechanism of higher pod-set and fertility within pod of NILs with E1e3e4 genotype.
Similar to the results of Takahashi and Asanuma (1996), weight of seeds in NILs with T was higher than NILs with t only under chilling treatments and the number of pods per plant was not different among the NILs (Tables 4 and 5). In 2002, weight of seed per plant in NILs with t was slightly higher than NILs with T under control conditions whereas NILs with T produced 32% higher weight of seeds compared to NILs with t under chilling treatments. In 2003, weight of seed per plant was not different among NILs for pubescence color whereas NILs with T produced 30% higher weight of seeds compared to NILs with t under chilling treatments. Probably, the dominant T allele is associated with chilling tolerance by improving seed-filling ability under chilling conditions. This is consistent with the observation of Takahashi and Asanuma (1996) that nitrogen-fixing ability of a NIL with T was higher than that of a NIL with t under chilling treatment. In this study, number of seeds per pod in NILs with T was higher than NILs with t under chilling treatments in both years (Tables 4 and 5). Higher assimilation ability of NILs with T may have increased number of seeds within pod.
Takahashi and Asanuma (1996) and Takahashi (1997) further reported that dominant T allele suppressed seed coat pigmentation and cracking in response to low temperatures. Dominant T allele may therefore be useful to ensure chilling tolerance in terms of both yield and quality of seeds when clear yellow seed coat and hilum color are not required.
Kitamishiro, a leading cultivar in Hokkaido from the late 1960s to early 1970s, is chilling tolerant in terms of seed yield. Kitamishiro belongs to MG1 and has an allelic combination of E1e3e4Ti-i (Saindon et al., 1990). This allelic combination of maturity and pubescence color genes may partly be responsible for chilling tolerance of Kitamishiro. Selection for the appropriate combination of maturity alleles may be effective in improving productivity as well as seed quality at high latitude regions.
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ACKNOWLEDGMENTS |
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Received for publication June 29, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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