The Journal of Heredity 2001:92(1)
© 2001 The American Genetic Association 92:89-92
Brief Communication |
Genetic and Linkage Analysis of Cleistogamy in Soybean
From the Legume Breeding Laboratory, National Agriculture Research Center, Kannondai 3-1-1, Tsukuba, Ibaraki, 305-8666 Japan (Takahashi), Tokachi Agricultural Experiment Station, Memuro, Hokkaido, Japan (Kurosaki and Yumoto), Faculty of Agriculture, Dankook University, Chonan, Korea (Han), and the Graduate School of Agriculture, Hokkaido University, Sapporo, Japan (Abe).
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Abstract |
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Early maturing cultivars of soybean [Glycine max (L.) Merr.] native to the shores of the Sea of Okhotsk (Sakhalin and Kuril Islands) and eastern Hokkaido (northern Japan) have been used in breeding for chilling tolerance. These cultivars have a strong tendency to produce cleistogamous flowers throughout their blooming period. This study was conducted to determine the genetic basis of cleistogamy in an early maturing cultivar, Karafuto-1, introduced from Sakhalin. Genetic analysis was performed using F1 plants, the F2 population, and 50 F3 families produced by crossing between Karafuto-1 and a chasmogamous cultivar, Toyosuzu. F1 plants had chasmogamous flowers, indicating that chasmogamy was dominant to cleistogamy. Analysis of F2 populations and F3 families generated segregation data that was close to a two-gene model with epistatic interactions, although a portion of the pooled F3 data on the frequency of chasmogamous segregants from cleistogamous families significantly deviated from the model. The results suggested that a minimum of two genes with epistatic effects were involved in the genetic control of cleistogamy. Furthermore, cleistogamy was associated with early flowering in the F2 and F3 populations. A gene for cleistogamy was linked to one of the recessive genes responsible for insensitivity to incandescent long daylength.
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Introduction |
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Cleistogamy, production of open (chasmogamous, CH) and closed (cleistogamous, CL) floral forms by one species, is widespread among the angiosperms. Lord (1981) classified cleistogamy into four categories: preanthesis cleistogamy in which pollination occurs followed by anthesis; pseudocleistogamy in which no morphological differences occur between CL and CH flowers other than a lack of expansion of petals and anthesis in CL flowers; complete cleistogamy in which species produce only CL flowers; and true cleistogamy in which floral dimorphism results from divergent developmental pathways in a single species or individual. Pseudocleistogamy is occasionally induced by environmental factors such as drought and low temperatures (Uphof 1938).
Cleistogamy has been described in members of the genus, Glycine. Newell and Hymowitz (1980) and Kenworthy et al. (1989) reported cleistogamy of Glycine tabacina, G. tomentella, and G. arenaria. Cleistogamy has also been observed in cultivated soybean [Glycine max (L.) Merr.] and its wild relative [G. soja Sieb. & Zucc.]. Soybean usually produces both CH and CL flowers on an individual plant; fertilization occurs without opening of petals in CL flowers. Thus, based on the classification of Lord (1981), soybean is pseudocleistogamous.
The production of CH and CL flowers on individual plants depends on developmental stages. Miyashita et al. (1999) studied the dynamics of CH and CL flowers in five G. max cultivars and two G. soja populations. The soybean cultivars produced primarily CH flowers at the early stage of flowering, whereas CL flowers were produced almost exclusively at the later stage. Among these cultivars, the proportion of CL flowers produced by an individual plant ranged from 22.6 to 66.5% of the total number of flowers.
In contrast, early maturing landraces (maturity group 000–00) native to the shores of the Sea of Okhotsk (Sakhalin and Kuril Islands) and eastern Hokkaido (northern Japan) usually produce only CL flowers when cultivated in Hokkaido. In these cultivars, fertilization occurs without opening of petals, or without any appearance of petals at anthesis. However, they have been observed to produce CH flowers at the early flowering stage during years with high temperatures.
The early maturing cleistogamous land-races have been used in breeding for chilling tolerance in Japan and Sweden (Holmberg 1973; Sanbuichi 1979). Soybeans are sensitive to low temperatures at various stages from germination to maturation (Raper and Kramer 1987). Low temperatures at the flowering stage, which is most sensitive to chilling stress, induce flower and pod abortion (Holmberg 1973; Hume and Jackson 1981, Takahashi and Asanuma 1996) or discolored and cracked seed coats (Sunada and Ito 1982; Takahashi and Abe 1994, 1999). Minimum temperatures required for good flowering and pod set varied among cultivars (Holmberg 1973; Hume and Jackson 1981). The early maturing landraces and some of their descendants, such as the cultivar Fiskeby V, have somewhat lower critical temperatures, and flowering and seed formation were generally not interrupted by low temperatures (Holmberg 1973; Hume and Jackson 1981).
Cleistogamy may be advantageous under severe environmental conditions because the energetic investment in fertilization costs (i.e. sepals, petals, pollen, and nectar) of CL flowers appears to be considerably lower than CH flowers. Schemske (1978) evaluated the energetic costs of CH and CL flowers in pseudocleistogamous plant species, Impatiens pallida, and found that CH flowers had an energetic investment of more than 100 times higher than that of CL flowers. Further, CL flowers possibly shelter pollen from chilling temperatures at fertilization because chilling temperatures hindered pollen formation, anther dehiscence, and fertilization in soybean (Goto and Yamamoto 1972). Detailed physiological and genetic studies are necessary to evaluate possible roles of cleistogamy in relation to chilling tolerance. This study was conducted to investigate the genetic basis of cleistogamy in the early maturing soybean cultivars.
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Materials and Methods |
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Plant Material
A CL cultivar, Karafuto-1, was pollinated by a CH cultivar, Toyosuzu, in 1997 and 1998. Toyosuzu is a cultivar (maturity group II) developed at the Tokachi Agricultural Experiment Station that has gray pubescence and a yellow hilum (IIrrtt). Toyosuzu has chasmogamous flowers irrespective of environmental conditions, except for the later stage of flowering. Karafuto-1 is a pure line selected from an early maturing landrace introduced from Sakhalin (maturity group 00); it has brown pubescence and brown hilum (i-ii-irrTT). Karafuto-1 has cleistogamous flowers throughout flowering time, except for years with high temperatures. The hybridization of F1 plants was ascertained by brown pubescence color.
Genetic and Linkage Analysis
Twenty seeds from each parent and 120 F2 seeds were sown in a field at Tokachi Agricultural Experiment Station, Memuro, Hokkaido, Japan (42°53'N, 143°05'E) on May 20, 1998. Twenty seeds from each parent, 4 F1 seeds, and 20 seeds from each of 50 F3 families were sown at the same location on May 19, 1999. Seeds of parents were sown in duplicate and thinned after emergence. Cleistogamy of each plant was determined by assessing flowers that fertilized at the first date of anthesis (R1; Fehr et al. 1971), because parental differences in floral forms were evident at an early period of flowering. The date of anthesis in CL plants was visually evaluated by the size of flower buds, and it was confirmed by comparing sizes of developing pods between CL and CH plants 5 days after anthesis. Floral forms were classified as follows; CH included open flowers with fully expanded petals, whereas CL included flowers that did not open fully, that is, those with slightly expanded petals that protruded out of calyxes, or those whose petals did not come out of calyxes.
Flowering response to incandescent long daylength (ILD) was also evaluated for parents and 98 F3 families at Hokkaido University, Sapporo, Hokkaido, Japan (43°03'N, 141°20'E) in 1999. Toyosuzu is sensitive to ILD and flowering was retarded under ILD, whereas Karafuto-1 is insensitive to ILD and its flowering was not affected by daylength. Twenty seeds for each parent and 98 F3 families were planted in a field where natural daylength was extended to 20 h using incandescent lamps. The response to ILD for individual plants was evaluated by growth stages at 60 days after planting. Classification of growth stage followed Fehr et al. (1971).
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Results and Discussion |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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Inheritance of Cleistogamy
Table 1 shows temperatures from June 26 to July 25 in 1998 and 1999 at Memuro. All 10 plants of Karafuto-1 and Toyosuzu had CL and CH flowers at anthesis in both years. Date of anthesis of the F2 plants in 1998 and that of the F3 plants in 1999 ranged from July 15 to 25 and July 11 to 21, respectively. High temperatures from July 21, 1999, apparently had no affect on cleistogamy.
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Due to virus infection, only two F1 plants and 98 F2 plants grew normally. The two F1 plants had CH flowers, indicating that chasmogamy was dominant to cleistogamy (Table 2). Ninety-eight F2 plants were classified into 77 CH and 21 CL plants. The result agreed with the dominance relationship observed in the F1 plants. F2 segregation fitted both a ratio of 3:1 under a single recessive gene model (
2 = 0.67, .4 < P < .5) and a ratio of 13:3 under a single recessive gene (a) and a dominant gene (B) model, in which the former is epistatic to the latter (2 = 0.46, .4 < P < .5).
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Segregation was further evaluated using 10 plants each from 50 F3 families including 9 F3 families derived from F2 CL plants and 41 F3 families derived from F2 CH plants. Four of the nine CL families produced only CL plants, whereas the remaining five families segregated into CH and CL plants. This result evidently contradicts the single-gene control model, but it seemed to fit a segregation ratio of 0:2:1 for CH-fixed family:segregating family (aaBb):CL-fixed family (aaBB) under the two-gene model with epistatic interaction (
2 = 0.50, .7 < P < .8). On the other hand, the 41 families derived from CH F2 plants produced 28 CH-fixed families and 13 segregating families, with the segregation ratio in agreement with the expected ratio of 7:6:0 ratio for CH-fixed family (AA–, Aabb, aabb):segregating family (AaBB, AaBb):CL-fixed family (2 = 3.44, .1 < P < .2).
Taking into account a possible bias resulting from the small number of plants tested in each F3 family, segregation of cleistogamy in F3 was evaluated on an individual-plant basis. A total of 495 plants could be evaluated due to lack of germination in some of the F3 families. The 41 CH families produced 376 CH plants and 30 CL plants, closely fitting the expected number of 367 CH plants (47/52) and 39 CL plants (5/52) (2 = 2.30, .1 < P < .2). The nine CL families, however, produced a surplus of CH segregants that was significantly different from the expected number of 15 (1/6) based on the model (2 = 29.9, P < .01). These results suggested that cleistogamy in Karafuto-1 is not controlled by a single gene, and a minimum of two genes with epistatic effects may be involved in genetic control. However, the effects of different climatic conditions between the two years or the microclimatic differences due to the different flowering dates within the segregating populations were not factored in the present study. Neither penetrance nor expressivity were taken into account as factors influencing the expression of cleistogamy in the segregating populations, although parents exhibited constant and clear differences in our experiments. A further characterization of cleistogamy and/or experiments under controlled environments may therefore be necessary to exactly determine the contributions of genetic and environmental factors on the expression of cleistogamy in Karafuto-1.
Association of Cleistogamy with ILD Insensitivity
Toyosuzu is sensitive to ILD and its flowering was retarded under ILD, whereas Karafuto-1 is insensitive to ILD and its flowering was not delayed. At 60 days after planting, Karafuto-1 reached R4, whereas Toyosuzu still remained vegetative. F3 plants were classified into the following three types: Karafuto-1 type, Toyosuzu type, and intermediate type whose growth stage reached R2–R3. Segregation of intermediate type in F3 families may indicate that dominance of genes for ILD sensitivity of Toyosuzu was not complete, unlike E3 or E4 (Buzzell 1971; Buzzell and Voldeng 1980). Of the 98 F3 families tested, 88 families with a minimum of 15 plants each were classified into seven classes: (1) family fixed for Karafuto-1 type, (2) family segregating for Karafuto-1 and intermediate type, (3) family segregating for all of the three types, (4) family segregating for Karafuto-1 and Toyosuzu type, (5) family fixed for intermediate type, (6) family segregating for Toyosuzu and intermediate type, and (7) family fixed for Toyosuzu type (Table 3). When all segregating or intermediate phenotypes (2–6) are considered as one phenotypic class, the observed number of Karafuto-1 class, segregating or intermediate class, and Toyosuzu class was close to a two-gene (1:14:1) model (2 = 5.45, .05 < P < .1), suggesting the involvement of two recessive genes in ILD insensitivity of Karafuto-1. As shown in Table 3, all of the 8 F3 families fixed for ILD insensitivity were derived from CL F2 plants, while all of the 10 F3 families fixed for ILD sensitivity were derived from CH F2 plants. Segregation of cleistogamy and ILD insensitivity was thus significantly associated (2 = 40.1, P < .005). The results strongly suggested that one of the genes for ILD insensitivity was closely linked with a gene for cleistogamy. CL plants flowered 3 days earlier than CH plants in both F2 and F3 populations probably due to the close linkage (Figure 1).
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Seven loci have so far been reported to control time to flowering and maturity in soybean: E1 and E2 (Bernard 1971), E3 (Buzzell 1971), E4 (Buzzell and Voldeng 1980), E5 (McBlain and Bernard 1987), J (Ray et al. 1995), and an unnamed gene for ILD response (e(t)) (Abe et al. 1998). The E3, E4, and e(t) loci are known to be involved in the initiation of flowering under ILD. The recessive alleles e3 and e4 or e(t) jointly confer insensitivity to ILD (Abe et al. 1998; Buzzell 1971; Buzzell and Voldeng 1980). When combined with e3 and e4, E1 markedly retards flowering under ILD or natural daylength relative to e1 (Cober et al. 1996). Of these genes, E1 was linked to T with a recombination frequency of 3.9% (Weiss 1970). The association between the genotype at the T locus and ILD insensitivity in the F2 population was not significant (data not shown), suggesting that E1 may not be a gene responsible for the varietal differences in ILD insensitivity. Linkage analysis using DNA markers may be useful to identify the maturity gene associated with cleistogamy.
This study revealed that cleistogamy in an early maturing soybean cultivar was primarily genetic and a few major genes were involved. Near-isogenic lines regarding cleistogamy should be developed to help clarify the relationship between cleistogamy and chilling tolerance.
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Acknowledgments |
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We thank Dr. T. Narikawa for useful advice and Dr. Joseph G. Dubouzet (JIRCAS) for critical reading of the manuscript.
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Footnotes |
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Address correspondence to R. Takahashi at the address above or e-mail: masako{at}narc.affrc.go.jp.
Corresponding Editor: Reid G. Palmer
Received June 6, 2000
Accepted October 31, 2000
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References |
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