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CROP BREEDING, GENETICS & CYTOLOGY

Photoperiod-Insensitive Japanese Soybean Landraces Differ at Two Maturity Loci

Laboratory of Plant Genetics and Evolution, Graduate School of Agriculture, Hokkaido University, Sapporo, 060-8589, Japan

^* Corresponding author (jabe{at}res.agr.hokudai.ac.jp)

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Insensitivity of flowering to long daylengths is an important character in the adaptation of soybean [Glycine max (L.) Merrill] to higher latitudinal environments. The objective of this study was to identify and map the maturity genes for incandescent long daylength (ILD) insensitivity for two landraces, ‘Miharudaizu’ and ‘Sakamotowase’, which belong to different cultivar groups. Two F₉ recombinant inbred line (RIL) families were developed by means of a repetitive heterozygote selection method from the F₂ population of the cross between the two landraces. Linkage analyses with isozyme and simple sequence repeat (SSR) markers revealed that the maturity gene for ILD insensitivity from Miharudaizu was a recessive allele at the E4 locus on Molecular linkage group (MLG) I. The MLG I order of the E4 locus and four markers was determined as Satt239–Satt496–E4–Enp–Satt354. The maturity gene for ILD insensitivity from Sakamotowase was found to cosegregate with four tightly linked SSRs on MLG C2, and was determined as a recessive allele at the E1 locus. The genotypes at the E3 locus for both Miharudaizu and Sakamotowase were estimated as e3e3 on the basis of the responses of late-flowering near-isogenic lines to fluorescent long daylength (FLD). The results obtained in this study suggest that Miharudaizu has the genotype of E1E1e3e3e4e4 and Sakamotowase has the genotype of e1e1e3e3E4E4. Because the e1 allele cannot induce flowering in ILD in the presence of the E4 allele, an unknown gene may condition the ILD insensitivity of Sakamotowase, possibly while combined with the e1 allele.

Abbreviations: FLD, fluorescent long daylength • ILD, incandescent long daylength • ND, natural daylength • NIL, near-isogenic line • RIL, recombinant inbred line • SSR, simple sequence repeat

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
INSENSITIVITY OF FLOWERING to long daylength is an important character in the adaptation of soybean to higher latitudinal environments. A number of maturity genes that influence flowering under long daylength have been reported in soybean (Buzzell, 1971; Buzzell and Voldeng, 1980; Saindon et al., 1989; Cober and Voldeng, 2001a). Of these, the E3 locus has the most prominent effect on flowering under long daylengths with a wide range of red-to-far red quantum ratios (R:FR) (Cober et al., 1996a,b). Plants with the genotype of e3e3 condition insensitivity to fluorescent long daylength (FLD). FLD is a natural daylength that is extended to 20 h with cool white fluorescent lamps with high R:FR ratio (Buzzell, 1971). A recessive allele at the E4 locus is necessary for plants homozygous for the e3 allele to flower under ILD when natural daylength is extended to 20 h with incandescent lamps with low R:FR ratio (Buzzell and Voldeng, 1980; Saindon et al., 1989).

The E1 locus is also involved in the response of flowering to ILD (Cober et al., 1996b). The E1 and E4 loci, however, do not act additively on flowering and maturity in plants homozygous for the e3 allele (Saindon et al., 1990; Cober et al., 1996b). The three maturity loci E1, E3, and E4 differ in their responses to different light qualities (Cober et al., 1996b). The E1 locus was most sensitive to light quality, and the E1 allele delayed flowering of plants homozygous for the e3 and e4 alleles, particularly under ILD of low R:FR ratio. The E3 locus was the least sensitive, and the E4 locus showed intermediate sensitivity to light quality (Cober et al., 1996b).

Response to ILD is also influenced by the genotypes at the E7 locus, which is linked to the E1 locus at a distance of 6.2 cM (Cober and Voldeng, 2001a). The action of the e7 allele is hypostatic under the E1 allele and weakens the sensitivity of soybean with the genotype of e1e1e3e3e4e4 to ILD (Cober and Voldeng, 2001b).

ILD-insensitive soybean landraces established in Hokkaido, Japan, are classified into two cultivar groups, type I and type II, on the basis of the allelic profiles at 16 isozyme loci (Abe et al., 1991). The type I cultivar group consists of landraces that have been adapted to the cool summer of northern and northeastern Hokkaido and Sakhalin, where the frost-free season is limited to less than 130 d. Soybean cultivars of this group have a determinate growth habit (dt1), tawny pubescence (T), and yellow seed coat color with brown hilum (iⁱ). A number of leading cultivars have been developed via crossbreeding with ILD-insensitive landraces and pureline selections of the type I cultivar group. An ILD-insensitive cultivar, Kitamishiro, was one of the cultivars released from the cross with an ILD-insensitive pureline selection ‘Ohyachi 2’. Its genotype for three maturity loci was determined as E1E1e3e3e4e4 (Saindon et al., 1990). The type II cultivar group consists of landraces that have been cultivated as a short-season crop in wide areas of Japan and the Korean peninsula. Soybean cultivars of this group have a determinate growth habit and tawny pubescence, but vary in seed coat colors and patterns.

The F₂ progeny of a cross between landraces of the two cultivar groups Miharudaizu (type I) and Sakamotowase (type II) exhibited a transgressive segregation toward ILD-insensitive late flowering (Abe et al., 1998b). This was unexpected because the insensitivity of soybean to ILD is controlled only by the genotype of e3e3e4e4 (Buzzell and Voldeng, 1980; Saindon et al., 1989). Abe et al. (1998b) suggested that the two cultivar groups may differ in the genetic basis for ILD insensitivity. To determine the genetic basis of ILD insensitivity of both landraces, we have developed near-isogenic lines (NILs) for ILD insensitivity from the F₂ population of a cross between Miharudaizu and Sakamotowase using a repetitive heterozygote selection method. The objective of this study was to identify and map the maturity genes for ILD insensitivity for two landraces.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Four F₉ RIL families in which the maturity genes controlling ILD insensitivity segregated were developed by means of a repetitive heterozygote selection method from different F₂ plants (#9, #11, #130, and #136) of the cross between Miharudaizu and Sakamotowase (Abe et al., 1998b). Since ILD-insensitivity segregated as a recessive character, seed of three to four ILD-sensitive plants that segregated from a single heterozygous plant were used to advance the generations. In this study, two of the four RIL families (#9 and #130) were used for mapping and identification of the maturity genes.

Seed of family #9 were sown directly in an experimental field with outdoor lighting at Hokkaido University, Sapporo, Japan (43°25'N, 143°32'E), on 26 May 2000. Seed of family #130 were sown on paper towels on the same date, and after sampling a small piece of cotyledon for identification of the Enp genotype, the 3-d-old seedlings were individually transplanted into pots. Pots were put in the above field until the seedlings were transplanted two weeks later. Plants were spaced 0.2 m apart in rows 16 m long and 0.8 m between rows.

ILD was generated with 500-W incandescent lamps placed 2 m above the soil surface at 4-m intervals. From seedling emergence to 31 July, lights were turned on from 0200 to 0600h and from 1800 to 2200h. The natural daylength including twilight at Sapporo reached a maximum of 16.5 h. Under incandescent lamps, the red-to-far red quantum ratio (R:FR; 660:730) was 0.72, and the average photosynthetic photon flux at the canopy surfaces was 1 µmol photon s^-1 m^-2, as measured at night using a LI-COR quantum sensor (Model LI-1800C, LI-COR Inc., Lincoln, NE). The plants were checked every other day for the first appearance of an open flower (R1: Fehr et al., 1971) till the end of ILD treatment.

The progeny test was performed under ILD for all of the segregants tested for the two RIL families in 2001. Each plot was 3.0 m long, with a spacing of 0.5 m between rows and a seeding rate of 7 seeds m^-1. Fifteen to 20 plants were used in each entry for evaluation of ILD sensitivity. Plants that reached R3 to R5 were classified as ILD insensitive, and those that remained vegetative were classified as ILD sensitive, on the basis of their flowering phenology at the end of ILD treatment.

Parents and three NILs of ‘Harosoy’ for the e3 and e4 alleles (OT89-5, L62-667, and OT94-41) were also included in the analysis. The response of the three Harosoy NILs to ILD has been fully characterized (Cober et al., 1996b). Genotypes at the four maturity loci involved in ILD insensitivity were e1e1e3e3e4e4E7E7 for OT89-5, e1e1e3e3E4E4E7E7 for L62-667, and e1e1E3E3e4e4E7E7 for OT94-41 (Voldeng and Saindon, 1991; Voldeng et al., 1996; Cober and Voldeng, 2001a, b). Seed of the parents and the three NILs were sown on the same date (26 May 2000) as the sowing date of the segregating families in the ILD field and its neighboring natural daylength (ND) field which did not have outdoor lighting. Each plot was 2.0 m long, with 0.8 m between rows. Ten plants in each entry were checked every other day for R1.

DNA was extracted individually from leaves of the parents and progeny, as described by Doyle and Doyle (1990). Twenty-one SSR markers were chosen from three MLGs (C2, I, and L) (Cregan et al., 1999), in which the known maturity loci controlling ILD insensitivity were located. SSR analyses have been described previously (Abe et al., 2003b). The forward primers of the SSRs were labeled with either 6-FAM (blue), HEX (green), or NED (yellow) fluorescent dyes. Amplified products were separated by an ABI 377 sequencer (Perkin Elmer/Applied Biosystems, Foster City, CA), and their sizes were analyzed by GeneScan software (version 3.1, Perkin Elmer/Applied Biosystems, Foster City, CA). Analyses for Enp followed the method of Abe et al. (1992). Recombination values between maturity loci and markers were estimated using the maximum likelihood method (Allard, 1956), and were converted into genetic distances (centimorgan: cM) by the Kosambi function.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Development of NILs for ILD Insensitivity
Miharudaizu and Sakamotowase flowered 2 to 5 d later under ILD than under ND (Table 1). A Harosoy NIL with e3e3e4e4 (OT89-5) showed no delay in flowering under ILD. Flowering time of Harosoy NILs with e3e3E4E4 (L62-667) and E3E3e4e4 (OT94-41) under ND was a few days later. However, these two NILs did not develop any flower buds under ILD till the end of the experiment (66 d after sowing). The results obtained for the Harosoy NILs are thus consistent with the results of previous studies (Buzzell and Voldeng, 1980; Saindon et al., 1989; Cober et al., 1996a, b), which indicate that a double recessive genotype for the e3 and e4 alleles is essential to the flowering under ILD without a marked delay. Parents and OT89-5 produced pods of 20 mm or more (R3 to R5: Fehr et al., 1971) at the end of the ILD treatment.

Two F₉ RIL families (#9 and #130) were examined for the flowering phenology under ILD. Both families produced ILD-insensitive and ILD-sensitive segregants that fit the expected ratio of 1:3 for a single recessive gene controlling ILD insensitivity (Table 2). The progeny test was performed to confirm the monogenic inheritance in both families. Of the 152 plants segregating in family #9, 35 were classified as homozygous for ILD insensitivity, 78 as heterozygous, and 39 as homozygous for ILD sensitivity. Of the 137 plants segregating in family #130, 36 were classified as homozygous for ILD insensitivity, 66 as heterozygous, and 35 as homozygous for ILD sensitivity. The segregation ratio based on the progeny test fit the expected 1:2:1 ratio in family #9 (² = 0.316, df = 2, 0.75 < P < 0.90) and family #130 (² = 0.197, df = 2, 0.90 < P < 0.95). The seed obtained from two randomly selected F₁₀ plants, each homozygous for ILD insensitivity or sensitivity, were used as NILs for the maturity genes for ILD insensitivity (I) and sensitivity (S), respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Linkage maps of E1 and E4 soybean loci with isozyme and SSR markers. Figures in the right side of markers indicate composite locations from SoyBase.

Yamanaka et al. (2001a) found that a quantitative trait locus (QTL) with a large effect on R1 was located in MLG C2 in the cross between a Japanese cultivar, Misuzudaizu, and a Chinese accession, ‘Moshidou Gong 503’. On the basis of its map position, they deduced that the QTL corresponds to the E1 locus. By making a fine map of the region around the QTL with SSRs, Yamanaka et al. (2001b) revealed that the QTL was located between Satt365 and Satt489, which were located at a distance of 1.6 cM. Of the above two SSR markers, Satt489 showed a tight linkage to the maturity locus segregating in family #9. Our linkage analyses thus suggest that the maturity locus segregating in family # 9 is E1.

Genetic Basis of ILD Insensitivity in Miharudaizu and Sakamotowase
The results obtained in this study indicate that at least two loci, E1 and E4, segregated in the F₂ progeny of the cross between ILD-insensitive landraces of Hokkaido, Miharudaizu, and Sakamotowase. Since the e1 allele cosegregated with the marker allele from Sakamotowase, it may have been derived from Sakamotowase. Similarly, since the e4 allele cosegregated with the marker allele from Miharudaizu, it may have been derived from Miharudaizu. The results obtained in this study thus indicate that Miharudaizu probably has the genotype E1E1e4e4 and Sakamotowase probably has the genotype e1e1E4E4.

The insensitivity of flowering of soybean to ILD is conditioned by a double recessive homozygote at the E3 and E4 loci (Buzzell and Voldeng, 1980; Saindon et al., 1989). The genotypes at the E3 locus have not yet been determined for either parent or their derived NILs used in this study. Since only the E3 locus controls the insensitivity to FLD with high R:FR ratio (Buzzell, 1971), the responses of the parents, and the ILD-insensitive and ILD-sensitive NILs to FLD were evaluated to estimate the genotypes at the E3 locus. In addition to the parents and the ILD-insensitive NILs, neither of the two ILD-sensitive NILs (#9-S and #130-S) showed a marked delay in flowering under FLD with a R:FR ratio of 5.6, compared with ND. Therefore, the two sets of NILs most likely possess the e3 allele in common. The genotypes at three maturity loci, E1, E3, and E4, are thus estimated as E1E1e3e3e4e4 for Miharudaizu and ILD-insensitive NIL #130-I, e1e1e3e3E4E4 for Sakamotowase and ILD-insensitive NIL #9-I, and E1E1e3e3E4E4 for ILD-sensitive NILs #9-S and #130-S. The estimated genotype of Miharudaizu is therefore in agreement with the genotype of Kitamishiro, which was identified by Saindon et al. (1990). Kitamishiro was released via crossbreeding with Ohyachi 2, which belongs to the type I cultivar group, as does Miharudaizu (Abe et al., 1991). The type I cultivar group in Hokkaido may therefore have the genotype of E1E1e3e3e4e4 for ILD insensitivity.

There is a question about the genotype of Sakamotowase. A Harosoy NIL for the e3 allele, L62-667, possesses the genotype of e1e1e3e3E4E4, the same as the estimated genotypes of Sakamotowase and NIL #9-I in this study. As expected, L62-667 did not develop any flower buds during the ILD treatment used in this study. The e1 allele could not singly induce the ILD insensitivity in the presence of the E4 allele, as has been suggested by Cober et al. (1996a)( b). On the other hand, Cober and Voldeng (2001a) detected a new locus for response to ILD (E7), which is linked to the E1 locus at a genetic distance of 6.2 cM. The e7 allele weakens the sensitivity to ILD only under the genotype of e1e1e3e3e4e4 (Cober and Voldeng, 2001b). Therefore, one possibility is that the ILD insensitivity of Sakamotowase is controlled by a combination of the e1 and e7 alleles in the presence of the E4 allele. The action of the e7 allele on ILD insensitivity was evaluated for plants with the genotypes of E1E1e3e3e4e4 and e1e1e3e3e4e4 (Cober and Voldeng, 2001b), but it has not yet been determined for plants having the E4 allele. Another possibility is that an unknown gene which, singly or in combination with the e1 allele, induces plants homozygous for the e3 and E4 alleles to flower under ILD, is involved in the ILD insensitivity of Sakamotowase.

In summary, the present study clarified the genotypes of the two ILD-insensitive Japanese landraces of different origins at three maturity loci, E1, E3, and E4. Further studies are in progress to determine the genetic mechanism of the ILD insensitivity of Sakamotowase and NIL #9-I.

	ACKNOWLEDGMENTS

 
This research was supported by the Japan Society for the Promotion of Science (JSPS) (Grant No. 0766000). We appreciate Dr. R. L. Nelson at USDA-ARS Univ. of Illinois, and Dr. E. R. Cober and Dr. H. D. Voldeng at Plant Res. Ctr., Agric. Agri-Food Canada for supplying the seeds of the Harosoy near-isogenic lines. We are thankful to T. Misawa for his technical assistance.

Received for publication March 31, 2002.

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This Article Abstract Figures Only Full Text (PDF) Free Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in ISI Web of Science Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (7) Citing Articles via Google Scholar Google Scholar Articles by Abe, J. Articles by Shimamoto, Y. Search for Related Content PubMed Articles by Abe, J. Articles by Shimamoto, Y. Agricola Articles by Abe, J. Articles by Shimamoto, Y. Related Collections Crop Genetics Rice