A Dominant Locus, qbsc-1, Controls β Subunit Content of Seed Storage Protein in Soybean [Glycine max (L.) Merri.]

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1 Journal of Integrative Agriculture Advanced Online Publication: 2013 Doi: /S (13) A Dominant Locus, qbsc-1, Controls β Subunit Content of Seed Storage Protein in Soybean [Glycine max (L.) Merri.] WANG Jun *, LIU Lin *, GUO Yong, WANG Yong-hui, ZHANG Le, JIN Long-guo, GUAN Rong-xia, LIU Zhang-xiong, WANG Lin-lin, CHANG Ru-zhen and QIU Li-juan National Key Facility for Gene Resources and Genetic Improvement (NFCRI)/Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing, , P.R.China Abstract Soybean seed storage protein is one of the most important plant vegetable protein, and β subunit is of great significance to enhance soybean protein quality and processing property. F 2 segregated population and residual heterozygous lines (RHL) derived from the cross between Yangyandou (low level of β subunit) and Zhonghuang13 (Normal level of β subunit) were used for mapping of β subunit content. Our results showed that β subunit content was controlled by a single dominant locus, qbsc-1 (Beta subunit content), which was mapped to a region of 11.9cM on chromosome 20 in F 2 population of 85 individuals. This region was narrowed down to 2.5 cm between BARCSOYSSR_20_0997 and BARCSOYSSR_20_0910 in RHL with a larger population size of 246 individuals. There were 48 predicted genes within qbsc-1 region based on the reference genome (Glyma 1.0, Williams 82), including the two copies of β subunit coding gene CG4. An InDel marker developed from a TT insertion in one copy of CG4 promoter region in Yangyandou cosegregrated with BARCSOYSSR_20_0975 within qbsc-1 region, suggesting that this InDel marker maybe a useful marker for MAS. Key words: soybean seed storage protein, β subunit, QTL mapping 1 Introduction Soybean [Glycine max (L.) Merr.] is one of the most important resource of plant vegetable protein for human and animal feeds (Clarke and Wiseman 2000). Soybean storage protein accounts for approximately 40% of seed dry weight (Nelsen 1995; Wilson 1987), of which around 70% are composed of two major fractions, glycinin (11S) and β-conglycinin (7S) (Derbyshire et al. 1976). As the only vegetable protein resource containing all eight essential amino acids (Dudek 2001; Morrison and Hark 1999), soy protein has a relatively low level of sulfur-containing animo acids. 7S β-conglycinin is composed of α, α, β subunits, with a molecular weight of 76kDa, 72kDa, and 52kDa respectively (Thanh and Shibasaki 1977). β subunit, which has been proved to be one of the allergen source (Adachi et al. 2009; Krishnan et al. 2009), is the only subunit of major soybean seed storage protein does not contain any cystine, cysteine or methionine. On the other hand β subunit could enhance thermal stability and emulsifying property during the step of soy food processing (Utsumi et al. 1997; Mo et al. 2011). Correspondence QIU Li-juan

2 Therefore, discovering genes controlled β subunit content has become increasingly important for both soy protein quality and processing property. Deficient mutant has always been perfect materials for genetic study of soybean seed protein composition. In earlier studies, most of these mutants were characterized as lacking α, α, (α+β), (α +α) or (α +α+β), until recently a lot of varieties with low level of β subunit content has been identified from minicore collection of Chinese soybean germplasm or other collections, however quite few of them were β subunit-null (Kitamura and Kaizuma 1981; Kitamura et al. 1984; Ladin et al. 1984; Tsukada et al. 1986; Kaizuma et al. 1989; Odanaka and Kaizuma 1989; Takahashi et al. 1994; Liu et al. 2006; Wang et al. 2008). This was probably because the accumulation of β subunit was easily be affected by numerous stresses, including ABA, drought, and sulfur-nitrogen deficiency (Holowach et al. 1984; Murphy and Resurreccion 1984; Gayler and Sykes 1985; Bray and Beachy 1985; Kim et al. 1999; Blanuša et al. 2000). So far genetic studies related to β subunit content were largely based on overall analysis of 7S conglycinin or total soybean seed protein composition. A mutant QT2 obtained from wild soybean with 7S conglycinin-deficient was caused by mutation of with a single dominant gene Scg-1 (suppressor of β-conglycinin) (Hajika et al. 1996, 1998), and Scg-1 was later on mapped to chromosome 20 where tightly linked coding genes of α and β subunits located (Tsubokura et al. 2006a). A total of 15 genes termed CG1-CG15 were reported to encode 7S β-conglycinin, in which only few was assigned to α (CG1), α (CG2, CG3), and β subunit (CG4) (Harada et al. 1989). With the consideration that the coding genes of α and β subunits were well conserved with high sequence similarity, Teraishi et al. (2001) and Tsubokura et al. (2006a, b) hypothesized that sequence within Scg-1 locus could form a stem-loop structure and regulate the 7S β-conglycinin subunit genes by RNAi. Another mutant cgdef lacking all three subunits of 7S β-conglycinin simultaneously was proved to be controlled by a single recessive allele referred as cgdef (Kitagawa et al. 1991; Hayashi et al. 2000). The Cgdef gene was mapped to chromosome 19 (Linkage group L) at the position between the Satt523 and Sat_388 simple sequence repeat (SSR) markers in a F 2 segregating population (Hayashi et al. 2009). Pathee et al. (2004) identified 16 glycinin and conglycinin related QTLs, of which Satt461, Satt249 and Satt255 located on linkage group D2, J, and N respectively contributed to β subunit. Among these efforts to map certain gene governing 7S β-conglycinin or specially β subunit, no consensus conclusion could be drawn, may implied that complicated regulation network(s) might existed to accommodate 7S β-conglycinin accumulation. In this study, a landrace named Yangyandou with trace amount of β subunit was identified from minicore collection of Chinese soybean germplasm (Wang et al. 2008). The trace amount of β subunit was stably inherited in different years and different environments. In order to map the QTL controlling β subunit content, Yangyandou was used to cross with Zhonghuang 13, a widely planted cultivar in China, and developed F 2 and RHL populations. The results showed that the β subunit content was controlled by a dominant locus qbsc-1. Linkage analysis assigned qbsc-1 to a QTL region of 2.5 cm between markers BARCSOYSSR_20_0910 and BARCSOYSSR_20_0997 on chromosome 20. These results contribute the first step toward positional cloning of qbsc-1 and uncovering mechanism underlying the regulation of soy protein. 2

3 Results Genetic and correlation analysis of protein subunits in soybean To better understand the relationship between β subunit and other composition, F 2 segregated population of 85 individuals and RHL of 246 individuals derived from one F 2 line were developed by the cross between Yangyandou ( ) and Zhonghuang 13( ). Seven major components, namely Lox, α, α, β, A3 (acidic), Acidic and Basic, which composed the majority of seed protein were clearly separated on SDS-PAGE gel (Figure 1). There was an apparently continuous distributed variation in terms of relative content of each subunit (Table 1). The relative content of Lox, α, and β subunit in Yangyandou (parent 1) were lower than that of Zhonghuang13 (parent 2), while others were higher (Table 1). But the variation range of β, acidic and basic subunits were wider than that of the rest (Table 1). It was interesting to notice that progenies from F 2 segregating population (2008) showed a relatively larger variation range than that of RHL (2010) (Table 1). Correlation coefficient analysis of different protein subunits showed that a highly negative correlation [r=-0.697(f 2), r=-0.684(rhl) P<0.01] between β subunit and overall 11S glycinin (A3, acidic and basic) content, although only moderate correlation was observed between β subunit and any component of 11S glycinin (Table 2). 11S glycinin and 7S conglycinin content displayed a negative correlation as well (r=-0.844(f 2), r=-0.92(rhl), P<0.01, Table 2). In regarding to 11S:7S value, Yangyandou and Zhonghuang13 has a 11S:7S value of 1.28 and 2.54 respectively in 2008, but this value fluctuated from 1.20 to 2.47 in In both F 2 and RHL populations, 11S:7S value varied from 0.99 to 2.94 and from 1.19 to 3.23 respectively (Table 1). Mapping of β subunit content in F2 segregated population In the F 2 segregated population derived from the cross between Yangyandou ( ) and Zhonghuang 13( ), the distribution of relative β subunit content appeared to have two distinct peaks, the ratio of normal content (Peak 2) to low content (Peak 1) was 2.70:1, which was then attested to fit the single locus model with a segregation ratio of 3:1 by Chi-square test (X 2 =0.123, P=0.726) (Fig. 2-A), indicated that β subunit content was controlled by a single dominant QTL locus, qbsc-1. To obtain more information about qbsc-1, bulked segregant analysis was used to identify related SSR markers. The results of BSA showed that BARCSOYSSR_20_0972 on chromosome 20 was genetically cosegregated with β subunit content indicated that region around this marker was a candidate locus for qbsc-1. A linkage map with 7 SSR markers and a total length of 14.9 cm was established based on phenotype and genotype data obtained from 85 F 2 individuals. The qbsc-1 region for β subunit content was identified on this map based on composite interval mapping. The maximum LOD score was 23.79, which occurred at BARCSOYSSR_20_0997, and qbsc-1 was mapped to a region of 11.5 cm between markers BARCSOYSSR_20_1010 and BARCSOYSSR_20_0910, which could explain 70.68% of phenotypic variation on average (Figure 2b). Within this region three SSR markers namely BARCSOYSSR_20_0978, BARCSOYSSR_20_0975 and BARCSOYSSR_20_0972 was located on the 3

4 same position (1.3cM away from BARCSOYSSR_20_0997). The additive effect of qbsc-1 was 3.05%, and no dominance effect was observed. Mapping of qbsc-1 in RHL In order to determine the location of qbsc-1 more precisely, we phenotyped the RHL population and a similar segregation ratio of normal content (Peak 2) to low content (Peak 1) was observed to be 3.47:1(X 2 = , P=0.459) as that in F 2 population (Fig. 3-A). Based on 246 RHL individuals, we were able to construct a linkage map around qbsc-1 locus by using 7 SSR markers as in F 2 population except BARCSOYSSR_20_1050, because this marker had no polymorphism in RHL (Data not shown). Finally the qbsc-1 was mapped to a region of 2.5cM between BARCSOYSSR_20_0997 and BARCSOYSSR_20_0910 (Fig. 3-B). Within this region the three SSR markers located on the same genetic position (7.1 cm, Fig. 2-B) was found to be separated by intervals of 0.5cM and 0.9cM respectively (Fig. 3-B). The location of qbsc-1 was almost the same region as the map of F 2 population but peaked at BARCSOYSSR_20_0975 with a LOD score of 71.37, which could explain 72.75% of phenotypic variance (Fig. 3-B). However, all SSR markers located within this region were significantly correlated with β subunit content (Student t-test P-value < 0.001, Table 4). The additive effect of RHL was 3.59% which is slightly higher than that in F 2 population. The relationship between β subunit content and its coding genes Bioinformatics analysis showed that the 2.5 cm qbsc-1 region covering a physical distance of ~460 kb was predicted to comprise 48 gene models related to metabolism, defense response, signal transduction and cupin protein accumulation (Supplemental Table 1). Of these genes, Glyma20g and Glyma20g was predicted to be two copies of β subunit coding gene, CG4 (Harada et al. 1989). To define the relationship between the mapped qbsc-1 region and β subunit coding genes, the genomic sequence of these two genes was amplified from Yangyandou and Zhonghuang 13, and sequenced respectively. For CG4 copy 1 (Glyma20g ), nine SNPs (Single nucleotide polymorphism) and one InDel were identified within promoter, exon and intron region respectively in Yangyandou compared with that in Zhonghuang13 (Table 3). In promoter region, there were two nucleotide transition between cytosine (C) and thymidine (T) (-295, -663), and a TT insertion (-287), which was coincidently located in a SEF4 (Soybean Embryo Factor 4) binding motif RTTTTTR (R= A or G) (Allen et al. 1989). Two out of three SNPs in the exon region leaded to amino acid change (F96L, F197L), but not altered secondary and tertiary structure (Data not shown). There were only two SNPs occurred in non-coding region (intron 4 and 3 UTR) of CG4 copy 2 (Glyma20g ) (Table 3), which would not lead to any amino acid substitution. An InDel marker developed from the TT insertion (-287) in CG4 copy 1 promoter was co-located at same position as BARCSOYSSR_20_0975 on the map based on RHL (Fig. 3-B), and it showed a higher correlation with β subunit content compared to other SSR markers within this region (Table 4). To test the InDel marker, we evaluated 220 varieties in soybean mini-core collection by using this marker. The varieties with same band pattern as Yangyandou (L) was 1/5 of that with same band pattern as Zhonghuang13 4

5 (N) (Fig. 4-A). Varieties with L band has relatively lower β subunit content (12.6%) than varieties with N band (14.7%) (Fig. 4-B). The frequency of either varieties with L band or N band showed continuous distribution (Fig. 4-C). Discussion β subunit content is controlled by a single dominant locus, qbsc-1 Due to the lack of either spontaneous or mutagen-induced mutant with decreased amount of β subunit and the feature that β subunit content is easily to be affected by numerous factors (ABA, drought, sulfur-nitrogen deficiency et al.), mapping of β subunit content-related QTL lagged behind the study in α and α subunit (Holowach et al. 1984; Gayler and Sykes 1985; Bray and Beachy 1985; Kim et al. 1999; Blanuša et al. 2000). The identification of Yangyandou, a stably inherited landrace in various environment made it feasible for us to map β subunit content-related QTL directly. In order to eliminate environmental effect on β subunit accumulation, we chosed the F 2 and RHL from a single F 1 plant and a single F 3 RIL plant respectively to guarantee least environmentally induced variation within population, and planted these two populations at different time and in different place to reduce the environmental effects between populations (detailed described in Plant Materials). In our study, both F 2 and RHL population showed approximately 3:1 ratio of normal to low content of β subunit, consistently indicated that β subunit content was dominated by a single QTL, qbsc-1 (Figs. 2-A, 3-A). qbsc-1 was preliminarily mapped to a region of 11.5 cm based on F 2 population, and then narrowed down to 2.5cM between markers BARCSOYSSR_20_0997 and BARCSOYSSR_20_0972 in RHL (Fig. 3-B). It is not the first case to find that soybean seed protein composition was controlled by a single locus either dominant or recessive. Two mutants lacking all three subunits of 7S β-conglycinin (α, α and β subunit) was reported to be controlled by a single dominant gene Scg-1 and a single recessive gene Cgdef respectively (Kitagawa et al. 1991; Hayashi et al. 2000; Teraishi et al. 2001; Tsubokura et al. 2006a, b). The 2.5 cm of qbsc-1 region covered a physical distance of around 460kb, in which 48 gene models were included. Of these genes two genes were corresponding to two copies of β subunit coding gene CG4 (Harada et al. 1989; Li and Zhang 2011). Only a few base substitutions or insertion/deletion were identified in two copies of CG4 between Yangyandou and Zhonghuang13 (Table 3). Among them the InDel altered the flanking sequence of SEF4 (Soybean Embryo Factor 4) binding motif (RTTTTTR, R=A or G) (Allen et al. 1989), which would possibly lessen the tightness of SEF4 binding. SEF4 was one of four nuclear proteins which were characterized to be remarkably important in modulating seed storage protein gene expression pattern (Lessard et al. 1991). Genetic analysis in RHL demonstrated that the InDel was mapped to qbsc-1 region and collocated with BARCSOYSSR_20_0975 at the peak of LOD score (Fig. 3-B), suggested that the TT InDel mutation in CG4 copy 1 promoter region cannot be eliminated from candidate genes for β subunit content. Scg-1 locus was also reported to be located in the same chromosomal region as the α and β subunits, but it should be different from qbsc-1 as they controlled two absolutely different phenotype (Teraishi et al. 2001). 5

6 In the mini-core collection of soybean germplasm, the frequency of varieties with either L or N InDel band pattern were continuously distributed (Fig. 4-C). Higher frequency of N band was consistent with the observation that most varieties have normal content of β subunit (Fig. 4-A). The average β subunit content of varieties with L band was significantly lower than that with N band (Fig. 4-B), indicated that the InDel was efficient in marker assisted evaluation of varieties for β subunit content. But we also noticed that some varieties with N band have low content of β subunit (Fig. 4-C), implied that β subunit content was a complicated trait and other mechanisms might contribute to the regulation of β subunit accumulation. β subunit was a major factor affecting soybean seed protein composition In this study, we statistically demonstrated that a significant negative correlation existed between β subunit and 11S glycinin in both F 2 and RHL populations (Table 2), which is consistent with the correlation analysis results among different soybean germplasm collections (Ma et al. 2006; Wang et al. 2011). This phenomenon was in agreement with the reports that sulfur deficiency caused a 40% decrease in the level of 11S glycinin and a 3-fold increase in the β subunit of 7S β-conglycinin, and an increase in 11S glycinin content and a suppressed accumulation of β subunit in cotyledon when exogenous methionine (Met) was applied to culture media in vitro (Holowach et al. 1984; Gayler and Sykes 1985). Relatively smaller variation was observed in α and α subunit content in both F 2 segregated population and RHL (Table 2). This is probably because α and α subunit accumulated one or two weeks earlier than β subunit but soon reached maximum about 30 days after flowering (Hill and Breidenbach 1974). These results might also imply that β subunit was a major factor affecting soybean seed protein composition. As soybean is the only vegetable protein resource containing all eight essential amino acids but with a relatively low level of sulfur-containing amino acids (Morrison and Hark 1999; Dudek 2001). Concerted efforts have been focused on increasing soybean seed protein content rich in sulfur-containing amino acids by various strategies including traditional breeding and elevating proteins rich in sulfur amino acids mediated by transgenic technique. But each strategy has its advantages and disadvantages (Krishnan 2005). β subunit is void of cysteine while 11S glycinin is rich in sulfur-containing amino acids (Utsumi et al. 1997). In this study, the negative correlated accumulation type between β subunit and 11S glycinin resulted in a wide distribution range of 11S:7S value in either F 2 or RHL progenies, which provide abundant material for different purpose oriented breeding. To summarize, lower level of β subunit content in soybean seed was found to be mainly controlled by a single dominant locus, qbsc-1, which was mapped to a region of 11.9cM in F 2 and this region was then narrowed down to 2.5cM in RHL derived from Yangyandou and Zhonghuang13. Among 48 genes within qbsc-1 region, β subunit coding genes were included. A TT InDel developed from CG4 (Glyma20g ) promoter cosegregated with phenotype and co-located with marker BARCSOYSSR_20_0975 in qbsc-1 region. This marker could be further utilized in marker assisted selection in breeding. Materials and Methods 6

7 Plant materials Soybean genotype Yangyandou and Zhonghuang 13 were used in this study. Yangyandou is a land race of Yunnan province selected from mini core collection of cultivated soybean (Qiu et al. 2009). Yangyandou has trace amount of β subunit and a 11S/7S value of as high as ~2.5. Zhonghuang13 is one of the most wildly planted cultivar in China. The cross between Yangyandou ( ) and Zhonghuang 13( ) was made during the summer season of F 1 seeds were planted in Hainan during winter of 2008, and consequently a total of 85 F 2 individuals were obtained. RHL comprised 246 individual seeds were derived from one individual plant from F 3 recombinant inbreeding lines planted in Beijing in Hybridization were confirmed by checking the segregation of relative content of β subunit for each F 2 and RHL seeds on SDS-PAGE. A total of 220 varieties from mini-core collection of soybean germplasm (Qiu et al. 2009) were used for validation of InDel marker developed. Four individual seeds of each variety were analyzed for β subunit content. SDS-PAGE and protein content calculation Five micrograms of soybean seed flour were grinded from each mature individual seed cotyledon by electric carving knife (Proxxon, #D-54518). Total soluble protein of soybean seed flour was dissolved in protein extraction buffer (0.05 mol L -1 Tris-base, 5 mol L -1 Urea, 0.2 mg L -1 Sodium Dodecyl Sulfonate, 0.025% bromophenol blue, ph8.0) contained 0.1 mol L -1 β-mercaptoethanol. Mixture was vortexing time by time and incubated at room temperature for 4 h, undissolved fat layer was removed by centrifuging at g for 10 min at 4, and aliquot of 5 μl supernatant with total soluble protein was ready for loading. Protein and polypeptides were separated by SDS-PAGE basically according to the description by Laemmli (1970). Gels were stained and destained in freshly prepared solution as described by Pathee (2004). Destatined gels were then enveloped by two separate cellophane after soaking in 5% glycerol for 1 h. Air dried gels were scanned by HP laserjet 3300 Scanner. Colorful gel graph were first transformed into 16-bit gray by Adobe Photoshop 7.0, then Quantity one (4.6.2) software were used to detect bands and determine band density (Biorad, # ). Seven major bands, namely Lox, α, α, β, A3, Acid and Basic band were manually detected and the volume of each band were calculated by Gauss-model algorithm based on band density. The percentage of relative volume to total volume of each sample were deemed as the relative content of each protein or polypeptide. DNA isolation, polymerase chain reaction and gel electrophoresis Genomic DNA of F 2 individual plants were extracted from young leaves by Genomic DNA Purification Kit (Fermentas, #K0512). Same method was used to isolate genomic DNA of RHL from soybean seed flour. Polymerase chain reaction (PCR) was performed basically in a 20-μL system comprise ~100 ng genomic DNA, 2 μl 10 PCR buffer, 3.75 mmol L -1 dntps mixture, 2μM mixture of forward and reverse primers, and 1 U of TaqQ (Doupson). Temperature program applied in the PCR was basically according to Pathee (2004) with an annealing 7

8 temperature of 55. PCR products were tested on a 6% denatured polyacrylamide gel electrophoresis (PAGE) (Pathee et al. 2004). Sequencing PCR amplification of two copies of CG4 in Yangyandou and Zhonghuang13 was performed basically the same system as mentioned above except that the Polymerase was replaced by ExTaq (TAKARA, #DDR100A) to guarantee the fidelity. Nested specific primers for CG4 copy 1 were listed in supplemental Table 2. PCR product was cloned to pmd 18-T vector (TAKARA, #D101A) then sequenced. Genetic mapping by SSR analysis Ten of extremely low content and high content of β subunit in F 2 population were selected respectively to create two distinct DNA pools for bulked segregant analysis. A total of 509 SSR markers from 20 soybean chromosomes (soymap 2.0) were used to screen locus related to β subunit content (Cregan et al. 1999; Song et al. 2004) in bulked DNA pools. When specific locus related to β subunit content were found a batch of 7 SSR markers located in the vicinity region of that locus from Beltsville Agriculture Research Center (BARCSOYSSR_1.0) were selected to construct a genetic map (Supplemental Table 3)(Song et al. 2010). Linkage map was constructed by Map Manager QTXb17 and Cartographer using composite interval mapping (Manly et al. 2001). A criterion of probability, equivalent to a LOD (likelyhood of odds) score of 2.5 was used as threshold line for QTL identification. Confidence interval region was defined as 1 LOD flanking the maximum LOD score. Acknowledgments This research was funded by National High Technology Research and Development Program of China (2012AA101106), National Basic Research Program of China (2009CB118404), Key Technologies Research and Development Program of China (2011BAD35B06) and National Transgenic Major Program (2008ZX ). Appreciations are extended to Long Yan, Jian Song and Huihui Li for support in data analysis, and to Nang Myint Phyu Sin Htwe for critical review of the manuscript. References Adachi A, Horikawa T, Shimizu H, Sarayama Y, Ogawa T, Sjolander S, Tanaka A, Moriyama T Soybean beta-conglycinin as the main allergen in a patient with food-dependent exercise-induced anaphylaxis by tofu: food processing alters pepsin resistance. Clinic & Experimental Allergy, 39, Allen R D, Bernier F, Lessard P A, Beachy R N Nuclear factors interact with a soybean beta-conglycinin enhancer. The Plant Cell, 1, Blanuša T, Stikić R, Vucelić-Radović B, Barać M, Veličković D Dynamics of seed protein biosynthesis in two soybean genotypes differing in drought susceptibility. Biologia Plantarum, 43,

9 Bray E A, Beachy R N Regulation by ABA of beta-conglycinin expression in cultured developing soybean cotyledons. Plant Physiology, 79, Cregan P B, Jarvik T, Bush A L, Shoemaker R C, Lark K G, Kahler A L, Kaya N, VanToai T T, Lohnes D G, Chung J, Specht J E An integrated genetic linkage map of the soybean genome. Crop Science, 39, Clarke E J, Wiseman J Developments in plant breeding for improved nutritional quality of soybeans I. protein and amino acids content. Journal of Agricultural Science, 134, Derbyshire E, Wright D J, Boulter D Legumin an vicilin, storage protein of legume seeds. Phytochemistry 15, Dudek S G Nutrition Essentials for Nursing Practice. 4th ed. Philadelphia: Lippincott. Gayler K R, Sykes G E β-conglycinins in developing soybean seeds. Plant Physiology, 67, Gayler K R, Sykes G E Effects of nutritional stress on the storage proteins of soybeans. Plant Physiology, 78, Hajika M, Takahashi M, Sakai S, Igita M A new genotype of 7S globulin (β-conglycinin) detected in wild soybean (Glycine soja Sieb. et Zucc.). Breed Science, 46, Hajika M, Takahashi M, Sakai S, Matsunaga R Dominant inheritance of a trait lacking β-conglycinin detected in a wild soybean line. Breed Science, 48, Harada J J, Barker S J, Goldberg R B Soybean beta-conglycinin genes are clustered in several DNA regions and are regulated by transcriptional and posttranscriptional processes. The Plant Cell, 1, Hayashi M, Nishioka M, Kitamura K, Harada K Identification of AFLP markers tightly linked to the gene for deficiency of the 7S globulin in soybean seed and characterization of abnormal phenotypes involved in the mutation. Breed Science, 50, Hayashi M, Kitamura K, Harada K Genetic mapping of Cgdef gene controlling accumulation of 7S globulin (beta-conglycinin) subunits in soybean seeds. Journal of Heredity, 100, Hill J E, Breidenbach R W Proteins of soybean seeds: II. Accumulation of the major protein components during seed development and maturation. Plant Physiology, 53, Holowach L P, Thompson J F, Madison J T Storage protein composition of soybean cotyledons grown in vitro in media of various sulfate concentrations in the presence and absence of exogenous l-methionine. Plant Physiology, 74, Kitagawa S, Ishimoto M, Kikuchi F, Kitamura K A characteristic lacking or decreasing remarkably 7S globulin subunits induced with γ ray irradiation in soybean seeds. Japanese Journal of Breeding, 41, Kim H, Hirai MY, Hayashi H, Chino M, Naito S, Fujiwara T Role of O-acetyl-L-serine in the coordinated regulation of the expression of a soybean seed storage-protein gene by sulfur and nitrogen nutrition. Planta, 209, Krishnan H B, Kim W S, Jang S, Kerley M S All three subunits of soybean β-conglycinin are potential food allergens. Journal of Agricultural Food Chemistry, 57,

10 Krishnan H B Engineering soybean for enhanced sulfur amino acid content. Crop Science, 45, Laemmli U K Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, Lessard P A, Allen R D, Bernier F, Crispino J D, Fujiwara T, Beachy R N Multiple nuclear factors interact with upstream sequences of differentially regulated beta-conglycinin genes. Plant Molecular Biology, 16, Li C, Zhang Y M Molecular evolution of glycinin and β-conglycinin gene families in soybean (Glycine max L. Merr.). Heredity, 106, Liu S, Ohta K, Dong C, Thanh V C, Ishimoto M, Qin Z, Hirata Y Genetic diversity of soybean (Glycine Max (L.) Merrill) 7S globulin protein subunits. Genetic Resource Crop Evolution, 53, Ma H, Wang X S, Liu C, Yu T, Hao X Y, Gao W R, He X L The content variation of 7S, 11S globulins and their subunits of seed storage protein 706 Chinese soybean germplasm. Soybean Science, 25, (in Chinese) Manly K F, Cudmore J R H, Meer J M Map Manager QTX, cross-platform software for genetic mapping. Mamm. Genome, 12, Meinke D W, Chen J, Beachy R N Expression of storage-protein genes during soybean seed development. Planta, 153, Mo X, Wang D, Sun X S Physicochemical properties of beta and alpha', alpha subunits isolated from soybean beta-conglycinin. Journal of Agricultural Food Chemistry, 59, Morrison G, Hark L Medical nutrition and disease, 2nd ed. Blackwell Science, Malden, MA Murphy P A, Resurreccion A P Varietal and environmental differences in soybean glycinin and beta-conglycinin content. Journal of Agricultural Food Chemistry, 32, Nielsen N C Soybean seed composition. In: Soybean: genetics, molecular biology & biotechnology, Biotechnology in Agriculture no 14. CAB International, Wallingford, U.K. Panthee D R, Kwanyuen P, Sams C E, West D R, Saxton A M, Pantalone V R Quantitative trait loci for β-conglycinin (7S) and glycinin (11S) fractions of soybean storage protein. Journal of American Oil Chemists Society, 81, Qiu L J, Li Y H, Guan R X, Liu Z X, Wang L X, Chang R Z Establishment, representative testing and research progress of soybean core collection and mini core collection. Acta Agronomica Sinica, 35, (in Chinese) Song Q J, Marek L F, Shoemaker R C, Lark K G, Concibido V C, Delannay X, Specht J E, Cregan P B A new integrated genetic linkage map of the soybean. Theoretical and Applied Genetics, 109, Song Q J, Jia G F, Zhu Y L, Grant D, Nelson R T, Hwang E Y, Hyten D L, Cregan P B Abundance of SSR motifs and development of candidate polymorphic SSR markers (BARCSOYSSR_1.0) in soybean. Crop Science, 50, Thanh V H, Shibasaki K β-conglycinin from soybean proteins. Isolation and immunological and physicochemical properties of the monomeric forms. Biochimica Biophysica Acta, 490,

11 Tsubokura Y, Hajika M, Harada K. 2006a. Molecular characterization of a β-conglycinin deficiency soybean. Euphytica, 150, Tsubokura Y, Hajika M, Harada K. 2006b. Molecular markers associated with β-conglycinin deficiency in soybean. Breed Science, 56, Utsumi S, Matsumura Y, Mori T Structure-function relationships of soy proteins. In: Food proteins and their applications. Marcel Dekker, Inc., New York Wang L L, Guan R X, Qi Z, Qiu L J, Luo S P Analysis of 11S/7S ratio between soybean mini core collection and cultivars. Journal of Plant Genetic Resource 9, (in Chinese) Wilson R F Seed metabolism. In: Soybeans: improvement, production and uses. Agronomy Monograph no. 16. Madison, Wisconsin: American Society of Agronomy. Wang Y P, Li G Q, Guo S J, Wang P Variation of protein subunits of soybean germplasms of different eco-types in Shanxi. Acta Ecologica Sinica, 31, (in Chinese) Figure Legends Fig. 1 Accumulation pattern of seed storage protein fraction of F2 segregated individuals derived from Yangyandou (lane 1) Zhonghuang 13(lane 2) in SDS-PAGE. Lane 3-10 showed some representative individuals. Fig. 2 Frequency distribution of β subunit content (a) and linkage map constructed based on F2 population (b) derived from Yangyandou (parent 1) Zhonghuang13 (parent 2). Putative qbsc-1 locus was marked by black arrow, Marker in red indicated the peak of LOD score. Dark gray represented a qbsc-1 locus region with high confidence. Fig. 3 Frequency distribution of β subunit content (a) and linkage map constructed based on RHL population (b) derived from Yangyandou (parent 1) Zhonghuang13 (parent 2). Putative qbsc-1 locus was marked by black arrow. Markers on the peak of LOD score were presented in red. qbsc-1 region with high confidence were shadowed in dark gray. Fig. 4 Evaluation of β subunit content in soybean mini-core germplasm collection by InDel. (a) Variety number with L and N band pattern (L: variety with same InDel band pattern as Yangyandou; N: variety with same InDel band pattern as Zhonghuang13); (b) Average β subunit content of varieties with different InDel band pattern; (c) Frequency distribution of varieties with different InDel band pattern. 11

12 Table 1. Descriptive statistics of each protein or polypeptide content in F2 and RHL population Traits Population type Min a Max b Mean SD c P d 1 P e 2 Lox F RHL α' F RHL α F RHL β F RHL S F RHL A3 F RHL Acidic F RHL Basic F RHL S F RHL S/7S F RHL a Min=minimum; b Max=maximum; c SD= standard deviation; d P 1=parent 1 (Yangyandou); e P2=parent 2 (Zhonghuang13) Table 2 Correlation coefficients between different protein fraction and 11S/7S in F2 segregated population(left lower) and RHL (upper right) F2 Lox α' α β A3 Acidic Basic 7S 11S 11S:7S RHL Lox *** 0.21 *** *** *** 0.17 *** *** *** Lox α' *** *** *** *** *** *** *** α' α *** *** *** *** *** *** α β *** *** *** *** 0.79 *** *** *** β A *** *** *** *** *** *** *** A3 Acidic ** *** *** ** ** *** *** *** Acidic Basic *** *** * ** *** *** *** *** Basic 7S *** *** ** *** *** *** 7S 11S *** *** *** *** *** *** *** *** 11S 11S:7S * *** *** * *** *** *** *** 1 11S:7S *,**,*** represent significant at a probability of 0.10,0.05, and 0.01 respectively. 12

13 Table 3 Sequencing statistics of β subunit coding genes among Yangyandou and Zhonghuang 13 Region Position a CG4 copy1(glyma20g ) CG4 copy2(glyma20g ) Yangyandou Zhonghuang13 Yangyandou Zhonghuang13 Promoter -663 C T T C TT Exon C (L b ) T (F c ) - - Intron G C G C - - Exon G (L) T (F) - - Intron A T G A 1439 C A - - Exon A T - - 3'UTR (untranslated region) C T a relative position in comparison to TSS (transcription starting site); b Leucine; c Phenylalanine Table 4. Statistic analysis of different markers within qbsc-1 region in RHL Marker L a M ± S b (%) N c M ± S (%) Student t-test BARCSOYSSR_20_ ± ± E-37 BARCSOYSSR_20_ ± ± E-45 InDel ± ± E-45 BARCSOYSSR_20_ ± ± E-45 BARCSOYSSR_20_ ± ± E-40 a L:individuals with same band pattern of Yangyandou; b M ± S: Mean ± standard deviation of average content of β subunit; c N:individuals with same band pattern as Zhonghuang 13 Fig. 1 Accumulation pattern of seed storage protein fraction of F 2 segregated individuals derived from Yangyandou (lane 1) Zhonghuang 13 (lane 2) in SDS-PAGE. Lane 3-10 showed some representative individuals. 13

14 Fig. 2 Frequency distribution of β subunit content (a) and linkage map constructed based on F 2 population (b) derived from Yangyandou (parent 1) Zhonghuang 13 (parent 2). Putative qbsc-1 locus was marked by black arrow, Marker in red indicated the peak of LOD score. Dark gray indicated a qbsc-1 locus region with high confidence. Fig. 3 Frequency distribution of β subunit content (a) and linkage map constructed based on RHL population (b) derived from Yangyandou (parent 1) Zhonghuang13 (parent 2). Putative qbsc-1 locus was marked by black arrow. Markers on the peak of LOD score were presented in red. qbsc-1 region with high confidence were shadowed in dark gray. 14

15 Fig. 4 Evaluation of β subunit content in soybean mini-core germplasm collection by InDel. (a) Variety number with L and N band pattern (L: variety with same InDel band pattern as Yangyandou; N: variety with same InDel band pattern as Zhonghuang13); (b) Average β subunit content of varieties with different InDel band pattern; (c) Frequency distribution of varieties with different InDel band pattern. Supplemental materials: Supplemental Table 1. Functional annotation of gene models between BARCSORSSR_20_0972 and BARCSOYSSR_20_0997 Supplemental Table 2. Primer sequences for CG4 copy1 and copy2 Supplemental Table 3. Primer sequences for SSR markers and InDel for linkage map construction 15