Relationship between Mutations of the Pectin Methylesterase Gene in

Article pubs.acs.org/JAFC

Relationship between Mutations of the Pectin Methylesterase Gene in Soybean and the Hardness of Cooked Beans Kyoko Toda,*,† Kaori Hirata,† Ryoichi Masuda, Takeshi Yasui, Tetsuya Yamada, Koji Takahashi, Taiko Nagaya, and Makita Hajika NARO Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki 305-8518 Japan S Supporting Information *

ABSTRACT: Hardness of cooked soybeans [Glycine max (L). Merr.] is an important attribute in food processing. We found one candidate gene, Glyma03g03360, to be associated with the hardness of cotyledons of cooked soybeans, based on a quantitative trait locus and fine-scale mapping analyses using a recombinant inbred line population developed from a cross between two Japanese cultivars, “Natto-shoryu” and “Hyoukei-kuro 3”. Analysis of the DNA sequence of Glyma03g03360, a pectin methylesterase gene homologue, revealed three patterns of mutations, two of which result in truncated proteins and one of which results in an amino acid substitution. The truncated proteins are presumed to lack the enzymatic activity of Glyma03g03360. We classified 24 cultivars into four groups based on the sequence of Glyma03g03360. The texture analysis using the 22 cultivars grown in different locations indicated that protein truncation of Glyma03g03360 resulted in softer cotyledons of cooked soybeans, which was further confirmed by texture analysis performed using F2 populations of a cross between “Enrei” and “LD00-3309”, and between “Satonohohoemi” and “Sakukei 98”. A positive correlation between hardness and calcium content implies the possible effect of calcium binding to pectins on the hardness of cooked soybean cotyledons. KEYWORDS: calcium, pectin methylesterase, seed hardness, soybean

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(Ha3) on chromosome 16.6 Previously, we identified two significantly stable QTLs, qHbs3-1 and qHbs6-1, on chromosomes 3 and 6, respectively, for hardness of cooked soybean cotyledons.7 Japanese soybean cultivars, “Natto-shoryu” and “Hyoukei-kuro 3”, and a recombinant inbred line (RIL) population derived from a cross between them were used for our previous study. Natto-shoryu showed more than twice the breaking stress of the cotyledon as that of Hyoukei-kuro 3. Contributions of the two QTLs to total phenotypic variance of the hardness of cooked soybean cotyledons were estimated using F5 and F6 generations, which were grown in 2010 and 2011, respectively. The contributions of qHbs3-1 were 47.4% and 44.3% for 2010 and 2011, respectively, and those of qHbs61 were 15.8% and 11.6% for the two years, respectively.7 Soybean seeds are composed of three major components: the cotyledon, embryonic axis, and seed coat (testa). Among them, the cotyledon probably has the predominant effect on the hardness of the cooked bean, because it is a major part of the seed. Moreover, the hardness of whole beans has shown a significant positive correlation with that of the embryo (cotyledon and embryonic axis, r = 0.976, P < 0.001).5 Thus, the qHbs3-1 was presumed to encode a gene which has a major effect on the hardness of cooked beans. In this study, one candidate gene, Glyma03g03360, which is a pectin methylesterase (PME) gene homologue, was identified to be associated with the hardness of cooked soybean cotyledons by fine-scale mapping of the qHbs3-1 region. We

INTRODUCTION Soybean [Glycine max (L). Merr.] is a major crop worldwide. It is used globally in oil production and as a feed grain; it is also used locally in processed foods, such as tofu, cooked beans (nimame), fermented steamed beans (natto), and fermented steamed bean pastes (miso). Soyfood has been consumed in Asia for over 1000 years and is becoming increasingly popular in other countries. The hardness of cooked soybeans is one of the most important factors for not only cooked beans but also natto and miso, which require a boiling or steaming process for softening beans before fermentation.1−3 Softer cooked beans are preferable for these food products. Harder beans require longer and stronger heating conditions, resulting in a darker color and unfavorable tastes (based on personal communication with manufacturers). Moreover, hard beans result in an undesirable texture and an unfavorable ammoniac flavor in natto.1 Makabe (2006) reported that cooked seed hardness is positively correlated with the quantity of calcium ions in the cooking solution.4 Negative correlation of the hardness with the amount of dissolved polysaccharides or boron during cooking of the seeds was also reported,2,4 but the seed component that determines cooked seed hardness remains to be identified. Genetic mechanisms have not been elucidated either, although differences in cooked seed hardness have been observed between cultivars.3,5 With recent advances in molecular techniques, it has become easier to manipulate quantitative trait loci (QTL). Using simple sequence repeat (SSR) markers, Zhang et al. identified two QTL, Ha1 and Ha2, for cooked seed hardness on chromosomes 16 and 1, respectively.3 Their group has confirmed the Ha2 region and has identified a new QTL © XXXX American Chemical Society

Received: June 15, 2015 Revised: August 31, 2015 Accepted: September 2, 2015

A

DOI: 10.1021/acs.jafc.5b02896 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Article

Journal of Agricultural and Food Chemistry also studied the relationship between the calcium content and hardness of cooked soybean cotyledons. PMEs are commonly present in plants and microorganisms such as fungi and bacteria.8,9 Evidence indicates that pectins are synthesized and methylesterified in the Golgi and thereafter secreted into the cell wall in a highly methylesterified state.10−12 Subsequently, they are deesterified by wall-associated PMEs that convert the methoxyl groups into carboxyl groups on the polygalacturonic acid chain and release both methanol and protons. It appears that most plant PMEs deesterify pectins in a blockwise fashion, e.g., linearly along the chain, which favors pectin gelation and concomitant cell wall stiffening due to the formation of cooperative Ca2+ crossbridges between free carboxyl groups of adjacent pectin chains.13 During the cooking process, it has been postulated that middle lamella pectin of the cell walls is depolymerized by βelimination of methyl-esterified polygalacturonic acids, promoting an increase in cell separation, and this softens the tissues.14 In legume seeds, tissue softening seems to be achieved mainly through the middle lamella polymer solubilization, which allows cell separation, producing a mealy texture.15 However, no report has been found on the effect of endogenous PME on the hardness of cooked beans. In this report, we discuss the role of an endogenous PME gene on the hardness of cooked beans, which has been demonstrated here in the soybean.

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Table 1. Soybean Cultivars and Locations Used in This Study cultivar

MATERIALS AND METHODS

Materials. An RIL population derived from a cross between two Japanese cultivars, Natto-shoryu and Hyoukei-kuro 3, was prepared according to a previously described method.7 To survey the candidate genetic region and validate the effect of a major QTL (qHbs3-1), we selected a heterozygous plant of the fifth filial generation (F5) from the RIL population using DNA markers in the region proximal to the QTL. The residual heterozygous line (RHL)16 in the F5 generation was self-fertilized, and the resultant F6 plants were harvested individually in 2011. Furthermore, F9 seeds showing different recombinant genotypes around the QTL region were obtained by self-fertilization and DNA-marker analysis, and F9 plants were grown in the experimental field. Seeds were harvested from 1−7 F9 plants for each genotype and were used for texture analysis of cooked beans. Another two F2 populations derived from a cross between two cultivars, “Enrei” and “LD00-3309” and between “Satonohohoemi” and “Sakukei 98”, were used to confirm the effect of the candidate gene in this report. Soybeans were planted and grown in the experimental field at NARO Institute of Crop Science (NICS), Ibaraki, Japan, in 2013 according to the method previously described.7 The sowing date of Natto-shoryu, Hyoukei-kuro 3, and their progenies was June 25, and that of other cultivars was June 18. Soybean cultivars grown in different locations in 2013 were collected from Kumamoto (Southern area of Japan) and Akita (Northern region of Japan) prefectures as described in Table 1. The sowing date in Akita was June 4, and those in Kumamoto were July 2, 9, or 10. Mature seeds were harvested in October or November, according to the maturing time. Moisture contents of the seeds were from 8 to 12% (Table S1). Soybean seeds were stored at 4 °C until use. The hardness of the cooked soybean cotyledons was analyzed from April 4 to June 18 in 2014. Texture Analysis of Cooked Beans. Cooked beans were prepared and the breaking stress of the cotyledons was measured according to methods described previously.7 Briefly, 30 seeds were soaked in 100 mL of deionized water at 20 °C for 22 ± 2 h. Imbibed seeds were boiled for 10 min in 200 mL of deionized water in a 500 mL tall glass beaker (HARIO Ltd., Tokyo, Japan) using a hot stirrer (IKA RCT basic, IKA japan K.K., Osaka, Japan). After cooking, the seeds were cooled in water at room temperature and the water was drained well. The seed coat and embryo axis were removed from the

genotype of Glyma03g03360

Akishirome

B

Enrei

B

Fukuibuki

A

Fukuminori

A′

Fukuyutaka

A′

Himeshirazu

B

Hyoukei-kuro 3 Kosuzu

B A

Kotoyutaka

B

LD00-3309 Natto-shoryu Oosuzu

A A B

Ryuuhou

B

Sachiyutaka

A′

Sakukei 98 Satonohohoemi

A′ B

Shuurei Suzukaren

B′ A′

Suzukari

A

Suzuyutaka

A

Tachinagaha

B

Tachiyutaka

B

Tamadaikoku

B′

Tamahomare

A

location NICS, Ibaraki, Japan KARC,† Kumamoto, Japan NICS, Ibaraki, Japana NICS, Ibaraki, Japanb KARC,† Kumamoto, Japan TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan KARC,† Kumamoto, Japan NICS, Ibaraki, Japan KARC,† Kumamoto, Japan NICS, Ibaraki, Japan KARC,† Kumamoto, Japan NICS, Ibaraki, Japan TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan KARC,† Kumamoto, Japan NICS, Ibaraki, Japan NICS, Ibaraki, Japan NICS, Ibaraki, Japan TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan KARC,† Kumamoto, Japan NICS, Ibaraki, Japan NICS, Ibaraki, Japana NICS, Ibaraki, Japanb TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan NICS, Ibaraki, Japan KARC,† Kumamoto, Japan TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan KARC,† Kumamoto, Japan TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan TARC,‡ Akita, Japana TARC,‡ Akita, Japanb TARC,‡ Akita, Japana TARC,‡ Akita, Japanb NICS, Ibaraki, Japan

† NARO Kyushu Okinawa Agricultiral Research center. ‡NARO Tohoku Agricultiral Research Center. Different letters mean different field plots.

B


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Journal of Agricultural and Food Chemistry

Figure 1. Locations of DNA markers (closed circles) and two genes (arrows in black; Glyma03g03350 and Glyma03g03360) on chromosome 3. Physical positions on the chromosome are indicated above. SSR_03g03340D is located downstream of Glyma03g03340 (an arrow in gray). BARCSOYSSR_03_0175 (BSS_03_0175) is located upstream of Glyma03g03390 (an arrow in gray). boiled seeds; then, the two cotyledons were separated from each other. The puncture strength was measured using a Rheoner RE-3305S texture analyzer (Yamaden Corp., Tokyo, Japan) fitted with a cylindrical probe (3 mm outside diameter) and a 2 kg capacity load cell. The adaxial (flat) side of a piece of cotyledon was placed facing downward on the stage of the analyzer, and the hardness was measured. The probe was operated at a distance of 2.45 mm and a speed of 1 mm/s. Puncture strength was determined as maximum force (pascals). The average score for more than 40 samples was used as an index of cooked seed hardness for each sample. The seed hardness measured by this method was positively correlated with the hardness of embryos (r = 0.918, p < 0.001) and that of whole beans (r = 0.926, p < 0.001) measured by the AACC approved method17 after 40 min of cooking. The 100-seed weight was calculated from the number and weight of air-dried seeds before soaking, and the water absorption ratio was calculated as the ratio of the seed weight after soaking to that before soaking. DNA Isolation and Marker Analysis. Genomic DNA was isolated from young leaves of F2 plants, F9 plants, and cultivars (see Materials) using a previously described method.7 Leaves were crushed in 400 μL of DNA extraction buffer (0.1 M Tris−HCl, pH 8.0; 0.05 M EDTA, pH 8.0; 0.5 M NaCl; 43.3 mM SDS; 10 mM dithiothreitol) with a SHAKE MASTER ball mill (Inter Bio Techno, Ltd., Tokyo, Japan), and homogenates were centrifuged at 2750g in a Himac CF 5RX centrifuge (Hitachi, Ltd., Tokyo, Japan) for 10 min. An aliquot of the supernatant was mixed with 100 μL of 5 M potassium acetate buffer and incubated at 5 °C for 10 min. The samples were centrifuged again at 2750g at 5 °C for 10 min. After centrifugation, 300 μL of the supernatant was gently mixed with 300 μL of isopropyl alcohol and incubated at 5 °C for 10 min. The total DNA was precipitated and segregated by centrifugation at 2750g at 5 °C for 10 min, then washed with 500 μL of 99.5% ethyl alcohol, and suspended in distilled water. The four SSR markers used in this study were BARCSOYSSR_03_0165, BARCSOYSSR_03_0172, BARCSOYSSR_03_0175, and BARCSOYSSR_03_0185, all located on chromosome 3.18 These markers are described in Table S2 and can be obtained from Soybase (http://soybase.org/).19 An SSR marker l o c a t e d b e t w e e n B A R C S O YSSR_0 3_0 172 an d B ARCSOYSSR_03_0175 was designed and named “SSR_03g03340D”. Polymerase chain reaction (PCR) was performed, and the products were analyzed by electrophoresis according to the methods described previously.7 Amplification was performed with an initial incubation for 2 min at 95 °C, then 33 cycles of 1 min at 92 °C, 1 min at 58 or 60 °C (depending on the primers used), and 1 min at 68 °C, followed by a final incubation for 5 min at 72 °C. PCR products of SSR_03g03340D were determined using the LabChip GX electrophoresis system (PerkinElmer, MA, US). DNA Sequencing. DNA fragments containing the open reading frame (ORF) regions of Glyma03g03350 and Glyma03g03360 were amplified by PCR using genomic DNA as a template. Information about predicted ORF regions was obtained from Phytozome (http://

www.phytozome.net/). The primers used for amplification are described in Table S3. PCR was performed in a 20 μL reaction volume using LA Taq Hot Start Version according to the manufacturer’s instruction (TAKARA, Shiga, Japan). Amplification was performed using the same conditions as described above. For DNA sequence analysis, DNA fragments were purified with a MonoFas DNA purification kit I (GL Sciences Inc., Tokyo, Japan) after electrophoresis using agarose gel. Sequencing reactions of purified PCR products were done with Big Dye terminators (Applied Biosystems, Foster City, CA). DNA sequences were determined with an ABI 3730xl automated sequencer (Applied Biosystems). cDNA Synthesis and RT-PCR. Total RNA extracts were prepared using RNeasy Plant Mini Kit (Qiagen, Tokyo, Japan), followed by treatment with DNaseI using Turbo DNA-free Kit (Life Technologies Japan, Tokyo, Japan). For each sample, 1.5 μg of extracted RNA was reverse-transcribed using SuperScript Vilo cDNA Synthesis Kit (Life Technologies Japan). The RT-PCR mixture contained 1 μL of cDNA synthesis reaction mixture, 450 nM of each primer, and 10 μL of 2× KAPA2G Robust HS RM with dye (NIPPON Genetics Co, Ltd., Tokyo, Japan). Amplification was performed with an initial incubation for 3 min at 95 °C, then 33 cycles of 15 s at 95 °C; 10 s at 56, 58, or 60 °C (depending on the primers used); and 10 s at 72 °C. Measurement of Calcium and Magnesium. Soybeans were ground into powder and 0.25 g of the sample was placed in a porcelain crucible for ashing at 500 °C for 16 h. After ashing, samples were extracted with 1 N HCl and diluted to 100 mL total. The contents of calcium and magnesium in the diluted samples were determined using ICP-AES Optima 4300 DV (PerkinElmer Japan Co., Ltd., Kanagawa, Japan). Statistical Analysis. Statistical analysis was performed using the SPSS software package (version 22, IBM Japan, Tokyo, Japan).

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RESULTS AND DISCUSSION A qHbs3-1 Candidate Gene and DNA Sequence Analysis. In our previous study, the analysis using progeny of the RHL derived from a cross between two Japanese cultivars, Natto-shoryu and Hyoukei-kuro 3, delimited the candidate region for qHbs3-1 between BARCSOYSSR_03_0165 and BARCSOYSSR_03_0185, a physical distance of approximately 330 kb according to the information from Phytozome (Figure 1).7 For further analysis to identify the gene responsible for qHbs3-1, F7 and F8 RHL progenies showing different recombinant genotypes of BARCSOYSSR_03_0165, BARCSOYSSR_03_0172, BARCSOYSSR_03_0175, and BARCSOYSSR_03_0185 were selected and the F9 RHL progenies were obtained by selffertilization. An SSR marker SSR_03g03340D and a cleaved amplified polymorphic sequence (CAPS) marker “BBCAPS” were further designed (Figure 1, Table S2). The breaking stress of cotyledons was compared among RHL progenies showing C


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Table 2. Comparison of the Breaking Stress of Progenies of the RHL Derived from a Cross between Two Japanese Cultivars, Natto-shoryu and Hyoukei-kuro 3, Showing Different Genotypes of BARCSOYSSR_03_0165, BARCSOYSSR_03_0172, SSR_03g03340D, BARCSOYSSR_03_0175, BARCSOYSSR_03_0185, and BBCAPSa

a

A indicates the same genotype as Natto-shoryu. B indicates the same genotype as Hyoukei-kuro 3. Breaking stress values more than 1200 kPa are shown in white type with a black background. *, ***: Values are significantly different from the B genotype at the 0.05 or 0.001 level, respectively. ns No siginificant difference was detected.

Table S3. Only the sequence in the third exon of Glyma03g03360 showed a difference between Natto-shoryu and Hyoukei-kuro 3: a single DNA substitution detected by BBCAPS, resulting in a truncated protein in Hyoukei-kuro 3 (Figure 2a,b). Glyma03g03360 is predicted to be a pectin methylesterase (PME) with an inhibitor domain at the N terminus20,21 according to Soybase. Glyma03g03360 of Hyoukei-kuro 3 is presumed to lack amino acids that are catalytically important (Figure 2a,b).22 PMEs deesterify pectins in the cell wall, converting methoxyl groups into carboxyl groups on the polygalacturonic acid chain and releasing both methanol and protons. The carboxyls can be cross-linked by calcium, which structurally rigidifies the cell wall.23,24 Transcripts of Glyma03g03360 were detected in embryo and seed coats at 45 days after anthesis (DAA) from Natto-shoryu by RT-PCR (Figure 3a). The transcripts were also detected in embryo from Hyoukei-kuro 3, although the band that was observed was fainter than that for Natto-shoryu (Figure 3b). It has been reported that mutation in the coding region affected the stability of mRNA and reduced the amount of transcripts, because premature termination of gene translation occasionally leads to transcript instability.25 It is also possible that the amount of transcripts was affected by mutation in the promoter or flanking regions of Glyma03g03360. These results suggested that deesterification of pectins in the cell wall

different genotypes of BARCSOYSSR_03_0165, BARCSOYSSR_03_0172, SSR_03g03340D, BARCSOYSSR_03_0175, BARCSOYSSR_03_0185, and BBCAPS. The graphical genotypes of RHL progenies and their breaking stress values indicated that qHbs3-1 is located in the region between SSR_03g03340D and BARCSOYSSR_03_0175, where 2 genes, Glyma03g03350 and Glyma03g033360, were presumed to be encoded (Soybase, Table 2 and Figure 1). BBCAPS, which is located between SSR_03g03340D and BARCSOYSSR_03_0175, detects a single base substitution of Glyma03g03360 in Hyoukei-kuro 3 (Figures 1 and 2a, Table S2). The PCR products using BBCAPS primers were digested with a DNA restriction enzyme Bst1107I, which detects the GTATAC sequence of Glyma03g03360 in Natto-shoryu (Figure 2b). All RHL progenies showing the same genotype as Hyoukei-kuro 3 at BBCAPS showed higher breaking stress values than that of Hyoukei-kuro 3, probably due to the effect of qHbs6-1, because all the RHL progenies examined showed the same genotypes as Natto-shoryu at SSR markers around the qHbs6-1 region, Satt 100, Satt 277, and Satt 307.7 Minor QTLs7 may potentially affect the breaking stress values of RHL progenies. The DNA sequence of the ORFs of the two genes Glyma03g03350 and Glyma03g03360 was analyzed. Primers used for amplification of exons of these genes are described in D


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Figure 2. (a) Predicted amino acid sequence of Glyma03g03360 of Natto-shoryu (A genotype). Quadrilaterals indicate the position of catalytically important residues.22 Glyma03g03360 of the B genotype lacks the sequence in gray. Glyma03g03360 of the B′ genotype lacks the sequence in gray and black. Underlined T indicates a single base substitution of the A′ genotype. (b) Comparison of DNA and predicted amino acid sequences of Glyma03g03360 between genotypes A and B. The nucleotides included in a Bst1107I restriction site are underlined. (c) Comparison of DNA and predicted amino acid sequences of Glyma03g03360 between genotypes A and A′. (d) Comparison of DNA and predicted amino acid sequences of Glyma03g03360 between genotypes A and B′.

even though the intensities of the bands were different among samples (Figure S1). Cultivar Differences in the Cooked Seed Hardness and Genotype of Glyma03g03360. Genotypes of Glyma03g03360 and breaking stress of cotyledons were further analyzed using 24 cultivars grown at different locations in 2013 (Table 1). CAPS analysis using the BBCAPS marker in the 24 cultivars resulted in three patterns of DNA fragments (Figure 4): one shows a single, main band designating Hyoukei-kuro 3; another shows two main bands and a minor band designating Natto-shoryu; and the last shows only two main bands. The sizes of the larger main bands were different between the latter two fragment patterns. DNA sequence analysis indicated that four polymorphisms exist in Glyma03g03360 among the 24 cultivars, and we named them A, A′, B, and B′. Polymorphisms A and B are the same as that of Natto-shoryu and Hyoukei-kuro 3, respectively. Polymorphism A′, which shows the third pattern by BBCAPS, contains a single base substitution in exon 3, resulting in an amino acid substitution from threonine to serine (Figure 2a,c). Because this amino acid is not predicted to be important for enzymatic activity,22 Glyma03g03360 of this group is presumably active. Polymorphism B′, which also shows the third pattern by BBCAPS and the same pattern as polymorphism A′, contains a single base deletion in exon 1, resulting in a truncated protein. This protein is also presumed to lack amino acids that are catalytically important. (Figure 2a,d). The upper primer sequence of BBCAPS is located in the intron of Glyma03g03360 between the second and third exons, and the lower primer sequence is located in the third exon. The expected sizes of Bst1107I restriction fragments from the sequence data are 434 bp and 233 bp for Natto-shoryu. The fragment pattern shown in Figure 4 agrees with the sequence data. The size difference in the larger main bands between A, and A′ and B′ genotypes is probably due to the difference in the

Figure 3. (a) RT-PCR analysis of RNA isolated from leaves (lanes 1− 3), embryos 21 days after anthesis (DAA; lanes 4−6), embryos 45 DAA (lanes 7−9), and immature seed coats 45 DAA (lanes 10−12) from Natto-shoryu. RNAs were isolated from three independent tissues. Specific primers for Glyma03g03360 (upper) and actin control (lower) were used. (b) RT-PCR analysis of RNA isolated from embryos 45 DAA from Natto-shoryu (lanes 1−3) and Hyoukei-kuro 3 (lanes 4−6). RNAs were isolated from three independent tissues. Specific primers for Glyma03g03360 (upper) and actin control (lower) were used.

is preceded by the activation of Glyme03g03360 at the late stage of seed development, in both the embryo and seed coat. Glyma03g03350 is a methionine aminopeptidase-like protein. In living organisms, proteins are synthesized with methionine as the first residue, which is specifically removed from the most mature proteins.26−28 The free amino-terminal methionine is removed by methionine aminopeptidase. Soybase indicates that Glyma03g03350 is expressed in all tissues examined, including the leaf, flower, pod, seed, root, and nodule. Transcripts of Glyma03g03350 were detected in embryos both from Natto-shoryu and from Hyoukei-kuro 3, E


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Figure 4. Products of BBCAPS from 24 soybean cultivars. M indicates DNA marker (phiX 174/HaeIII). The migration of size markers is shown to the left of the gel.

Phenotypic and Genomic Variation of an F2 Population from Enrei and LD00-3309, and Satonohohoemi and Sakukei 98. The breaking stress of cotyledons and genotypes of Glyma03g03360 were analyzed using F 2 populations from a cross between Enrei (B genotype) and LD00-3309 (A genotype), and between Satonohohoemi (B genotype) and Sakukei 98 (A′ genotype). The averages of the breaking stress values were 1467 and 1059 kPa for progenies from Enrei and LD00-3309 with an A genotype and those with a B, respectively. The breaking stress of progenies from Enrei and LD00-3309 showing the A genotype was significantly higher than that of the progenies showing the B genotype (p < 0.05, Figure 6a). The breaking stress of progenies from Satonohohoemi and Sakukei 98 showing an A′ genotype was significantly higher than that of the progenies showing a B genotype (p < 0.05, Figure 6b). Moreover, all the progenies of the A′ genotype showed breaking stress values higher than those of the B genotype. From these results, we concluded that Glyma03g03360, a PME homologue, is most certainly qHbs3-1. Effect of Seed Size, Water Absorption Ratio, and Calcium Content on the Cooked Seed Hardness. Negative correlations between cooked seed hardness and 100-seed weight or degree of water absorption have been reported.29,30 However, no significant negative correlation between the breaking stress and 100-seed weight or water absorption ratio was detected using RHL populations from Natto-shoryu and Hyoukei-kuro 3 (n = 24), which showed no significant difference in the 100-seed weight and the water absorption ratio between progenies of the A genotype and those of the B genotype (Table 3). In contrast, statistical analysis using soybean samples described in Table 1 indicated that the 100-seed weight and water absorption ratio are significantly correlated with the breaking stress value (r = −0.408 and −0.391, respectively; p < 0.01, Table 4). Soybeans with the A and A′ genotypes showed 100-seed weight and water absorption ratio significantly lower than those with B and B′. The averages of the 100-seed weight were 24.69 and 33.24 g for A + A′ genotypes and B + B′ genotypes, respectively. The

intron sequence. The genotypes of Glyma03g03360 for the full 24 cultivars are described in Table 1 and Figure 4. The breaking stress values of cotyledons were compared using the 22 cultivars (without Natto-shoryu and Hyoukei-kuro 3) grown in different locations described in Table 1. The averages of these values were 1572, 1568, and 1456 kPa for A or A′ genotypes in Ibaraki (NICS), Kumamoto (KARC), and Akita (TARC), respectively. The averages of the values were 979, 1117, and 870 kPa for B or B′ genotypes in Ibaraki, Kumamoto, and Akita, respectively (Figure 5). Statistical

Figure 5. Averages of the breaking stress for A, A′, B, and B′ genotypes. Different letters indicate a significant difference at the 0.05 level by Tukey’s test. Bars indicate SD.

analysis indicated that the breaking stress of A and A′ genotypes is significantly different from that of B and B′ genotypes in each location. However, there was no significant difference shown between the A genotype and the A′, or between the B genotype and the B′ (data not shown). These results indicated that Glyma03g03360 significantly affects the cooked seed hardness in many soybean cultivars. F


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Figure 6. (a) Breaking stress of cotyledons of F2 progenies from a cross between Enrei and LD00-3309. Black bars indicate the progenies with an A genotype, while white bars indicate progenies with a B genotype. (b) Breaking stress of cotyledons of F2 progenies from a cross between Satonohohoemi and Sakukei 98. Black bars indicate the progenies with an A′ genotype, while white bars indicate progenies with a B genotype.

100-seed weight or the water absorption ratio and breaking stress is that Glyma03g03360 may have an effect on the 100seed weight and the water absorption ratio, which are also affected by other factors. Another possibility is that a gene close to the qHbs3-1 may have an effect on the 100-seed weight and the water absorption ratio, with no difference in amino-acid sequence and expression between Natto-shoryu and Hyoukeikuro 3. In our previous study, no QTL was identified in the same chromosomal region as that of qHbs3-1 for 100-seed weight or water absorption ratio using an RIL population derived from a cross between Natto-shoryu and Hyoukei-kuro 3, although the 100-seed weight of Hyoukei-kuro 3 was more than 4 times higher than that of Natto-shoryu.7 It has been reported that a PME inhibitor plays a role in the early stage of radicle emergence.31 Glyma03g03360 likely has a role in the rigidification of the cell wall and elongation of the cell through PME activity, which may partly affect 100-seed weight and water absorption ratio. Seed weight, also characterized as seed mass or seed size generally, has been widely accepted as a complex trait controlled by polygenes.32 Hemicellulose content of seed coat fractions, particularly xylans, has been reported to be correlated with water uptake ratio and occurrence of hard seeds (stone seeds).33 Calcium content was significantly correlated with breaking stress using soybean samples described in Table 1 (r = 0.424, p < 0.01, Table 4). Soybeans of A and A′ genotypes showed a correlation between calcium content and breaking stress (r = 0.702, p < 0.001, Table 4 and Figure 7) higher than those of B and B′ genotypes (r = 0.457 p < 0.01, Table 4, and Figure 7). These results imply that the Ca2+ crossbridges between free carboxyl groups of adjacent pectin chains play an important role in the cooked seed hardness of soybeans. B and B′ genotypes also showed a significant correlation between calcium content and breaking stress, probably due to the expression of the other PME genes. According to SoyBase, more than 100 PME-like

Table 3. Correlation of 100-Seed Weight and Water Absorption Ratios and the Breaking Stress Values and the Average Values of 100-Seed Weight and Water Absorption Ratio for RHL Progenies between Natto-shoryu and Hyoukei-kuro 3 correlation with the breaking stress values 100-seed weight water absorption ratio

average for each genotype of Glyma03g03360

0.44*

19.14 g (A genotype)

−0.041

20.49 g (B genotype) 2.55 (A genotype) 2.53 (B genotype)

*

Significant at the 0.05 level.

Table 4. Correlation of Calcium and Magnesium Contents, 100-Seed Weight, Water Absorption Ratios, and the Breaking Stress Values between the Groups of Cultivars Showing A, A′, B, and B′ Genotypes

100-seed weight water absorption ratio Ca content Mg content

all samples

A+A′ genotypes

B+B′ genotypes

(n = 54)

(n = 22)

(n = 32)

−0.408** −0.391** 0.424** 0.309*

−0.015 −0.235 0.702*** 0.245

−0.225 0.273 0.457** 0.132

*,*,***: Significant at the 0.05, 0.01, or 0.001 level, respectively.

averages of the water absorption ratio were 2.26 and 2.33 for A + A′ genotypes and B + B′ genotypes, respectively. No significant correlation between the breaking stress and 100-seed weight or water absorption ratio was detected within the A + A′ genotypes and within the B + B′ genotypes. One possible explanation for the discordance in the relationship between the G


Article


marker-assisted selection and for the evaluation of seed quality, because it is otherwise time-consuming and laborious to evaluate cooked seed hardness. RT-PCR indicated that Glyma03g03360 is expressed both in embryos and seed coats (Figure 3), suggesting the possible effect of Glyma03g03360 on the texture of the seed coat, although it was not analyzed in this study. The mechanisms underlying how PME activity affects cooked seed hardness still remain to be clarified, although previous studies have shown that the demethylesterification of pectins has dramatic consequences on the rheological properties of the cell wall.14,39 Hardness after cooking is also important for other beans, such as carioca bean,40 fava bean,41 and chickpea.42 Further studies may help reveal ways to improve the quality of soyfoods and of food products of other beans.

Figure 7. Correlation between calcium content and breaking stress of cotyledons of cultivars with an A or A′ genotype (black squares) and those with a B or B′ genotype (white rhombuses).

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genes are found in soybean. Magnesium was also significantly correlated with breaking stress (r = 0.309, p < 0.05, Table 3). Magnesium can cross-link the carboxyl groups of pectins in a similar manner. Magnesium content showed less correlation with breaking stress than calcium, probably due to less variation of magnesium content than that of calcium content (Table 5).

* Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b02896. 100-seed weight, water absorption ratio, breaking stress, Ca content, Mg content, and moisture content in soybeans listed in Table 1 (Table S1); information about SSR and CAPS markers (Table S2); information about primers used for DNA amplification and RT-PCR (Table S3); RT-PCR analysis of RNA isolated from embryos 45 DAA from Natto-shoryu and Hyoukei-kuro 3 (Figure S1) (PDF)

Table 5. Average, Standard Deviations (SDs), and Coefficients of Variations (CVs) of Calcium and Magnesium Contents

Ca content (mg/gdw)

Mg content (mg/gdw)

average

all samples

A+A′ genotypes

B+B′ genotypes

(n = 54)

(n = 22)

(n = 32)

2.13

2.19

2.09

ASSOCIATED CONTENT

S

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AUTHOR INFORMATION

Corresponding Author SD CV average

0.52 0.25 2.48

0.53 0.24 2.54

0.52 0.25 2.44

*E-mail: kyokot@affrc.go.jp. Tel: +81-29-838-8503.

SD CV

0.20 0.08

0.22 0.09

0.17 0.07

Notes

Author Contributions †

K.T. and K.H.: These authors contributed equally to this work.

The authors declare no competing financial interest.

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Sajjaanantakul et al. demonstrated that β-elimination of pectins was primary responsible for the heat degradation at pH 6.1 and that the rate of the β-eliminative cleavage of the prepared pectin depended on the degree of esterification.34 It has been proposed that a decrease in degree of methylesterification necessitates both endogenous and exogenous calcium to form Ca2+ crossbridges between the free carboxyl groups of adjacent pectin molecules.35 Because Ca2+ and Mg2+ are bound to protein and/or phytate,36−38 other mechanisms in the relationship between the calcium and/or magnesium contents and the hardness of cotyledons may possibly be involved. Further studies are required to clarify the relationship between the calcium content in soybean and the degree of methylesterification. In this study, we conclude that Glyma03g03360, a PME homologue, is qHbs3-1, a stable QTL for hardness of cooked soybean cotyledons. The contribution of the F5 generation grown in 2010 to the total phenotypic variance of qHbs3-1 was 47.4% and that of the F6 generation grown in 2011 was 44.3%.7 No QTL was identified in the same chromosomal region as that of qHbs3-1 for 100-seed weight, also implying that Glyma03g03360 is useful for the improvement of soybean cultivars through breeding, because larger seeds are preferable for nimame, while smaller seeds are preferable for natto. DNA markers used in this study would be useful both for DNA

ACKNOWLEDGMENTS The authors are grateful to the Hyogo Prefectural Research Center of Agriculture, Forestry and Fisheries for providing “Hyoukei-kuro 3” seeds. The authors are also grateful to the Nagano Vegetable and Ornamental Crops Experiment Station for providing “Enrei,” “Tachinagaha,” and “Tamadaikoku” seeds.

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ABBREVIATIONS USED CAPS, cleaved amplified polymorphic sequence; CV, coefficients of variations; DAA, days after anthesis; F2, second filial generation; F5, fifth filial generation; F6, sixth filial generation; F7, seventh filial generation; F8, eighth filial generation; F9, ninth filial generation; ORF, open reading frame; PCR, polymerase chain reaction; PME, pectin methylesterase; QTL, quantitative trait locus; RIL, recombinant inbred line; SD, standard deviations; SSR, simple sequence repeat

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