Assessing genetic diversity and geographical differentiation in a global collection of wild soybean (Glycine soja Sieb. et Zucc.) and assigning a mini-core collection.

Wild soybean (Glycine soja), the ancestor of the cultivated soybean (G. max), is a crucial resource for capturing the genetic diversity of soybean species. In this study, we used a set of 78 genome-wide microsatellite markers to analyse the genetic diversity and geographic differentiation patterns in a global collection of 2,050 G. soja accessions and a mini-core collection of G. max stored in two public seed banks. We observed a notable reduction in the genetic diversity of G. max compared with G. soja and identified a close phylogenetic relationship between G. max and a G. soja subpopulation located in central China. Furthermore, we revealed substantial genetic divergence between northern and southern subpopulations, accompanied by diminished genetic diversity in the northern subpopulations. Two clusters were discovered among the accessions from north-eastern China-one genetically close to those from South Korea and Southern Japan, and another close to those from Amur Oblast, Russia. Finally, 192 accessions were assigned to a mini-core collection of G. soja, retaining 73.8% of the alleles detected in the entire collection. This mini-core collection is accessible to those who need it, facilitating efficient evaluation and utilization of G. soja genetic resources in soybean breeding initiatives.

Phosphate starvation response precedes abscisic acid response under progressive mild drought in plants.

Drought severely damages crop production, even under conditions so mild that the leaves show no signs of wilting. However, it is unclear how field-grown plants respond to mild drought. Here, we show through six years of field trials that ridges are a useful experimental tool to mimic mild drought stress in the field. Mild drought reduces inorganic phosphate levels in the leaves to activate the phosphate starvation response (PSR) in soybean plants in the field. Using Arabidopsis thaliana and its mutant plants grown in pots under controlled environments, we demonstrate that PSR occurs before abscisic acid response under progressive mild drought and that PSR plays a crucial role in plant growth under mild drought. Our observations in the field and laboratory using model crop and experimental plants provide insight into the molecular response to mild drought in field-grown plants and the relationship between nutrition and drought stress response.

A novel QTL associated with tolerance to cold-induced seed cracking in the soybean cultivar Toyomizuki.

Low temperatures after flowering cause seed cracking (SC) in soybean. Previously, we reported that proanthocyanidin accumulation on the dorsal side of the seed coat, controlled by the I locus, may lead to cracked seeds; and that homozygous IcIc alleles at the I locus confer SC tolerance in the line Toiku 248. To discover new genes related to SC tolerance, we evaluated the physical and genetic mechanisms of SC tolerance in the cultivar Toyomizuki (genotype II). Histological and texture analyses of the seed coat revealed that the ability to maintain hardness and flexibility under low temperature, regardless of proanthocyanidin accumulation in the dorsal seed coat, contributes to SC tolerance in Toyomizuki. This indicated that the SC tolerance mechanism differed between Toyomizuki and Toiku 248. A quantitative trait loci (QTL) analysis of recombinant inbred lines revealed a new, stable QTL related to SC tolerance. The relationship between this new QTL, designated as qCS8-2, and SC tolerance was confirmed in residual heterozygous lines. The distance between qCS8-2 and the previously identified QTL qCS8-1, which is likely the Ic allele, was estimated to be 2-3 Mb, so it will be possible to pyramid these regions to develop new cultivars with increased SC tolerance.

Triterpenoids in aerenchymatous phellem contribute to internal root aeration and waterlogging adaptability in soybeans.

Soybeans (Glycine max) develop newly differentiated aerenchymatous phellem (AP) in response to waterlogging stress. AP is formed in the hypocotyl and root, thus contributing to internal aeration and adaptation to waterlogging for several legumes. Extensive accumulation of triterpenoids - lupeol and betulinic acid - has been identified in AP. However, their physiological roles in plants remain unclarified. Lupeol is converted from 2,3-oxidosqualene by lupeol synthase (LUS) and oxidized to betulinic acid. Notably, soybeans have two LUS genes (GmLUS1 and GmLUS2). Functional analysis was performed to reveal the biological and physiological functions of triterpenoids in AP using lus mutants. The AP cells of lus1 mutant lacked triterpenoid accumulation and epicuticular wax. Lupeol and betulinic acid were the major components of epicuticular wax and contributed to tissue hydrophobicity and oxygen transport to the roots. Tissue porosity in AP was lower in the lus1 mutant than in the wild-type, which resulted in reduced oxygen transport to the roots via AP. This reduction in oxygen transport resulted in shallow root systems under waterlogged conditions. Triterpenoid accumulation in AP contributes to effective internal aeration and root development for adaptation to waterlogging, suggesting the significance of triterpenoids in improving waterlogging tolerance.

Identification and validation of quantitative trait loci associated with seed yield in soybean.

In soybean [Glycine max (L.) Merrill], the genetic analysis of seed yield is important to aid in the breeding of high-yielding cultivars. Seed yield is a complex trait, and the number of quantitative trait loci (QTL) involved in seed yield is high. The aims of this study were to identify QTL associated with seed yield and validate their effects on seed yield using near-isogenic lines. The QTL analysis was conducted using a recombinant inbred line population derived from a cross between Japanese cultivars 'Toyoharuka' and 'Toyomusume', and eight seed yield-associated QTL were identified. There were significant positive correlations between seed yield and the number of favorable alleles at QTL associated with seed yield in the recombinant inbred lines for three years. The effects of qSY8-1, a QTL promoting greater seed yield, was validated in the Toyoharuka background. In a two-year yield trial, the 100-seed weight and seed yield of Toyoharuka-NIL, the near-isogenic line having the Toyomusume allele at qSY8-1, were significantly greater than those of Toyoharuka (106% and 107%, respectively) without any change for days to flowering and maturity. Our results suggest that qSY8-1 was not associated with maturity genes, and contributed to the 100-seed weight.

Improving Quantitative Traits in Self-Pollinated Crops Using Simulation-Based Selection With Minimal Crossing.

Genomic selection and marker-assisted recurrent selection have been applied to improve quantitative traits in many cross-pollinated crops. However, such selection is not feasible in self-pollinated crops owing to laborious crossing procedures. In this study, we developed a simulation-based selection strategy that makes use of a trait prediction model based on genomic information to predict the phenotype of the progeny for all possible crossing combinations. These predictions are then used to select the best cross combinations for the selection of the given trait. In our simulated experiment, using a biparental initial population with a heritability set to 0.3, 0.6, or 1.0 and the number of quantitative trait loci set to 30 or 100, the genetic gain of the proposed strategy was higher or equal to that of conventional recurrent selection method in the early selection cycles, although the number of cross combinations of the proposed strategy was considerably reduced in each cycle. Moreover, this strategy was demonstrated to increase or decrease seed protein content in soybean recombinant inbred lines using SNP markers. Information on 29 genomic regions associated with seed protein content was used to construct the prediction model and conduct simulation. After two selection cycles, the selected progeny had significantly higher or lower seed protein contents than those from the initial population. These results suggest that our strategy is effective in obtaining superior progeny over a short period with minimal crossing and has the potential to efficiently improve the target quantitative traits in self-pollinated crops.

Investigation of the Genetic Diversity of a Rice Core Collection of Japanese Landraces using Whole-Genome Sequencing.

The Rice Core Collection of Japanese Landraces (JRC) consisting of 50 accessions was developed by the genebank at the National Agriculture and Food Research Organization (NARO) in 2008. As a Japanese landrace core collection, the JRC has been used for many research projects, including screening for different phenotypes and allele mining for target genes. To understand the genetic diversity of Japanese Landraces, we performed whole-genome resequencing of these 50 accessions and obtained a total of 2,145,095 single nucleotide polymorphism (SNPs) and 317,832 insertion-deletions (indels) by mapping against the Oryza sativa ssp. japonica Nipponbare genome. A JRC phylogenetic tree based on 1,394 representative SNPs showed that JRC accessions were divided into two major groups and one small group. We used the multiple genome browser, TASUKE+, to examine the haplotypes of flowering genes and detected new mutations in these genes. Finally, we performed genome-wide association studies (GWAS) for agronomical traits using the JRC and another core collection, the World Rice Core Collection (WRC), comprising 69 accessions also provided by the NARO genebank. In leaf blade width, a strong peak close to NAL1, a key gene for the regulation of leaf width, and, in heading date, a peak near HESO1 involved in flowering regulation were observed in GWAS using the JRC. They were also detected in GWAS using the combined JRC + WRC. Thus, JRC and JRC + WRC are suitable populations for GWAS of particular traits.

Site-directed mutagenesis by biolistic transformation efficiently generates inheritable mutations in a targeted locus in soybean somatic embryos and transgene-free descendants in the T1 generation.

The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is being rapidly developed for mutagenesis in higher plants. Ideally, foreign DNA introduced by this system is removed in the breeding of edible crops and vegetables. Here, we report an efficient generation of Cas9-free mutants lacking an allergenic gene, Gly m Bd 30K, using biolistic transformation and the CRISPR/Cas9 system. Five transgenic embryo lines were selected on the basis of hygromycin resistance. Cleaved amplified polymorphic sequence analysis detected only two different mutations in e all of the lines. These results indicate that mutations were induced in the target gene immediately after the delivery of the exogenous gene into the embryo cells. Soybean plantlets (T0 plants) were regenerated from two of the transgenic embryo lines. The segregation pattern of the Cas9 gene in the T1 generation, which included Cas9-free plants, revealed that a single copy number of transgene was integrated in both lines. Immunoblot analysis demonstrated that no Gly m Bd 30K protein accumulated in the Cas9-free plants. Gene expression analysis indicated that nonsense mRNA decay might have occurred in mature mutant seeds. Due to the efficient induction of inheritable mutations and the low integrated transgene copy number in the T0 plants, we could remove foreign DNA easily by genetic segregation in the T1 generation. Our results demonstrate that biolistic transformation of soybean embryos is useful for CRISPR/Cas9-mediated site-directed mutagenesis of soybean for human consumption.

Whole-genome sequence diversity and association analysis of 198 soybean accessions in mini-core collections.

We performed whole-genome Illumina resequencing of 198 accessions to examine the genetic diversity and facilitate the use of soybean genetic resources and identified 10 million single nucleotide polymorphisms and 2.8 million small indels. Furthermore, PacBio resequencing of 10 accessions was performed, and a total of 2,033 structure variants were identified. Genetic diversity and structure analysis congregated the 198 accessions into three subgroups (Primitive, World, and Japan) and showed the possibility of a long and relatively isolated history of cultivated soybean in Japan. Additionally, the skewed regional distribution of variants in the genome, such as higher structural variations on the R gene clusters in the Japan group, suggested the possibility of selective sweeps during domestication or breeding. A genome-wide association study identified both known and novel causal variants on the genes controlling the flowering period. Novel candidate causal variants were also found on genes related to the seed coat colour by aligning together with Illumina and PacBio reads. The genomic sequences and variants obtained in this study have immense potential to provide information for soybean breeding and genetic studies that may uncover novel alleles or genes involved in agronomically important traits.

Characterization of the genomic sequence data around common cutworm resistance genes in soybean (Glycine max) using short- and long-read sequencing methods.

The common cutworm (CCW, Spodopteraab litura Fabricius) is one of the pests that most severely infect soybean (Glycine max L. Merr.). In a previous report, quantitative trait loci (QTL) analysis of CCW resistance using a recombinant inbred line derived from a cross between a susceptible cultivar 'Fukuyutaka' and a resistant cultivar 'Himeshirazu', identified two antixenosis resistance QTLs, CCW-1 and CCW-2. To reveal sequence variation between the aforementioned two cultivars, whole genome resequencing was performed using Illumina HiSeq2000 (75,632,747 and 91,540,849 reads). The generated datasets can be used for fine mapping and gene isolation of CCW-1 and CCW-2 as well as for revealing more detailed genetic differences between 'Fukuyutaka' and 'Himeshirazu' .

Functional Divergence of G and Its Homologous Genes for Green Pigmentation in Soybean Seeds.

The degradation of chlorophyll in mature soybean seeds is closely related to the development of their yellow color. In this study, we examined G, its homologue G-like (GL), and their mutant alleles and investigated the relationship between these genes and chlorophyll accumulation in the seed coats of mature seeds. Transient expression of G and GL proteins fused with green fluorescent protein revealed that both were localized in plastids. Overexpression of G resulted in the accumulation of chlorophyll in the seed coats and cotyledons of mature seeds, indicating that high expression levels of G result in chlorophyll accumulation that exceeds its metabolism in the seeds of yellow soybean. Analysis of near isogenic lines at the G locus demonstrated a significant difference in the chlorophyll content of the seed coats and cotyledons of mature seeds when G and mutant g alleles were expressed in the d1d2 stay-green genetic background, indicating that the G protein might repress the SGR-independent degradation of chlorophyll. We examined the distribution of mutant alleles at the G and GL loci among cultivated and wild soybean germplasm. The g allele was widely distributed in cultivated soybean germplasm, except for green seed coat soybean lines, all of which contained the G allele. The gl alleles were much fewer in number than the g alleles and were mainly distributed in the genetic resources of cultivated soybean from Japan. None of the landraces and breeding lines investigated in this study were observed to contain both the g and gl alleles. Therefore, in conclusion, the mutation of the G locus alone is essential for establishing yellow soybeans, which are major current soybean breeding lines.

A cellulose synthase-derived enzyme catalyses 3-O-glucuronosylation in saponin biosynthesis.

Triterpenoid saponins are specialised metabolites distributed widely in the plant kingdom that consist of one or more sugar moieties attached to triterpenoid aglycones. Despite the widely accepted view that glycosylation is catalysed by UDP-dependent glycosyltransferase (UGT), the UGT which catalyses the transfer of the conserved glucuronic acid moiety at the C-3 position of glycyrrhizin and various soyasaponins has not been determined. Here, we report that a cellulose synthase superfamily-derived glycosyltransferase (CSyGT) catalyses 3-O-glucuronosylation of triterpenoid aglycones. Gene co-expression analyses of three legume species (Glycyrrhiza uralensis, Glycine max, and Lotus japonicus) reveal the involvement of CSyGTs in saponin biosynthesis, and we characterise CSyGTs in vivo using Saccharomyces cerevisiae. CSyGT mutants of L. japonicus do not accumulate soyasaponin, but the ectopic expression of endoplasmic reticulum membrane-localised CSyGTs in a L. japonicus mutant background successfully complement soyasaponin biosynthesis. Finally, we produced glycyrrhizin de novo in yeast, paving the way for sustainable production of high-value saponins.

A Soybean Deletion Mutant That Moderates the Repression of Flowering by Cool Temperatures.

Ambient growing temperature and photoperiod are major environmental stimuli that summer annual crops use to adjust their reproductive phenology so as to maximize yield. Variation in flowering time among soybean (Glycine max) cultivars results mainly from allelic diversity at loci that control photoperiod sensitivity and FLOWERING LOCUS T (FT) orthologs. However, variation in the thermal regulation of flowering and its underlying mechanisms are poorly understood. In this study, we identified a novel mutant (ef1) that confers altered thermal regulation of flowering in response to cool ambient temperatures. Mapping analysis with simple sequence repeat (SSR) markers located the mutation in the upper part of chromosome 19, where no QTL for flowering has been previously reported. Fine-mapping and re-sequencing revealed that the mutation was caused by deletion of a 214 kbp genomic region that contains 11 annotated genes, including CONSTANS-LIKE 2b (COL2b), a soybean ortholog of Arabidopsis CONSTANS. Comparison of flowering times under different photo-thermal conditions revealed that early flowering in the mutant lines was most distinct under cool ambient temperatures. The expression of two FT orthologs, FT2a and FT5a, was dramatically downregulated by cool temperature, but the magnitude of the downregulation was lower in the mutant lines. Cool temperatures upregulated COL2b expression or delayed peak expression, particularly at the fourth trifoliate-leaf stage. Intriguingly, they also upregulated E1, a soybean-specific repressor of FT orthologs. Our results suggest that the ef1 mutation is involved in thermal regulation of flowering in response to cool ambient temperature, and the lack of COL2b in the mutant likely alleviates the repression of flowering by cool temperature. The ef1 mutant can be used as a novel gene resource in breeding soybean cultivars adapted to cool climate and in research to improve our understanding of thermal regulation of flowering in soybean.

Suppressed Methionine γ-Lyase Expression Causes Hyperaccumulation of S-Methylmethionine in Soybean Seeds.

Several soybean (Glycine max) germplasms, such as Nishiyamahitashi 98-5 (NH), have an intense seaweed-like flavor after cooking because of their high seed S-methylmethionine (SMM) content. In this study, we compared the amounts of amino acids in the phloem sap, leaves, pods, and seeds between NH and the common soybean cultivar Fukuyutaka. This revealed a comparably higher SMM content alongside a higher free Met content in NH seeds, suggesting that the SMM-hyperaccumulation phenotype of NH soybean was related to Met metabolism in seeds. To investigate the molecular mechanism behind SMM hyperaccumulation, we examined the phenotype-associated gene locus in NH plants. Analyses of the quantitative trait loci in segregated offspring of the cross between NH and the common soybean cultivar Williams 82 indicated that one locus on chromosome 10 explains 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a transposon insertion into the intron of a gene, Glyma.10g172700, is associated with the SMM-hyperaccumulation phenotype. The Glyma.10g172700-encoded recombinant protein showed Met-γ-lyase (MGL) activity in vitro, and the transposon-insertion mutation in NH efficiently suppressed Glyma.10g172700 expression in developing seeds. Exogenous administration of Met to sections of developing soybean seeds resulted in transient increases in Met levels, followed by continuous increases in SMM concentrations, which was likely caused by Met methyltransferase activity in the seeds. Accordingly, we propose that the SMM-hyperaccumulation phenotype is caused by suppressed MGL expression in developing soybean seeds, resulting in transient accumulation of Met, which is converted into SMM to avoid the harmful effects caused by excess free Met.

Reconstruction of the Evolutionary Histories of UGT Gene Superfamily in Legumes Clarifies the Functional Divergence of Duplicates in Specialized Metabolism.

Plant uridine 5'-diphosphate glycosyltransferases (UGTs) influence the physiochemical properties of several classes of specialized metabolites including triterpenoids via glycosylation. To uncover the evolutionary past of UGTs of soyasaponins (a group of beneficial triterpene glycosides widespread among Leguminosae), the UGT gene superfamily in Medicago truncatula, Glycine max, Phaseolus vulgaris, Lotus japonicus, and Trifolium pratense genomes were systematically mined. A total of 834 nonredundant UGTs were identified and categorized into 98 putative orthologous loci (POLs) using tree-based and graph-based methods. Major key findings in this study were of, (i) 17 POLs represent potential catalysts for triterpene glycosylation in legumes, (ii) UGTs responsible for the addition of second (UGT73P2: galactosyltransferase and UGT73P10: arabinosyltransferase) and third (UGT91H4: rhamnosyltransferase and UGT91H9: glucosyltransferase) sugars of the C-3 sugar chain of soyasaponins were resulted from duplication events occurred before and after the hologalegina-millettoid split, respectively, and followed neofunctionalization in species-/ lineage-specific manner, and (iii) UGTs responsible for the C-22-O glycosylation of group A (arabinosyltransferase) and DDMP saponins (DDMPtransferase) and the second sugar of C-22 sugar chain of group A saponins (UGT73F2: glucosyltransferase) may all share a common ancestor. Our findings showed a way to trace the evolutionary history of UGTs involved in specialized metabolism.

Identification of a cytochrome P450 hydroxylase, CYP81E22, as a causative gene for the high sensitivity of soybean to herbicide bentazon.

KEY MESSAGE: A frame shift invoked by a single-base deletion in the gene encoding a cytochrome P450 hydroxylase, CYP81E22, causes the loss of bentazon detoxification function in soybean. Bentazon is an effective herbicide in soybean cultivation applied at post-emergence stages for control of several broadleaf weeds. However, some soybean cultivars are highly sensitive to bentazon and are killed upon application. In this study, the gene related to the high sensitivity of soybean cultivars to bentazon was mapped to chromosome 16, and its location was narrowed down to a 257-kb region where three cytochrome P450 genes were located. In these genes, a single-base deletion of cytosine was detected in the coding region of Glyma.16G149300, CYP81E22, at + 1465 bp downstream from the translation start codon, leading to a frame shift in the open reading frame and creating a premature stop codon. This stop codon resulted in the loss of more than half of the P450, and consequently, the remaining molecule failed to form a functioning protein. This single-base deletion was common among the highly sensitive cultivars screened from the soybean mini-core collection and other previously reported highly sensitive cultivars. Furthermore, we screened plant lines from the targeting-induced local lesions in genomes library of the soybean cultivar Enrei based on a modelled 3D structure of CYP81E22. The lines with mutations in Glyma.16G149300 were highly sensitive to bentazon, which provides strong evidence that Glyma.16G149300 is the gene responsible for high sensitivity to bentazon.

Two novel quantitative trait loci affecting the variation in leaf photosynthetic capacity among soybeans.

There is a large variation in CO2 assimilation rate per unit of leaf area (A) within or among crop species, which can be exploited to improve A by elucidating the mechanisms underlying such variation. The objective of the present study is to elucidate the genetic factors affecting the variation in leaf photosynthetic capacity among soybeans. Here, we conducted field experiments over three years, using Enrei, a leading variety in Japan, Peking, a landrace from China and the chromosome segment substitution lines derived from their progenies. The gas exchange measurements were conducted to evaluate A among soybean. Peking showed higher A than Enrei after the flowering in all the years. The genetic analysis identified two novel quantitative trait loci (QTLs) related to variation in A, which were located on chromosome 13 (qLPC13) and 20 (qLPC20). The Peking allele at qLPC13 increased A by 8.3 % in the Enrei genetic background, while the Peking allele at qLPC20 decreased A by 15.3 %. The present study is the first report on QTLs affecting a genotypic variation in leaf photosynthetic capacity among field-grown soybeans. The identification of the causal genes in these QTLs can provide a novel strategy to enhance leaf photosynthetic capacity with soybean breeding.

Evaluation of the resistance effect of QTLs derived from wild soybean (Glycine soja) to common cutworm (Spodoptera litura Fabricius).

Common cutworm (CCW) is a serious herbivorous insect pest of soybean. Previously, we conducted an antixenosis bioassay (measuring feeding preference) with CCW using recombinant inbred lines (RILs) derived from a cross between a wild soybean (Glycine soja) collected in Hiroshima prefecture (JP110755) and the leading cultivar, Fukuyutaka. The analysis revealed quantitative trait loci (QTLs) for antixenosis resistance, qRslx3 and qRslx4. In the present study we developed another RIL population using Fukuyutaka and a different G. soja, collected in Kumamoto prefecture (G406). An analysis revealed an antixenosis resistance QTL on chromosome 7, and the resistant allele of the QTL was derived from G406. The chromosomal position of the QTL was almost the same as that of CCW-2, a previously-reported antibiosis resistance QTL for CCW, detected in a F2 population derived from a cross between Fukuyutaka and a resistant cultivar Himeshirazu. These QTLs could be the same locus; however, G406 and Himeshirazu are likely to possess different alleles, because Himeshirazu allele exhibits no antixenosis effect. We expect that pyramiding of the resistance QTLs derived from G. soja will contribute to the development of CCW resistant cultivars.

Soybean antiviral immunity conferred by dsRNase targets the viral replication complex.

Eukaryotic positive-strand RNA viruses replicate their genomes in membranous compartments formed in a host cell, which sequesters the dsRNA replication intermediate from antiviral immune surveillance. Here, we find that soybean has developed a way to overcome this sequestration. We report the positional cloning of the broad-spectrum soybean mosaic virus resistance gene Rsv4, which encodes an RNase H family protein with dsRNA-degrading activity. An active-site mutant of Rsv4 is incapable of inhibiting virus multiplication and is associated with an active viral RNA polymerase complex in infected cells. These results suggest that Rsv4 enters the viral replication compartment and degrades viral dsRNA. Inspired by this model, we design three plant-gene-derived dsRNases that can inhibit the multiplication of the respective target viruses. These findings suggest a method for developing crops resistant to any target positive-strand RNA virus by fusion of endogenous host genes.

Symbiotic incompatibility between soybean and Bradyrhizobium arises from one amino acid determinant in soybean Rj2 protein.

Cultivated soybean (Glycine max) carrying the Rj2 allele restricts nodulation with specific Bradyrhizobium strains via host immunity, mediated by rhizobial type III secretory protein NopP and the host resistance protein Rj2. Here we found that the single isoleucine residue I490 in Rj2 is required for induction of symbiotic incompatibility. Furthermore, we investigated the geographical distribution of the Rj2-genotype soybean in a large set of germplasm by single nucleotide polymorphism (SNP) genotyping using a SNP marker for I490. By allelic comparison of 79 accessions in the Japanese soybean mini-core collection, we suggest substitution of a single amino acid residue (R490 to I490) in Rj2 induces symbiotic incompatibility with Bradyrhizobium diazoefficiens USDA 122. The importance of I490 was verified by complementation of rj2-soybean by the dominant allele encoding the Rj2 protein containing I490 residue. The Rj2 allele was also found in Glycine soja, the wild progenitor of G. max, and their single amino acid polymorphisms were associated with the Rj2-nodulation phenotype. By SNP genotyping against 1583 soybean accessions, we detected the Rj2-genotype in 5.4% of G. max and 7.7% of G. soja accessions. Distribution of the Rj2-genotype soybean plants was relatively concentrated in the temperate Asian region. These results provide important information about the mechanism of host genotype-specific symbiotic incompatibility mediated by host immunity and suggest that the Rj2 gene has been maintained by environmental conditions during the process of soybean domestication.