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.

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.

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.

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.

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.

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.

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.

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.

Lotus japonicus Triterpenoid Profile and Characterization of the CYP716A51 and LjCYP93E1 Genes Involved in Their Biosynthesis In Planta.

Lotus japonicus is an important model legume plant in several fields of research, such as secondary (specialized) metabolism and symbiotic nodulation. This plant accumulates triterpenoids; however, less information regarding its composition, content and biosynthesis is available compared with Medicago truncatula and Glycine max. In this study, we analyzed the triterpenoid content and composition of L. japonicus. Lotus japonicus accumulated C-28-oxidized triterpenoids (ursolic, betulinic and oleanolic acids) and soyasapogenols (soyasapogenol B, A and E) in a tissue-dependent manner. We identified an oxidosqualene cyclase (OSC) and two cytochrome P450 enzymes (P450s) involved in triterpenoid biosynthesis using a yeast heterologous expression system. OSC9 was the first enzyme derived from L. japonicus that showed α-amyrin (a precursor of ursolic acid)-producing activity. CYP716A51 showed triterpenoid C-28 oxidation activity. LjCYP93E1 converted ß-amyrin into 24-hydroxy-ß-amyrin, a metabolic intermediate of soyasapogenols. The involvement of the identified genes in triterpenoid biosynthesis in L. japonicus plants was evaluated by quantitative real-time PCR analysis. Furthermore, gene loss-of-function analysis of CYP716A51 and LjCYP93E1 was conducted. The cyp716a51-mutant L. japonicus hairy roots generated by the genome-editing technique produced no C-28 oxidized triterpenoids. Likewise, the complete abolition of soyasapogenols and soyasaponin I was observed in mutant plants harboring Lotus retrotransposon 1 (LORE1) in LjCYP93E1. These results indicate that the activities of these P450 enzymes are essential for triterpenoid biosynthesis in L. japonicus. This study increases our understanding of triterpenoid biosynthesis in leguminous plants and provides information that will facilitate further studies of the physiological functions of triterpenoids using L. japonicus.

High-Throughput Screening and Characterization of a High-Density Soybean Mutant Library Elucidate the Biosynthesis Pathway of Triterpenoid Saponins.

Triterpenes (C30) constitute one of the diverse class of natural products with potential applications in food, cosmetic and pharmaceutical industries. Soyasaponins are oleanane-type triterpenoids widespread among legumes and particularly abundant in soybean seeds. They have associated with various pharmacological implications and undesirable taste properties of soybean-based food products. Uncovering the biosynthetic genes of soyasaponins will provide new opportunities to control the pathway for human benefits. However, the pathway of soyasaponin biosynthesis has not been fully elucidated in part because of a paucity of natural mutants. Here, we applied a structured high-density soybean mutant library for the forward genetic screening of triterpenoid biosynthesis. The seed soyasaponin polymorphism in the mutant library was evaluated using a high-throughput thin-layer chromatography and liquid chromatography tandem mass spectrometry analysis. This screening identified 35 mutants (3.85% of 909 mutant lines) with seven unusual soyasaponin phenotypes (Categories 1-7), which was greater than the number of natural mutants reported previously (22 mutants, 0.18% of ∼12,428 accessions). Nine unique intermediates of soyasaponin biosynthesis were identified and their chemical structures were estimated based on their MS/MS fragment patterns. Based on published information, 19 mutants could be associated with loss of function of four individual soyasaponin biosynthesis genes identified through expressed sequence tag mining or positional cloning, whereas the remaining 16 mutants were novel and may facilitate discovery of the unknown biosynthetic genes of soyasaponins. Our approach and library may help to identify new phenotype materials and causative genes associated with specialized metabolite production and other traits.

Mapping versatile QTL for soybean downy mildew resistance.

KEY MESSAGE: Three versatile QTL for soybean downy mildew resistance in Japan were detected using five RIL populations and confirmed using recombinant fixed pairs or a backcrossed line. Downy mildew reduces soybean seed quality and size. It is a problem in Japan, where 90% of soybean grown is used as food. In the USA, 33 downy mildew races have been reported, but race differentiation in Japan is unclear. To identify quantitative trait loci (QTL) for downy mildew resistance effective in the Kanto and Tohoku regions, we performed QTL analysis using five populations of recombinant inbred lines (RILs) originated from 'Natto-shoryu' × 'Tachinagaha' (NT), 'Natto-shoryu' × 'Suzumaru', 'Satonohohoemi' × 'Fukuibuki' (SF), 'Kinusayaka' × 'COL/Akita/2009/TARC/1,' and 'YR-82' × 'Harosoy' over a 4-year period (2014-2017). We evaluated spontaneously developed symptoms of the RILs and applied 112-233 polymorphic markers to each population. Out of 31 QTL detected, we found five on chromosome 3 in three populations and another five on chromosome 7 in three populations. Other QTL were detected in one population, nine of them in different years. In the NT population, two QTL were detected in a 3.0-Mb region on chromosome 7 and in an 8.1-Mb region on chromosome 18 by evaluating nine recombinant fixed pairs in both Kanto and Tohoku regions. In the SF population, a QTL on chromosome 8 was detected in both regions. This QTL was introduced into the 'Satonohohoemi' background by backcrossing, and its effect was confirmed in both regions. In summary, two QTL on chromosomes 7 and 18 from the NT population and one QTL on chromosome 8 from the SF population were confirmed to be effective in both Tohoku and Kanto regions.

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.

Isolation and characterization of induced mutants in the gene associated with seed cadmium accumulation in soybean.

Food contamination by cadmium (Cd) is a serious threat to human health. Thus, it is imperative to prevent Cd accumulation in staple crops like soybean. The development of low Cd accumulating cultivars is an effective solution. To this end, it is essential to identify the gene(s) controlling seed Cd accumulation. Although Glyma.09G055600 (GmHMA3) seems to be associated with Cd accumulation in soybean, it has not been established if it is responsible for seed Cd accumulation. In the present study, the effect of GmHMA3 on seed Cd accumulation in soybean was validated using three independent GmHMA3 mutants isolated from an ethyl methanesulfonate-induced soybean mutant library. Each of mutant had an amino acid substitution in GmHMA3 and segregating progenies were developed by crossing the original cultivar with each of the three mutants. The relationship between these three mutations and seed Cd accumulation was investigated. While two of them significantly increased seed Cd accumulation corresponding to previous reports of a natural missense mutation in GmHMA3, the other slightly decreased seed Cd accumulation. Overall, these results indicate that GmHMA3 is responsible for seed Cd accumulation in soybean.

Effect of change from a determinate to a semi-determinate growth habit on the yield and lodging resistance of soybeans in the northeast region of Japan.

Although an indeterminate growth habit is attractive to develop high-yield soybean varieties with higher number of pods (Glycine max (L). Merr.), lodging in indeterminate varieties remains a problem in Japan. As the semi-determinate varieties have shorter main stem length than the indeterminate varieties, this trait can be useful to improve varieties with high yield and low lodging risk. We introduced the genes Dt1 and Dt2, which regulate stem growth habit, into three determinate varieties by backcrossing and evaluated the resulting effects on yield and lodging tendency under four different growing environments. The yield and lodging degree of the semi-determinate and indeterminate lines were higher and more severe than those of the determinate lines. Despite the lower overall lodging score, the semi-determinate lines had marginally lower overall yield than that of the indeterminate lines. However, the effect of introduction of semi-determinate traits on yield and lodging degree was different in the three backgrounds, with the yield of semi-determinate lines being the highest and the difference in lodging degree between the semi-determinate and determinate lines being under 1.0 in one background. Therefore, semi-determinate growth habit has potential to develop high yielding varieties with low lodging risk.

Erratum: Quantitative trait loci associated with short inter-node length in soybean.

[This corrects the article on p. 554 in vol. 68, PMID: 30697116.].

Metabolic switching of astringent and beneficial triterpenoid saponins in soybean is achieved by a loss-of-function mutation in cytochrome P450 72A69.

Triterpenoid saponins are major components of secondary metabolites in soybean seeds and are divided into two groups: group A saponins, and 2,3-dihydro-2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP) saponins. The aglycone moiety of group A saponins consists of soyasapogenol A (SA), which is an oxidized ß-amyrin product, and the aglycone moiety of the DDMP saponins consists of soyasapogenol B (SB). Group A saponins produce a bitter and astringent aftertaste in soy products, whereas DDMP saponins have known health benefits for humans. We completed map-based cloning and characterization of the gene Sg-5, which is responsible for SA biosynthesis. The naturally occurring sg-5 mutant lacks group A saponins and has a loss-of-function mutation (L164*) in Glyma15g39090, which encodes the cytochrome P450 enzyme, CYP72A69. An enzyme assay indicated the hydroxylase activity of recombinant CYP72A69 against SB, which also suggested the production of SA. Additionally, induced Glyma15g39090 mutants (R44* or S348P) lacked group A saponins similar to the sg-5 mutant, indicating that Glyma15g39090 corresponds to Sg-5. Endogenous levels of DDMP saponins were higher in the sg-5 mutant than in the wild-type lines due to the loss of the enzyme activity that converts SB to SA. Interestingly, the genomes of palaeopolyploid soybean and the closely related common bean carry multiple Sg-5 paralogs in a genomic region syntenic to the soybean Sg-5 region. However, SA did not accumulate in common bean samples, suggesting that Sg-5 activity evolved after gene duplication event(s). Our results demonstrate that metabolic switching of undesirable saponins with beneficial saponins can be achieved in soybean by disabling Sg-5.

Isolation and Characterization of the Soybean Sg-3 Gene that is Involved in Genetic Variation in Sugar Chain Composition at the C-3 Position in Soyasaponins.

Soyasaponins are specialized metabolites present in soybean seeds that affect the taste and quality of soy-based foods. The composition of the sugar chains attached to the aglycone moiety of soyasaponins is regulated by genetic loci such as sg-1, sg-3 and sg-4. Here, we report the cloning and characterization of the Sg-3 gene, which is responsible for conjugating the terminal (third) glucose (Glc) at the C-3 sugar chain of soyasaponins. The gene Glyma.10G104700 is disabled in the sg-3 cultivar, 'Mikuriya-ao', due to the deletion of genomic DNA that results in the absence of a terminal Glc residue on the C-3 sugar chain. Sg-3 encodes a putative glycosyltransferase (UGT91H9), and its predicted protein sequence has a high homology with that of the product of GmSGT3 (Glyma.08G181000; UGT91H4), which conjugates rhamnose (Rha) to the third position of the C-3 sugar chain in vitro. A recombinant Glyma.10G104700 protein could utilize UDP-Glc as a substrate to conjugate the third Glc to the C-3 sugar chain, and introducing a functional Glyma.10G104700 transgene into the mutant complemented the sg-3 phenotype. Conversely, induction of a premature stop codon mutation in Glyma.10G104700 (W270*) resulted in the sg-3 phenotype, suggesting that Glyma.10G104700 was Sg-3. The gmsgt3 (R339H) mutant failed to accumulate soyasaponins with the third Rha at the C-3 sugar chain, and the third Glc and Rha conjugations were both disabled in the sg-3 gmsgt3 double mutant. These results demonstrated that Sg-3 and GmSGT3 are non-redundantly involved in conjugation of the third Glc and Rha at the C-3 sugar chain of soyasaponins, respectively.

Simultaneous site-directed mutagenesis of duplicated loci in soybean using a single guide RNA.

KEY MESSAGE: Using a gRNA and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two GmPPD loci in soybean. Mutations in GmPPD loci were confirmed in at least 33% of T2 seeds. The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated endonuclease 9 (Cas9) system is a powerful tool for site-directed mutagenesis in crops. Using a single guide RNA (gRNA) and Agrobacterium-mediated transformation, we performed simultaneous site-directed mutagenesis of two homoeologous loci in soybean (Glycine max), GmPPD1 and GmPPD2, which encode the orthologs of Arabidopsis thaliana PEAPOD (PPD). Most of the T1 plants had heterozygous and/or chimeric mutations for the targeted loci. The sequencing analysis of T1 and T2 generations indicates that putative mutation induced in the T0 plant is transmitted to the T1 generation. The inheritable mutation induced in the T1 plant was also detected. This result indicates that continuous induction of mutations during T1 plant development increases the occurrence of mutations in germ cells, which ensures the transmission of mutations to the next generation. Simultaneous site-directed mutagenesis in both GmPPD loci was confirmed in at least 33% of T2 seeds examined. Approximately 19% of double mutants did not contain the Cas9/gRNA expression construct. Double mutants with frameshift mutations in both GmPPD1 and GmPPD2 had dome-shaped trifoliate leaves, extremely twisted pods, and produced few seeds. Taken together, our data indicate that continuous induction of mutations in the whole plant and advancing generations of transgenic plants enable efficient simultaneous site-directed mutagenesis in duplicated loci in soybean.

The effect of stem growth habit on single seed weight and seed uniformity in soybean (Glycine max (L.) Merrill).

The timing of flower formation and length of the seed-filling period of indeterminate growth soybean varieties vary more than those of determinate varieties (Glycine max (L.) Merrill). These variations have been hypothesized to affect single seed weight and its uniformity which determine the processing quality of soybean used in foods. We derived near isogenic lines (NILs) with different growth characteristics from an indeterminate line (donor parent) and three determinate lines with heavy seeds (recurrent parents), and evaluated the effects of growth habit on seed weight and its uniformity. Each NIL population consisting of five indeterminate and five determinate BC4F4 lines tested at two locations in two different years with two replications. Split-plot analysis of variance, with main-plot and sub-plot being cross combination and growth habit, respectively, showed that indeterminate varieties had slightly heavier seeds than determinate varieties and that there was no significant difference in uniformity of single seed weights. The effects of growth habit on seed uniformity was related to genetic background, but differences between the two growth characteristics were less than the differences among genetic background. This indicates that indeterminate growth habit did not much influence seed weight or its uniformity.

Identification and dissection of single seed weight QTLs by analysis of seed yield components in soybean.

Single seed weight (SSW), or seed size, is a seed yield components (SYC) in soybean, and it is suggested that the genetic factors regulating SSW are involved in the control of other SYCs. The quantitative trait loci (QTLs) for SSW and their effects on the other SYCs were investigated using a recombinant inbred line population derived from typical small- and large-seeded cultivars that were cultivated in two different environments. QTL analysis detected four environmentally stable QTLs for SSW, two of which coincided with the defined loci, qSw17-1 and Ln. The effects of the other loci, qSw12-1 and qSw13-1, were confirmed by analyzing residual heterozygous line progenies derived from the recombinant population. These four QTL regions were also involved in the control of an additional SYC, namely the large-seeded allele at each locus that reduced either the number of pods per plant or the number of ovules per pod. These results suggest the presence of at least two different regulatory mechanisms for SSW. Isolation of genes responsible for these QTLs provides an important tool in the understanding and utilization of SSW diversity for soybean breeding.