Reaction norm for genomic prediction of plant growth: modeling drought stress response in soybean.

KEY MESSAGE: We proposed models to predict the effects of genomic and environmental factors on daily soybean growth and applied them to soybean growth data obtained with unmanned aerial vehicles. Advances in high-throughput phenotyping technology have made it possible to obtain time-series plant growth data in field trials, enabling genotype-by-environment interaction (G × E) modeling of plant growth. Although the reaction norm is an effective method for quantitatively evaluating G × E and has been implemented in genomic prediction models, no reaction norm models have been applied to plant growth data. Here, we propose a novel reaction norm model for plant growth using spline and random forest models, in which daily growth is explained by environmental factors one day prior. The proposed model was applied to soybean canopy area and height to evaluate the influence of drought stress levels. Changes in the canopy area and height of 198 cultivars were measured by remote sensing using unmanned aerial vehicles. Multiple drought stress levels were set as treatments, and their time-series soil moisture was measured. The models were evaluated using three cross-validation schemes. Although accuracy of the proposed models did not surpass that of single-trait genomic prediction, the results suggest that our model can capture G × E, especially the latter growth period for the random forest model. Also, significant variations in the G × E of the canopy height during the early growth period were visualized using the spline model. This result indicates the effectiveness of the proposed models on plant growth data and the possibility of revealing G × E in various growth stages in plant breeding by applying statistical or machine learning models to time-series phenotype data.

Apoplast-Localized ß-Glucosidase Elevates Isoflavone Accumulation in the Soybean Rhizosphere.

Plant specialized metabolites (PSMs) are often stored as glycosides within cells and released from the roots with some chemical modifications. While isoflavones are known to function as symbiotic signals with rhizobia and to modulate the soybean rhizosphere microbiome, the underlying mechanisms of root-to-soil delivery are poorly understood. In addition to transporter-mediated secretion, the hydrolysis of isoflavone glycosides in the apoplast by an isoflavone conjugate-hydrolyzing ß-glucosidase (ICHG) has been proposed but not yet verified. To clarify the role of ICHG in isoflavone supply to the rhizosphere, we have isolated two independent mutants defective in ICHG activity from a soybean high-density mutant library. In the root apoplastic fraction of ichg mutants, the isoflavone glycoside contents were significantly increased, while isoflavone aglycone contents were decreased, indicating that ICHG hydrolyzes isoflavone glycosides into aglycones in the root apoplast. When grown in a field, the lack of ICHG activity considerably reduced isoflavone aglycone contents in roots and the rhizosphere soil, although the transcriptomes showed no distinct differences between the ichg mutants and wild-types (WTs). Despite the change in isoflavone contents and composition of the root and rhizosphere of the mutants, root and rhizosphere bacterial communities were not distinctive from those of the WTs. Root bacterial communities and nodulation capacities of the ichg mutants did not differ from the WTs under nitrogen-deficient conditions either. Taken together, these results indicate that ICHG elevates the accumulation of isoflavones in the soybean rhizosphere but is not essential for isoflavone-mediated plant-microbe interactions.

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.

Two tightly linked genes coding for NAD-dependent malic enzyme and dynamin-related protein are associated with resistance to Cercospora leaf spot disease in cowpea (Vigna unguiculata (L.) Walp.).

Cercospora leaf spot (CLS) caused by Cercospora canescens is an important disease of cowpea (Vigna unguiculata). A previous study using an F2 population [CSR12906 (susceptible) × IT90K-59-120 (resistant)] identified a major QTL qCLS9.1 for resistance to CLS. In this study, we finely mapped and identified candidate genes of qCLS9.1 using an F3:4 population of 699 individuals derived from two F2:3 individuals segregating at qCLS9.1 from the original population. Fine mapping narrowed down the qCLS9.1 for the resistance to a 60.6-Kb region on cowpea chromosome 10. There were two annotated genes in the 60.6-Kb region; Vigun10g019300 coding for NAD-dependent malic enzyme 1 (NAD-ME1) and Vigun10g019400 coding for dynamin-related protein 1C (DRP1C). DNA sequence analysis revealed 12 and 2 single nucleotide polymorphisms (SNPs) in the coding sequence (CDS) and the 5' untranslated region and TATA boxes of Vigun10g019300 and Vigun10g019400, respectively. Three SNPs caused amino acid changes in NAD-ME1 in CSR12906, N299S, S488N and S544N. Protein prediction analysis suggested that S488N of CSR12906 may have a deleterious effect on the function of NAD-ME1. Gene expression analysis demonstrated that IT90K-59-120 and CSR12906 challenged with C. canescens showed different expression in both Vigun10g019300 and Vigun10g019400. Taken together, these results indicated that Vigun10g019300 and Vigun10g019400 are the candidate genes for CLS resistance in the cowpea IT90K-59-120. Two derived cleaved amplified polymorphic sequence markers were developed to detect the resistance alleles at Vigun10g019300 and Vigun10g019400 in IT90K-59-120.

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.

Impacts of genomic research on soybean improvement in East Asia.

It has been commonly accepted that soybean domestication originated in East Asia. Although East Asia has the historical merit in soybean production, the USA has become the top soybean producer in the world since 1950s. Following that, Brazil and Argentina have been the major soybean producers since 1970s and 1990s, respectively. China has once been the exporter of soybean to Japan before 1990s, yet she became a net soybean importer as Japan and the Republic of Korea do. Furthermore, the soybean yield per unit area in East Asia has stagnated during the past decade. To improve soybean production and enhance food security in these East Asian countries, much investment has been made, especially in the breeding of better performing soybean germplasms. As a result, China, Japan, and the Republic of Korea have become three important centers for soybean genomic research. With new technologies, the rate and precision of the identification of important genomic loci associated with desired traits from germplasm collections or mutants have increased significantly. Genome editing on soybean is also becoming more established. The year 2019 marked a new era for crop genome editing in the commercialization of the first genome-edited plant product, which is a high-oleic-acid soybean oil. In this review, we have summarized the latest developments in soybean breeding technologies and the remarkable progress in soybean breeding-related research in China, Japan, and the Republic of Korea.

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.

Construction of genetic linkage map and genome dissection of domestication-related traits of moth bean (Vigna aconitifolia), a legume crop of arid areas.

The moth bean (Vigna aconitifolia), possibly the most primitive crop of the genus Vigna, is a highly drought- and heat-resistant legume grown in arid areas. Moth bean domestication involved phenotypic changes, including reduction of seed dormancy and pod shattering, increased organ size, and earlier flowering and maturity. However, the genetics of the domestication process in moth bean is not known. In this study, we constructed a genetic linkage map for moth bean and used the map to identify quantitative trait loci (QTL) for domestication-related traits of an F2 population of 188 individuals produced from a cross of wild moth bean (TN67) and cultivated moth bean (ICPMO056). The genetic linkage map comprised 11 linkage groups (LG) of 172 simple sequence repeat markers and spanned a total length of 1016.8 centiMorgan (cM), with an average marker distance of 7.34 cM. A comparative genome analysis showed high genome synteny between moth bean and mungbean (Vigna radiata), adzuki bean (Vigna angularis), rice bean (Vigna umbellata), and yardlong bean (Vigna unguiculata). In total, 50 QTLs and 3 genes associated with 20 domestication-related traits were identified. Most of the QTLs belonged to five LGs (1, 2, 4, 7, and 10). Key traits related to domestication such as seed dormancy and pod shattering were controlled by large-effect QTLs (PVE > 20%) with one or two minor QTLs, whereas all other traits were controlled by one-seven minor QTLs, apart from seed weight, which was controlled by one major and seven minor QTLs. These results suggest that a small number of mutations with large phenotypic effects have contributed to the domestication of the moth bean. Comparative analysis of QTLs with related Vigna crops revealed that there are several domestication-related large-effect QTLs that had not been used in moth bean domestication. This study provides a basic genetic map and identified genome regions associated with domestication-related traits, which will be useful for the genetic improvement of the moth bean and related Vigna species.

Identification and characterization of a major QTL underlying soybean isoflavone malonylglycitin content.

Isoflavones in soybean seeds are responsible for plant-microbe interactions and defend against pathogens, and are also beneficial to human health. We used two biparental populations and mini core collection of soybean germplasm to identify and validate QTLs underlying the content of isoflavone components. We identified a major QTL, qMGly_11, which regulates the content of malonylglycitin, on chromosome Gm11, in populations bred from parents with high, low, and null glycitein contents. qMGly_11 explained 44.5% of phenotypic variance in a population derived from a cross between 'Aokimame' (high) and 'Fukuyutaka' (low) and 79.9% of that in a population between 'Kumaji-1' (null) and 'Fukuyutaka' (low). The effect was observed only in the hypocotyl. We further confirmed the effect of qMGly_11 in a mini-core collection, where it explained 57.1% of the genetic diversity of glycitin production and 56.5% of malonylglycitin production. qMGly_11 increased the contents of glycitin and malonylglycitin at the expense of daidzin and malonyldaidzin in all analyzed populations. We discuss the gene responsible for this QTL and the availability of the null allele for metabolic engineering of soybean seed isoflavones.

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.

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.

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

Manipulating the genetic control of plant height is essential in soybean breeding to increase yield through the enlargement of the plant size while preventing lodging. A Japanese soybean germplasm, Y2, has distinctively shorter inter-node lengths than those of recently developed Japanese cultivars and is expected to provide new variation to prevent lodging. A quantitative trait loci (QTL) analysis for plant height-related traits was conducted using F2 individuals derived from a cross between the elite Japanese cultivar Fukuyutaka and Y2. A major QTL for average inter-node length (AIL) and plant height was identified on chromosome 13 and named qSI13-1 (QTL for short inter-node on chromosome 13). The Y2 allele of qSI13-1 was partially dominant for plant height. qSI13-1 exhibited no effect on either days to flowering or number of main stem nodes. The AILs and plant heights of the near-isogenic lines containing the Y2 allele of qSI13-1 in the genetic background of Fukuyutaka were significantly less than those of Fukuyutaka. No significant differences between the near-isogenic lines and Fukuyutaka were observed for seed yield and flowering date, indicating that qSI13-1 will be useful in developing cultivars with short plant heights without having negative effects on yield potential and days to flowering.

Evaluation of Resistance to Phytophthora sojae in Soybean Mini Core Collections Using an Improved Assay System.

Stem and root rot disease caused by Phytophthora sojae is devastating to soybean crops worldwide. Developing host resistance to P. sojae, considered the most effective and stable means to control this disease, is partly hampered by limited germplasm resources. In this study, we first modified conventional methods for a P. sojae resistance assay to a simpler and more cost-effective version, in which the P. sojae inoculum was mixed into the soil and the resistance was evaluated by survival rate (%) of soybean seedlings. This rating had significant correlations (P < 0.01) with the reduction in root fresh weight and the visual root rot severity. Applying this method to evaluate P. sojae resistance in soybean mini core collections comprising either 79 accessions originating from Japan (JMC) or 80 accessions collected around the world (WMC) revealed a wide variation in resistance among the individual varieties. In total, 38 accessions from the JMC and 41 from the WMC exhibited resistance or moderate resistance to P. sojae isolate N1 (with virulence to Rps1b, 3c, 4, 5, and 6), with ≥50% survival. Of these, 26 from the JMC and 29 from the WMC showed at least moderate resistance to P. sojae isolate HR1 (vir Rps1a-c, 1k, 2, 3a-c, 4-6, and 8). Additionally, 24 WCS accessions, in contrast to only 6 from the JMC, exhibited 100% survival after being challenged with both the N1 and HR1 isolates, suggesting a biogeographical difference between the two collections. We further verified two JMC varieties, Daizu and Amagi zairai 90D, for their resistance to an additional four P. sojae isolates (60 to 100% survival), which may provide new and valuable genetic sources for P. sojae resistance breeding in soybean.

Identification of quantitative trait loci for flowering time by a combination of restriction site-associated DNA sequencing and bulked segregant analysis in soybean.

Soybean (Glycine max) has a paleopolyploid genome, and many re-sequencing experiments to characterize soybean genotypes have been conducted using next-generation sequencing platforms. The accumulation of information about single nucleotide polymorphisms (SNPs) throughout the soybean genome has accelerated identification of genomic regions related to agronomically important traits through association studies. However, although many efficient mapping techniques that use next-generation sequencing are available, the number of practical approaches to identify genes/loci is still limited. In this study, we used a combination of restriction site-associated DNA sequencing (RAD-seq) and bulk segregant analysis (BSA) to identify quantitative trait locus (QTLs) for flowering time in a segregating population derived from a cross between Japanese soybean cultivars. Despite the homogeneous genetic background of the parents, over 7000 SNPs were identified and can be used to detect QTLs by RAD-seq BSA analysis. By comparing genotype frequency between early and late-flowering bulks from the F3 segregating population, we identified a QTL on Gm10, which corresponds to the previously identified E2 locus, and a QTL on Gm04, which is close to the E8 locus. Out of these SNPs, more than 2000 were easily converted to conventional DNA markers. Our approach would improve the efficiency of genetic mapping.

Confirmation of the pleiotropic control of leaflet shape and number of seeds per pod by the Ln gene in induced soybean mutants.

Most soybean cultivars possess broad leaflets; however, a recessive allele on the Ln locus is known to cause the alteration of broad to narrow leaflets. The recessive allele ln has also been considered to increase the number of seeds per pod (NSP) and has the potential to improve yield. Recently, Gm-JAG1 (Glyma20g25000), a gene controlling Ln, has been shown to complement leaf shape and silique length in Arabidopsis mutants. However, whether Gm-JAG1 is responsible for those traits in soybean is not yet known. In this study, we investigated the pleiotropic effect of soybean Ln gene on leaflet shape and NSP by using two independent soybean Gm-jag1 mutants and four ln near isogenic lines (NILs). The leaflet shape was evaluated using a leaf image analysis software, SmartLeaf, which was customized from SmartGrain. The leaflets of both the Gm-jag1 mutants were longer and narrower than those of the wild-type plants. Interestingly, the image analysis results clarified that the perimeter of the mutant leaflets did not change, although their leaflet area decreased. Furthermore, one mutant line with narrow leaflets showed significantly higher NSP than that in the wild (or Ln) genotype, indicating that soybean Ln gene pleiotropically controls leaflet shape and NSP.

Multiple organ gigantism caused by mutation in VmPPD gene in blackgram (Vigna mungo).

Seed size is one of the most important traits in leguminous crops. We obtained a recessive mutant of blackgram that had greatly enlarged leaves, stems and seeds. The mutant produced 100% bigger leaves, 50% more biomass and 70% larger seeds though it produced 40% less number of seeds. We designated the mutant as multiple-organ-gigantism (mog) and found the mog phenotype was due to increase in cell numbers but not in cell size. We also found the mog mutant showed a rippled leaf (rl) phenotype, which was probably caused by a pleiotropic effect of the mutation. We performed a map-based cloning and successfully identified an 8 bp deletion in the coding sequence of VmPPD gene, an orthologue of Arabidopsis PEAPOD (PPD) that regulates arrest of cell divisions in meristematic cells. We found no other mutations in the neighboring genes between the mutant and the wild type. We also knocked down GmPPD genes and reproduced both the mog and rl phenotypes in soybean. Controlling PPD genes to produce the mog phenotype is highly valuable for breeding since larger seed size could directly increase the commercial values of grain legumes.

Construction of a high-density mutant library in soybean and development of a mutant retrieval method using amplicon sequencing.

BACKGROUND: Functions of most genes predicted in the soybean genome have not been clarified. A mutant library with a high mutation density would be helpful for functional studies and for identification of novel alleles useful for breeding. Development of cost-effective and high-throughput protocols using next generation sequencing (NGS) technologies is expected to simplify the retrieval of mutants with mutations in genes of interest. RESULTS: To increase the mutation density, seeds of the Japanese elite soybean cultivar Enrei were treated with the chemical mutagen ethyl methanesulfonate (EMS); M2 seeds produced by M1 plants were treated with EMS once again. The resultant library, which consisted of DNA and seeds from 1536 plants, revealed large morphological and physiological variations. Based on whole-genome re-sequencing analysis of 12 mutant lines, the average number of base changes was 12,796 per line. On average, 691 and 35 per line were missense and nonsense mutations, respectively. Two screening strategies for high resolution melting (HRM) analysis and indexed amplicon sequencing were designed to retrieve the mutants; the mutations were confirmed by Sanger sequencing as the final step. In comparison with HRM screening of several genes, indexed amplicon sequencing allows one to scan a longer sequence range and skip screening steps and to know the sequence information of mutation because it uses systematic DNA pooling and the index of NGS reads, which simplifies the discovery of mutants with amino acid substitutions. CONCLUSIONS: A soybean mutant library with a high mutation density was developed. A high mutation density (1 mutation/74 kb) was achieved by repeating the EMS treatment. The mutation density of our library is sufficiently high to obtain a plant in which a gene is nonsense mutated. Thus, our mutant library and the indexed amplicon sequencing will be useful for functional studies of soybean genes and have a potential to yield useful mutant alleles for soybean breeding.