Genetic and Phenotypic Characterization of Soybean Landraces Collected from the Zhejiang Province in China.

The soybean is an important feed, industrial raw material, and food crop in the world due to its rich components. There is a long history of soybean cultivation with different types and rich resources in the Zhejiang province of China. It is important to understand genetic diversity as well as phenotypic variation for soybean breeding. The objective of this study was to analyze both genetic and phenotypic characteristics of the 78 soybean landraces collected, and to explore a potential advantage of germplasm resources for further application. These 78 autumn-type soybean landraces have been propagated, identified, and evaluated in both 2021 and 2022. There were agronomic, quality, and genetic variations according to the comprehensive analyses. There was a good consistency between seed size and seed coat color. There were significant differences of seed protein, fat, and sugar contents based upon the seed coat color. These soybean landraces were genotyped using 42 simple sequence repeat markers and then clustered into two groups. The two groups had a consistency with the seed coat color. This study gave us a combined understanding of both the phenotypic variation and the genetic diversity of the soybean landraces. Therefore, the reasonable crossing between different soybean types is highly recommended.

A novel strand-specific RNA-sequencing protocol using dU-adaptor-assembled Tn5.

Strand-specific RNA-seq is a powerful tool for the discovery of novel transcripts, annotation of genomes, and profiling of gene expression levels. Tn5 transposase has been successfully applied in massive-scale sequencing projects; in particular, Tn5 adaptor modification is used in epigenetics, genomic structure, and chromatin visualization. We developed a novel dU-adaptor-assembled Tn5-mediated strand-specific RNA-sequencing protocol and compared this method with the leading dUTP method in terms of experimental procedure and multiple quality metrics of the generated libraries. The results showed that the dU-Tn5 method is easy to operate and generates a strand-specific RNA-seq library of comparable quality considering library complexity, strand-specificity, evenness, and continuity of annotated transcript coverage. We also evaluated the performance of the dU-Tn5 method in identifying nitrogen-responsive protein-coding genes and long non-coding RNAs in soybean roots. The results indicated that ~62-70% of differentially expressed genes detected from conventional libraries were also detected in dU-Tn5 libraries, indicating good agreement of our method with the current standard; moreover, their fold-changes were highly correlated (R>0.9). Thus, our method provides a promising 'do-it-yourself' stranded RNA-seq procedure for gene expression profiling.

Transcriptome and Metabolome Analysis of a Late-Senescent Vegetable Soybean during Seed Development Provides New Insights into Degradation of Chlorophyll.

(1) Background: Senescence represents the final stage of plant growth and development, which transfers nutrients to growing seeds and directly affects the yield and quality of crops. However, little is known about chlorophyll degradation in developing and maturing seeds, in contrast to leaf senescence; (2) Methods: RNA-Seq was used to analyze the differentially expressed genes of different late-senescent germplasms. A widely untargeted metabolic analysis was used to analyze differential metabolites. In addition, qRT-PCR was conducted to detect gene expression levels; (3) Results: Transcriptome analysis revealed that ZX12 seeds have a higher expression level of the chlorophyll synthesis genes in the early stage of maturity, compared with ZX4, and have a lower expression level of chlorophyll degradation genes in the late stage of maturity. Flavonoids were the primary differential metabolites, and ZX12 contains the unique and highest expression of three types of metabolites, including farrerol-7-O-glucoside, cyanidin-3-o-(6'-o-feruloyl) glucoside, and kaempferide-3-o-(6'-malonyl) glucoside. Among them, farrerol-7-O-glucoside and cyanidin-3-o-(6'-o-feruloyl) glucoside are flavonoid derivatives containing mono and dihydroxy-B-ring chemical structures, respectively; and (4) Conclusions: It is speculated that the two metabolites can slow down the degradation process of chlorophyll by scavenging oxygen-free radicals in the chloroplast.

Genome-Wide Comparison of Structural Variations and Transposon Alterations in Soybean Cultivars Induced by Spaceflight.

Space mutation causes genetic and phenotypic changes in biological materials. Transposon activation is an adaptive mechanism for organisms to cope with changes in the external environment, such as space mutation. Although transposon alterations have been widely reported in diverse plant species, few studies have assessed the global transposon alterations in plants exposed to the space environment. In this study, for the first time, the effects of transposon alterations in soybean caused by space mutation were considered. A new vegetable soybean variety, 'Zhexian 9' (Z9), derived from space mutation treatment of 'Taiwan 75' (T75), was genetically analyzed. Comparative analyses of these two soybean genomes uncovered surprising structural differences, especially with respect to translocation breakends, deletions, and inversions. In total, 12,028 structural variations (SVs) and 29,063 transposable elements (TEs) between T75 and Z9 were detected. In addition, 1336 potential genes were variable between T75 and Z9 in terms of SVs and TEs. These differential genes were enriched in functions such as defense response, cell wall-related processes, epigenetics, auxin metabolism and transport, signal transduction, and especially methylation, which implied that regulation of epigenetic mechanisms and TE activity are important in the space environment. These results are helpful for understanding the role of TEs in response to the space environment and provide a theoretical basis for the selection of wild plant materials suitable for space breeding.

Evolutionary and Regulatory Pattern Analysis of Soybean Ca2+ ATPases for Abiotic Stress Tolerance.

P2-type Ca2+ ATPases are responsible for cellular Ca2+ transport, which plays an important role in plant development and tolerance to biotic and abiotic stresses. However, the role of P2-type Ca2+ ATPases in stress response and stomatal regulation is still elusive in soybean. In this study, a total of 12 P2-type Ca2+ ATPases genes (GmACAs and GmECAs) were identified from the genome of Glycine max. We analyzed the evolutionary relationship, conserved motif, functional domain, gene structure and location, and promoter elements of the family. Chlorophyll fluorescence imaging analysis showed that vegetable soybean leaves are damaged to different extents under salt, drought, cold, and shade stresses. Real-time quantitative PCR (RT-qPCR) analysis demonstrated that most of the GmACAs and GmECAs are up-regulated after drought, cold, and NaCl treatment, but are down-regulated after shading stress. Microscopic observation showed that different stresses caused significant stomatal closure. Spatial location and temporal expression analysis suggested that GmACA8, GmACA9, GmACA10, GmACA12, GmACA13, and GmACA11 might promote stomatal closure under drought, cold, and salt stress. GmECA1 might regulate stomatal closure in shading stress. GmACA1 and GmECA3 might have a negative function on cold stress. The results laid an important foundation for further study on the function of P2-type Ca2+ ATPase genes GmACAs and GmECAs for breeding abiotic stress-tolerant vegetable soybean.

Transcriptomic and Metabolomic Analyses Reveal a Potential Mechanism to Improve Soybean Resistance to Anthracnose.

Anthracnose, caused by Colletotrichum truncatum, leads to large-scale reduction in quality and yield in soybean production. Limited information is available regarding the molecular mechanisms of resistance to anthracnose in soybean. We conducted a transcriptomic and targeted metabolomic analysis of pods from two soybean lines, "Zhechun No. 3" (ZC3) and ZC-2, in response to C. truncatum infection. Factors contributing to the enhanced resistance of ZC-2 to anthracnose compared with that of ZC3, included signal transduction (jasmonic acid, auxin, mitogen-activated protein kinase, and Ca2+ signaling), transcription factors (WRKY and bHLH), resistance genes (PTI1, RPP13, RGA2, RPS6, and ULP2B), pathogenesis-related genes (chitinase and lipid transfer protein), and terpenoid metabolism. Targeted metabolomic analysis revealed that terpenoid metabolism responded more promptly and more intensely to C. truncatum infection in ZC-2 than in ZC3. In vitro antifungal activity and resistance induction test confirmed that jasmonic acid, auxin signaling and terpenoids played important roles in soybean resistance to anthracnose. This research is the first study to explore the molecular mechanisms of soybean resistance to anthracnose. The findings are important for in-depth analysis of molecular resistance mechanisms, discovery of resistance genes, and to expedite the breeding of anthracnose-resistant soybean cultivars.

Biochar alleviated the toxicity of atrazine to soybeans, as revealed by soil microbial community and the assembly process.

Atrazine has a detrimental effect on soybean growth in corn-soybean rotation systems. A knowledge gap exists regarding how rhizosphere microbial interactions respond to atrazine stress, and specifically, whether they may alleviate the detriment of atrazine on soybeans, this serving as a target to alleviate the adverse impact. Biochar are widely used for remediation in herbicide contamination soil, however, little is known about how biochar fuels the microbiomes in rhizosphere to improve soybean performance. We investigated the response of the microbial community to atrazine stress with and without biochar application to soybean cultivation in a greenhouse experiment. Atrazine had detrimental effects on soybeans and nodules, reshaping the microbial community in both the bulk and rhizosphere soil. Biochar application was able to ameliorate atrazine effects on soybean and nodule activity, with an increase in competition among microbes in the soybean rhizosphere soils. Biochar favored the probiotics such as the bacteria Lysobacter, Paenarthrobacter, and Sediminibacterium in the rhizosphere soils. The relative abundance of Lysobacter exhibited strong-negative correlations with potential pathogens. Elastic net regression with bioindicators and environmental factors accurately predicted the residual content of atrazine in soil. Collectively, our results provide a practical strategy of using biochar to improve soil quality for corn-soybean rotation that is contaminated with residual atrazine. Overall, beneficial plant microbes and changes in microbial interactions and assembly processes in the soybean rhizosphere are capable of alleviating atrazine stress on soybean growth.

A Sequence Variation in GmBADH2 Enhances Soybean Aroma and Is a Functional Marker for Improving Soybean Flavor.

The vegetable soybean (Glycine max L. Merr.) plant is commonly consumed in Southeast Asian countries because of its nutritional value and desirable taste. A "pandan-like" aroma is an important value-added quality trait that is rarely found in commercial vegetable soybean varieties. In this study, three novel aromatic soybean cultivars with a fragrant volatile compound were isolated. We confirmed that the aroma of these cultivars is due to the potent volatile compound 2-acetyl-1-pyrroline (2AP) that was previously identified in soybean. A sequence comparison of GmBADH1/2 (encoding an aminoaldehyde dehydrogenase) between aromatic and non-aromatic soybean varieties revealed a mutation with 10 SNPs and an 11-nucleotide deletion in exon 1 of GmBADH2 in Quxian No. 1 and Xiangdou. Additionally, a 2-bp deletion was detected in exon 10 of GmBADH2 in ZK1754. The mutations resulted in a frame shift and the introduction of premature stop codons. Moreover, genetic analyses indicated that the aromatic trait in these three varieties was inherited according to a single recessive gene model. These results suggested that a mutated GmBADH2 may be responsible for the aroma of these three aromatic soybean cultivars. The expression and function of GmBADH2 in aromatic soybean seeds were confirmed by qRT-PCR and CRISPR/Cas9. A functional marker developed on the basis of the mutated GmBADH2 sequence in Quxian No. 1 and Xiangdou was validated in an F2 population. A perfect association between the marker genotypes and aroma phenotypes implied that GmBADH2 is a major aroma-conferring gene. The results of this study are potentially useful for an in-depth analysis of the molecular basis of 2-AP formation in soybean and the marker-assisted breeding of aromatic vegetable soybean cultivars.

Ultrasonic Waves Regulate Antioxidant Defense and Gluconeogenesis to Improve Germination From Naturally Aged Soybean Seeds.

Soybean seeds contain substantial triacylglycerols and fatty acids that are prone to oxidation during storage, contributing to the dramatic deterioration of seed vigor. This study reports an ultrasonic waves treatment (UWT), which is a physical method capable of promoting the germination ability of the aged soybean seeds by regulating the antioxidant defense and gluconeogenesis. Germination test revealed that UWT significantly increased the germination rate and seedlings' establishment of the soybean seeds stored for 12 months, although insignificantly impacting the vigor of fresh (stored for 1 month) and short-term stored (for 6 months) seeds. Further biochemical analysis revealed that UWT decreased the hydrogen peroxide (H2O2), O2⋅-, and malondialdehyde contents in the aged soybean seeds during early germination. Consistently, UWT prominently elevated the activities of superoxide dismutase, catalase, and acetaldehyde dehydrogenase, and also the corresponding gene expressions. Besides, the soluble sugar content of UWT was significantly higher than that of the untreated aged seeds. Analysis of enzyme activity showed UWT significantly upregulated the activities of several key enzymes in gluconeogenesis and the transcription levels of corresponding genes. Moreover, UWT enhanced the invertase activity within aged seeds, which was responsible for catalyzing sucrose hydrolysis for forming glucose and fructose. In summary, UWT improved germination and seedlings establishment of aged soybean seeds by regulating antioxidant defense and gluconeogenesis. This study expands the application of ultrasonication in agricultural production and further clarifies the physiological and molecular mechanisms of the aged seed germination, aiming to provide theoretical and practical guidance for seed quality and safety.

Development and Application of an In Vitro Method to Evaluate Anthracnose Resistance in Soybean Germplasm.

Anthracnose caused by Colletotrichum truncatum is a major fungal disease of soybean, especially vegetable soybean (edamame). Studies of this disease have mainly focused on resistance evaluation, but the primary methods used-in vivo inoculation of pods or plants under greenhouse or field conditions-have limitations with respect to accuracy, stability, scale, and environmental safety. In this study, we developed a method for inoculating pods in vitro by soaking in a mycelial suspension. We optimized the crucial components, including the mycelial suspension concentration (40 to 60 mg mL-1), the maturity of the sampled pods (15 days after flowering), and the post-inoculation incubation period (5 days). Application of the mycelial suspension by soaking rather than spraying improved the efficiency of inoculation and made large-scale evaluation possible. Using this method, we evaluated 589 soybean germplasm resources (275 cultivars, 233 landraces, and 81 wild accessions). We identified 25 highly resistant cultivars, 11 highly resistant landraces, but only one highly resistant wild accession. Our results will aid future research on soybean anthracnose resistance, including gene discovery, the elucidation of molecular mechanisms, and the breeding of resistant cultivars.

Comparative Analysis and Development of a Flavor Fingerprint for Volatile Compounds of Vegetable Soybean Seeds Based on Headspace-Gas Chromatography-Ion Mobility Spectrometry.

Evaluating the volatile compounds and characteristic fingerprints of the core cultivars of vegetable soybean would provide useful data for improving their aroma in the breeding programs. The present study used headspace-gas chromatography-ion mobility spectrometry (HS-GC-IMS) to evaluate the volatile compounds of vegetable soybean seeds at a specific growth stage. In total, 93 signal peaks were identified, 63 compounds qualitatively, with 14 volatile flavor compounds providing multiple signals. The 63 volatile compounds consisted of 15 esters, 15 aldehydes, 13 alcohols, 15 ketones, one acid, and four other compounds. The peak intensity of most of the volatile compounds varied greatly between the core cultivars. The alcohols and aldehydes determined the basic volatile flavor of the vegetable soybean seeds. Volatile flavors were determined by their respective esters, ketones, or other components. Characteristic fingerprints were found in some core vegetable soybean cultivars. Four cultivars (Xiangdou, ZHE1754, Zhexian 65018-33, and Qvxian No. 1) had pleasant aromas, because of their higher content of 2-acetyl-1-pyrroline (2-AP). A principal component analysis (PCA) was used to distinguish the samples based on the signal intensity of their volatile components. The results showed that the composition and concentration of volatile compounds differed greatly between the core cultivars, with the volatile flavor compounds of soybeans being determined by the ecotype of the cultivar, the direction of breeding selection, and their geographical origin. Characteristic fingerprints of the cultivars were established by HS-GC-IMS, enabling them to be used to describe and distinguish cultivars and their offspring in future breeding studies.

Transcriptome analysis identifies differentially expressed genes in the progenies of a cross between two low phytic acid soybean mutants.

Phytic acid (PA) is a major antinutrient that cannot be digested by monogastric animals, but it can decrease the bioavailability of micronutrients (e.g., Zn and Fe). Lowering the PA content of crop seeds will lead to enhanced nutritional traits. Low-PA mutant crop lines carrying more than one mutated gene (lpa) have lower PA contents than mutants with a single lpa mutant gene. However, little is known about the link between PA pathway intermediates and downstream regulatory activities following the mutation of these genes in soybean. Consequently, we performed a comparative transcriptome analysis using an advanced generation recombinant inbred line with low PA levels [2mlpa (mips1/ipk1)] and a sibling line with homozygous non-mutant alleles and normal PA contents [2MWT (MIPS1/IPK1)]. An RNA sequencing analysis of five seed developmental stages revealed 7945 differentially expressed genes (DEGs) between the 2mlpa and 2MWT seeds. Moreover, 3316 DEGs were associated with 128 metabolic and signal transduction pathways and 4980 DEGs were annotated with 345 Gene Ontology terms related to biological processes. Genes associated with PA metabolism, photosynthesis, starch and sucrose metabolism, and defense mechanisms were among the DEGs in 2mlpa. Of these genes, 36 contributed to PA metabolism, including 22 genes possibly mediating the low-PA phenotype of 2mlpa. The expression of most of the genes associated with photosynthesis (81 of 117) was down-regulated in 2mlpa at the late seed developmental stage. In contrast, the expression of three genes involved in sucrose metabolism was up-regulated at the late seed developmental stage, which might explain the high sucrose content of 2mlpa soybeans. Furthermore, 604 genes related to defense mechanisms were differentially expressed between 2mlpa and 2MWT. In this study, we detected a low PA content as well as changes to multiple metabolites in the 2mlpa mutant. These results may help elucidate the regulation of metabolic events in 2mlpa. Many genes involved in PA metabolism may contribute to the substantial decrease in the PA content and the moderate accumulation of InsP3-InsP5 in the 2mlpa mutant. The other regulated genes related to photosynthesis, starch and sucrose metabolism, and defense mechanisms may provide additional insights into the nutritional and agronomic performance of 2mlpa seeds.

Genome-Wide Variation Analysis of Four Vegetable Soybean Cultivars Based on Re-Sequencing.

Vegetable soybean is a type of value-added specialty soybean, served as a fresh vegetable or snack in China. Due to the difference from other types, it is important to understand the genetic structure and diversity of vegetable soybean for further utilization in breeding programs. The four vegetable cultivars, Taiwan-75, Zhexiandou No. 8, Zhexian No. 9 and Zhexian No. 10 are popular soybean varieties planted in Zhejiang province, and have large pods and intermediate maturity. The clustering showed a close relationship of these four cultivars in simple sequence repeat analysis. To reveal the genome variation of vegetable soybean, these four improved lines were analyzed by whole-genome re-sequencing. The average sequencing depth was 7X and the coverage ratio of each cultivar was at least more than 94%. Compared with the reference genome, a large number of single-nucleotide polymorphisms, insertion/deletions and structure variations were identified with different chromosome distributions. The average heterozygosity rate of the single-nucleotide polymorphisms was 11.99% of these four cultivars. According to the enrichment analysis, there were 23,371 genes identified with putative modifications, and a total of 282 genes were related to carbohydrate metabolic processes. These results provide useful information for genetic research and future breeding, which can facilitate the selection procedures in vegetable soybean breeding.

Quantitative proteomic analyses of two soybean low phytic acid mutants to identify the genes associated with seed field emergence.

BACKGROUND: Seed germination is essential to crop growth and development, and ultimately affects its harvest. It is difficult to breed soybeans low in phytic acid with a higher seed field emergence. Although additional management and selection could overcome the phytate reduction, the mechanisms of seed germination remain unknown. RESULTS: A comparative proteomic analysis was conducted between two low phytic acid (LPA) soybean mutants (TW-1-M and TW-1), both of which had a deletion of 2 bp in the GmMIPS1 gene. However, the TW-1 seeds showed a significantly lower field emergence compared to the TW-1-M. There were 282 differentially accumulated proteins (DAPs) identified between two mutants at the three stages. Among these DAPs, 80 were down-accumulated and 202 were up-accumulated. Bioinformatic analysis showed that the identified proteins were related to functional categories of oxidation reduction, response to stimulus and stress, dormancy and germination processes and catalytic activity. KEGG analysis showed that these DAPs were mainly involved in energy metabolism and anti-stress pathways. Based upon the conjoint analysis of DAPs with the differentially expressed genes (DEGs) previously published among three germination stages in two LPA mutants, 30 shared DAPs/DEGs were identified with different patterns, including plant seed protein, beta-amylase, protein disulfide-isomerase, disease resistance protein, pyrophosphate-fructose 6-phosphate 1-phosphotransferase, cysteine proteinase inhibitor, non-specific lipid-transfer protein, phosphoenolpyruvate carboxylase and acyl-coenzyme A oxidase. CONCLUSIONS: Seed germination is a very complex process in LPA soybean mutants. The TW-1-M and TW-1 showed many DAPs involved in seed germination. The differential accumulation of these proteins could result in the difference of seed field emergence between the two mutants. The high germination rate in the TW-1-M might be strongly attributed to reactive oxygen species-related and plant hormone-related genes. All these findings would help us further explore the germination mechanisms in LPA crops.

Whole genome-wide transcript profiling to identify differentially expressed genes associated with seed field emergence in two soybean low phytate mutants.

BACKGROUND: Seed germination is important to soybean (Glycine max) growth and development, ultimately affecting soybean yield. A lower seed field emergence has been the main hindrance for breeding soybeans low in phytate. Although this reduction could be overcome by additional breeding and selection, the mechanisms of seed germination in different low phytate mutants remain unknown. In this study, we performed a comparative transcript analysis of two low phytate soybean mutants (TW-1 and TW-1-M), which have the same mutation, a 2 bp deletion in GmMIPS1, but show a significant difference in seed field emergence, TW-1-M was higher than that of TW-1 . RESULTS: Numerous genes analyzed by RNA-Seq showed markedly different expression levels between TW-1-M and TW-1 mutants. Approximately 30,000-35,000 read-mapped genes and ~21000-25000 expressed genes were identified for each library. There were ~3900-9200 differentially expressed genes (DEGs) in each contrast library, the number of up-regulated genes was similar with down-regulated genes in the mutant TW-1and TW-1-M. Gene ontology functional categories of DEGs indicated that the ethylene-mediated signaling pathway, the abscisic acid-mediated signaling pathway, response to hormone, ethylene biosynthetic process, ethylene metabolic process, regulation of hormone levels, and oxidation-reduction process, regulation of flavonoid biosynthetic process and regulation of abscisic acid-activated signaling pathway had high correlations with seed germination. In total, 2457 DEGs involved in the above functional categories were identified. Twenty-two genes with 20 biological functions were the most highly up/down- regulated (absolute value Log2FC >5) in the high field emergence mutant TW-1-M and were related to metabolic or signaling pathways. Fifty-seven genes with 36 biological functions had the greatest expression abundance (FRPM >100) in germination-related pathways. CONCLUSIONS: Seed germination in the soybean low phytate mutants is a very complex process, which involves a series of physiological, morphological and transcriptional changes. Compared with TW-1, TW-1-M had a very different gene expression profile, which included genes related to plant hormones, antioxidation, anti-stress and energy metabolism processes. Our research provides a molecular basis for understanding germination mechanisms, and is also an important resource for the genetic analysis of germination in low phytate crops. Plant hormone- and antioxidation-related genes might strongly contribute to the high germination rate in the TW-1-M mutant.

Profiling and relationship of water-soluble sugar and protein compositions in soybean seeds.

Sugar and protein are important quality traits in soybean seeds for making soy-based food products. However, the investigations on both compositions and their relationship have rarely been reported. In this study, a total of 35 soybean germplasms collected from Zhejiang province of China, were evaluated for both water-soluble sugar and protein. The total water-soluble sugar (TWSS) content of the germplasms studied ranged from 84.70 to 140.91 mg/g and the water-soluble protein (WSP) content varied from 26.5% to 36.0%. The WSP content showed positive correlations with the TWSS and sucrose contents but negative correlations with the fructose and glucose contents. The clustering showed the 35 germplasms could be divided into four groups with specific contents of sugar and protein. The combination of water-soluble sugar and protein profiles provides useful information for future breeding and genetic research. This investigation will facilitate future work for seed quality improvement.

Identification and characterization of the soybean IPK1 ortholog of a low phytic acid mutant reveals an exon-excluding splice-site mutation.

Phytic acid (myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is an important constituent of soybean meal. Since phytic acid and its mineral salts (phytates) are almost indigestible for monogastrics, their abundance in grain food/feed causes nutritional and environmental problems; interest in breeding low phytic acid has therefore increased considerably. Based on gene mapping and the characteristics of inositol polyphosphates profile in the seeds of a soybean mutant line Gm-lpa-ZC-2, the soybean ortholog of inositol 1,3,4,5,6 pentakisphosphate (InsP(5)) 2-kinase (IPK1), which transforms InsP(5) into phytic acid, was first hypothesized as the candidate gene responsible for the low phytic acid alteration in Gm-lpa-ZC-2. One IPK1 ortholog (Glyma14g07880, GmIPK1) was then identified in the mapped region on chromosome 14. Sequencing revealed a G → A point mutation in the genomic DNA sequence and the exclusion of the entire fifth exon in the cDNA sequence of GmIPK1 in Gm-lpa-ZC-2 compared with its wild-type progenitor Zhechun No. 3. The excluded exon encodes 37 amino acids that spread across two conserved IPK1 motifs. Furthermore, complete co-segregation of low phytic acid phenotype with the G → A mutation was observed in the F(2) population of ZC-lpa x Zhexiandou No. 4 (a wild-type cultivar). Put together, the G → A point mutation affected the pre-mRNA splicing and resulted in the exclusion of the fifth exon of GmIPK1 which is expected to disrupt the GmIPK1 functionality, leading to low phytic acid level in Gm-lpa-ZC-2. Gm-lpa-ZC-2, would be a good germplasm source in low phytic acid soybean breeding.

Effects of two low phytic acid mutations on seed quality and nutritional traits in soybean (Glycine max L. Merr).

Reduction of phytic acid in soybean seeds has the potential to improve the nutritional value of soybean meal and lessen phosphorus pollution in large scale animal farming. The objective of this study was to assess the effect of two new low phytic acid (LPA) mutations on seed quality and nutritional traits. Multilocation/season comparative analyses showed that the two mutations did not affect the concentration of crude protein, any of the individual amino acids, crude oil, and individual saturated fatty acids. Among other traits, Gm-lpa-TW75-1 had consistently higher sucrose contents (+47.4-86.1%) and lower raffinose contents (-74.2 to -84.3%) than those of wild type (WT) parent Taiwan 75; Gm-lpa-ZC-2 had higher total isoflavone contents (3038.8-4305.4 microg/g) than its parent Zhechun # 3 (1583.6-2644.9 microg/g) in all environments. Further tests of homozygous F(3) progenies of the cross Gm-lpa-ZC-2 x Wuxing # 4 (WT variety) showed that LPA lines had a mean content of total isoflavone significantly higher than WT lines. This study demonstrated that two LPA mutant genes have no negative effects on seed quality and nutritional traits; they instead have the potential to improve a few other properties. Therefore, these two mutant genes are valuable genetic resources for breeding high quality soybean varieties.

Generation and characterization of two novel low phytate mutations in soybean (Glycine max L. Merr.).

Phytic acid (PA, myo-inositol 1, 2, 3, 4, 5, 6 hexakisphosphate) is important to the nutritional quality of soybean meal. Organic phosphorus (P) in PA is indigestible in humans and non-ruminant animals, which affects nutrition and causes P pollution of ground water from animal wastes. Two novel soybean [(Glycine max L. (Merr.)] low phytic acid (lpa) mutations were isolated and characterized. Gm-lpa-TW-1 had a phytic acid P (PA-P) reduction of 66.6% and a sixfold increase in inorganic P (Pi), and Gm-lpa-ZC-2 had a PA-P reduction of 46.3% and a 1.4-fold increase in Pi, compared with their respective non-mutant progenitor lines. The reduction of PA-P and increase of Pi in Gm-lpa-TW-1 were molar equivalent; the decrease of PA-P in Gm-lpa-ZC-2, however, was accompanied by the increase of both Pi and lower inositol phosphates. In both mutant lines, the total P content remained similar to their wild type parents. The two lpa mutations were both inherited in a single recessive gene model but were non-allelic. Sequence data and progeny analysis indicate that Gm-lpa-TW-1 lpa mutation resulted from a 2 bp deletion in the soybean D: -myo-inositol 3-phosphate synthase (MIPS1 EC 5.5.1.4) gene 1 (MIPS1). The lpa mutation in Gm-lpa-ZC-2 was mapped on LG B2, closely linked with microsatellite loci Satt416 and Satt168, at genetic distances of approximately 4.63 and approximately 9.25 cM, respectively. Thus this mutation probably represents a novel soybean lpa locus. The seed emergence rate of Gm-lpa-ZC-2 was similar to its progenitor line and was not affected by seed source and its lpa mutation. However, Gm-lpa-TW-1 had a significantly reduced field emergence when seeds were produced in a subtropic environment. Field tests of the mutants and their progenies further demonstrated that the lpa mutation in Gm-lpa-ZC-2 does not negatively affect plant yield traits. These results will advance understanding of the genetic, biochemical and molecular control of PA synthesis in soybean. The novel lpa mutation in Gm-lpa-ZC-2, together with linked simple sequence repeat (SSR) markers, will be of value for breeding productive lpa soybeans, with meal high in digestible Pi eventually to improve animal nutrition and lessen environmental pollution.