Mapping quantitative trait loci for root development under hypoxia conditions in soybean (Glycine max L. Merr.).

KEY MESSAGE: Greatest potential, QTLs for hypoxia and waterlogging tolerance in soybean roots were detected using a new phenotypic evaluation method. Waterlogging is a major environmental stress limiting soybean yield in wet parts of the world. Root development is an important indicator of hypoxia tolerance in soybean. However, little is known about the genetic control of root development under hypoxia. This study was conducted to identify quantitative trait loci (QTLs) responsible for root development under hypoxia. Recombinant inbred lines (RILs) developed from a cross between a hypoxia-sensitive cultivar, Tachinagaha, and a tolerant landrace, Iyodaizu, were used. Seedlings were subjected to hypoxia, and root development was evaluated with the value change in root traits between after and before treatments. We found 230 polymorphic markers spanning 2519.2 cM distributed on all 20 chromosomes (Chrs.). Using these, we found 11 QTLs for root length (RL), root length development (RLD), root surface area (RSA), root surface area development (RSAD), root diameter (RD), and change in average root diameter (CARD) on Chrs. 11, 12, 13 and 14, and 7 QTLs for hypoxia tolerance of these root traits. These included QTLs for RLD and RSAD between markers Satt052 and Satt302 on Chr. 12, which are important markers of hypoxia tolerance in soybean; those QTLs were stable between 2 years. To validate the QTLs, we developed a near-isogenic line with the QTL region derived from Iyodaizu. The line performed well under both hypoxia and waterlogging, suggesting that the region contains one or more genes with large effects on root development. These findings may be useful for fine mapping and positional cloning of gene responsible for root development under hypoxia.

Positional cloning and characterization reveal the molecular basis for soybean maturity locus E1 that regulates photoperiodic flowering.

The complex and coordinated regulation of flowering has high ecological and agricultural significance. The maturity locus E1 has a large impact on flowering time in soybean, but the molecular basis for the E1 locus is largely unknown. Through positional cloning, we delimited the E1 locus to a 17.4-kb region containing an intron-free gene (E1). The E1 protein contains a putative bipartite nuclear localization signal and a region distantly related to B3 domain. In the recessive allele, a nonsynonymous substitution occurred in the putative nuclear localization signal, leading to the loss of localization specificity of the E1 protein and earlier flowering. The early-flowering phenotype was consistently observed in three ethylmethanesulfonate-induced mutants and two natural mutations that harbored a premature stop codon or a deletion of the entire E1 gene. E1 expression was significantly suppressed under short-day conditions and showed a bimodal diurnal pattern under long-day conditions, suggesting its response to photoperiod and its dominant effect induced by long day length. When a functional E1 gene was transformed into the early-flowering cultivar Kariyutaka with low E1 expression, transgenic plants carrying exogenous E1 displayed late flowering. Furthermore, the transcript abundance of E1 was negatively correlated with that of GmFT2a and GmFT5a, homologues of FLOWERING LOCUS T that promote flowering. These findings demonstrated the key role of E1 in repressing flowering and delaying maturity in soybean. The molecular identification of the maturity locus E1 will contribute to our understanding of the molecular mechanisms by which a short-day plant regulates flowering time and maturity.

Phosphoproteome analysis of Lotus japonicus seeds.

In this study, we report the first dataset of phosphoproteins of the seeds of a model plant, Lotus japonicus. This dataset might be useful in studying the regulatory mechanisms of seed germination in legume plants. By proteomic analysis of seeds following water absorption, we identified a total of 721 phosphopeptides derived from 343 phosphoproteins in cotyledons, and 931 phosphopeptides from 473 phosphoproteins in hypocotyls. Kinase-specific prediction analyses revealed that different kinases were activated in cotyledons and hypocotyls. In particular, many peptides containing ATM-kinase target motifs, X-X-pS/pT-Q-X-X, were detected in cotyledons. Moreover, by real-time RT-PCR analysis, we found that expression of a homolog of ATM kinase is upregulated specifically in cotyledons, suggesting that this ATM-kinase homolog plays a significant role in cell proliferation in the cotyledons of L. japonicus seeds. The data have been deposited to the ProteomeXchange with identifier PXD000053 (http://proteomecentral.proteomexchange.org/dataset/PXD000053).

A thaumatin-like protein, Rj4, controls nodule symbiotic specificity in soybean.

Soybeans exhibit a nitrogen-fixing symbiosis with soil bacteria of the genera Bradyrhizobium and Ensifer/Sinorhizobium in a unique organ, the root nodule. It is well known that nodulation of soybean is controlled by several host genes referred to as Rj (rj) genes. Among these genes, a dominant allele, Rj4, restricts nodulation with specific bacterial strains such as B. elkanii USDA61 and B. japonicum Is-34. These incompatible strains fail to invade the host epidermal cells as revealed by observations using DsRed-labeled bacteria. Here, we describe the molecular identification of the Rj4 gene by using map-based cloning with several mapping populations. The Rj4 gene encoded a thaumatin-like protein (TLP) that belongs to pathogenesis-related (PR) protein family 5. In rj4/rj4 genotype soybeans and wild soybeans, we found six missense mutations and two consecutive amino acid deletions in the rj4 gene as compared with the Rj4 allele. We also found, using hairy root transformation, that the rj4/rj4 genotype soybeans were fully complemented by the expression of the Rj4 gene. Whereas the expression of many TLPs and other PR proteins is induced by biotic/abiotic stress, Rj4 gene expression appears to be constitutive in roots including root nodules.

Construction and targeted retrieval of specific clone from a non-gridded soybean bacterial artificial chromosome library.

Although a post-genomic era is emerging for many plants, the bacterial artificial chromosome (BAC) library is still a valuable tool for genomic studies and preservation of precious genetic resources. Construction of non-gridded BAC libraries would dramatically reduce cost and save storage space. A non-gridded BAC library composed of approximately 96,000 insert-containing clones in 80 pools with an average insert size of 75 kb was constructed. This library represented 5.2 genome equivalents. We successfully developed a unique procedure to retrieve positive clones from the non-gridded pools. With this retrieving protocol, the non-gridded library system can be adapted to different species and to serve various research needs.

Natural variation in the genes responsible for maturity loci E1, E2, E3 and E4 in soybean.

BACKGROUND AND AIMS: The timing of flowering has a direct impact on successful seed production in plants. Flowering of soybean (Glycine max) is controlled by several E loci, and previous studies identified the genes responsible for the flowering loci E1, E2, E3 and E4. However, natural variation in these genes has not been fully elucidated. The aims of this study were the identification of new alleles, establishment of allele diagnoses, examination of allelic combinations for adaptability, and analysis of the integrated effect of these loci on flowering. METHODS: The sequences of these genes and their flanking regions were determined for 39 accessions by primer walking. Systematic discrimination among alleles was performed using DNA markers. Genotypes at the E1-E4 loci were determined for 63 accessions covering several ecological types using DNA markers and sequencing, and flowering times of these accessions at three sowing times were recorded. KEY RESULTS: A new allele with an insertion of a long interspersed nuclear element (LINE) at the promoter of the E1 locus (e1-re) was identified. Insertion and deletion of 36 bases in the eighth intron (E2-in and E2-dl) were observed at the E2 locus. Systematic discrimination among the alleles at the E1-E3 loci was achieved using PCR-based markers. Allelic combinations at the E1-E4 loci were found to be associated with ecological types, and about 62-66 % of variation of flowering time could be attributed to these loci. CONCLUSIONS: The study advances understanding of the combined roles of the E1-E4 loci in flowering and geographic adaptation, and suggests the existence of unidentified genes for flowering in soybean.

Polymorphisms of E1 and GIGANTEA in wild populations of Lotus japonicus.

In plants, timing of flowering is an essential factor that controls the survival rates of descendants. The circadian clock genes E1 and GIGANTEA (GI) play a central role in transmitting signals to flowering locus T (FT) in leguminous plants. Lotus japonicus is a wild Japanese species that ranges from northern Hokkaido to the southern Ryukyus and exhibits a wide range in terms of the time between seeding and first flowering. In this study, we first identified LjGI and analyzed polymorphisms of LjE1 and LjGI among wild populations covering the entire distribution range of this species in Japan. LjGI had a coding sequence (CDS) length of 3495 bp and included 14 exons. The homologies of DNA and amino acid sequences between LjGI and GmGI were 89 and 88% (positive rate was 92%), respectively. LjE1 harbored five nucleic acid changes in a 552 bp CDS, all of which were nonsynonymous; four of the changes were located in the core function area. LjE1 alleles exhibited partial north-south differentiation and non-neutrality. In contrast, the LjGI harbored one synonymous and one nonsynonymous change. Thus, our study suggests that LjE1 may be involved in the control of flowering times, whereas LjGI may be under strong purifying selection.

The receptor-like kinase KLAVIER mediates systemic regulation of nodulation and non-symbiotic shoot development in Lotus japonicus.

In legumes, the number of symbiotic root nodules is controlled by long-distance communication between the shoot and the root. Mutants defective in this feedback mechanism exhibit a hypernodulating phenotype. Here, we report the identification of a novel leucine-rich repeat receptor-like kinase (LRR-RLK), KLAVIER (KLV), which mediates the systemic negative regulation of nodulation in Lotus japonicus. In leaf, KLV is predominantly expressed in the vascular tissues, as with another LRR-RLK gene, HAR1, which also regulates nodule number. A double-mutant analysis indicated that KLV and HAR1 function in the same genetic pathway that governs the negative regulation of nodulation. LjCLE-RS1 and LjCLE-RS2 represent potential root-derived mobile signals for the HAR1-mediated systemic regulation of nodulation. Overexpression of LjCLE-RS1 or LjCLE-RS2 did not suppress the hypernodulation phenotype of the klv mutant, indicating that KLV is required and acts downstream of LjCLE-RS1 and LjCLE-RS2. In addition to the role of KLV in symbiosis, complementation tests and expression analyses indicated that KLV plays multiple roles in shoot development, including maintenance of shoot apical meristem, vascular continuity, shoot growth and promotion of flowering. Biochemical analyses using transient expression in Nicotiana benthamiana revealed that KLV has the ability to interact with HAR1 and with itself. Together, these results suggest that the potential KLV-HAR1 receptor complex regulates symbiotic nodule development and that KLV is also a key component in other signal transduction pathways that mediate non-symbiotic shoot development.

Genetic variation in four maturity genes affects photoperiod insensitivity and PHYA-regulated post-flowering responses of soybean.

BACKGROUND: Absence of or low sensitivity to photoperiod is necessary for short-day crops, such as rice and soybean, to adapt to high latitudes. Photoperiod insensitivity in soybeans is controlled by two genetic systems and involves three important maturity genes: E1, a repressor for two soybean orthologs of Arabidopsis FLOWERING LOCUS T (GmFT2a and GmFT5a), and E3 and E4, which are phytochrome A genes. To elucidate the diverse mechanisms underlying photoperiod insensitivity in soybean, we assessed the genotypes of four maturity genes (E1 through E4) in early-flowering photoperiod-insensitive cultivars and their association with post-flowering responses. RESULTS: We found two novel dysfunctional alleles in accessions originally considered to have a dominant E3 allele according to known DNA markers. The E3 locus, together with E1 and E4, contained multiple dysfunctional alleles. We identified 15 multi-locus genotypes, which we subdivided into 6 genotypic groups by classifying their alleles by function. Of these, the e1-as/e3/E4 genotypic group required an additional novel gene (different from E1, E3, and E4) to condition photoperiod insensitivity. Despite their common pre-flowering photoperiod insensitivity, accessions with different multi-locus genotypes responded differently to the post-flowering photoperiod. Cultivars carrying E3 or E4 were sensitive to photoperiod for post-flowering characteristics, such as reproductive period and stem growth after flowering. The phytochrome A-regulated expression of the determinate growth habit gene Dt1, an ortholog of Arabidopsis TERMINAL FLOWER1, was involved in the persistence of the vegetative activity at the stem apical meristem of flower-induced plants under long-day conditions. CONCLUSIONS: Diverse genetic mechanisms underlie photoperiod insensitivity in soybean. At least three multi-locus genotypes consisting of various allelic combinations at E1, E3, and E4 conferred pre-flowering photoperiod insensitivity to soybean cultivars but led to different responses to photoperiod during post-flowering vegetative and reproductive development. The phyA genes E3 and E4 are major controllers underlying not only pre-flowering but also post-flowering photoperiod responses. The current findings improve our understanding of genetic diversity in pre-flowering photoperiod insensitivity and mechanisms of post-flowering photoperiod responses in soybean.

The ß-conglycinin deficiency in wild soybean is associated with the tail-to-tail inverted repeat of the α-subunit genes.

ß-conglycinin, a major seed protein in soybean, is composed of α, α', and ß subunits sharing a high homology among them. Despite its many health benefits, ß-conglycinin has a lower amino acid score and lower functional gelling properties compared to glycinin, another major soybean seed protein. In addition, the α, α', and ß subunits also contain major allergens. A wild soybean (Glycine soja Sieb et Zucc.) line, 'QT2', lacks all of the ß-conglycinin subunits, and the deficiency is controlled by a single dominant gene, Scg-1 (Suppressor of ß-conglycinin). This gene was characterized using a soybean cultivar 'Fukuyutaka', 'QY7-25', (its near-isogenic line carrying the Scg-1 gene), and the F2 population derived from them. The physical map of the Scg-1 region covered by lambda phage genomic clones revealed that the two α-subunit genes, a ß-subunit gene, and a pseudo α-subunit gene were closely organized. The two α-subunit genes were arranged in a tail-to-tail orientation, and the genes were separated by 197 bp in Scg-1 compared to 3.3 kb in the normal allele (scg-1). In addition, small RNA was detected in immature seeds of the mutants by northern blot analysis using an RNA probe of the α subunit. These results strongly suggest that ß-conglycinin deficiency in QT2 is controlled by post-transcriptional gene silencing through the inverted repeat of the α subunits.

Genetic and molecular bases of photoperiod responses of flowering in soybean.

Flowering is one of the most important processes involved in crop adaptation and productivity. A number of major genes and quantitative trait loci (QTLs) for flowering have been reported in soybean (Glycine max). These genes and QTLs interact with one another and with the environment to greatly influence not only flowering and maturity but also plant morphology, final yield, and stress tolerance. The information available on the soybean genome sequence and on the molecular bases of flowering in Arabidopsis will undoubtedly facilitate the molecular dissection of flowering in soybean. Here, we review the present status of our understanding of the genetic and molecular mechanisms of flowering in soybean. We also discuss our identification of orthologs of Arabidopsis flowering genes from among the 46,367 genes annotated in the publicly available soybean genome database Phytozome Glyma 1.0. We emphasize the usefulness of a combined approach including QTL analysis, fine mapping, and use of candidate gene information from model plant species in genetic and molecular studies of soybean flowering.

Rj (rj) genes involved in nitrogen-fixing root nodule formation in soybean.

It has long been known that formation of symbiotic root nodules in soybean (Glycine max (L.) Merr.) is controlled by several host genes referred to as Rj (rj) genes, but molecular cloning of these genes has been hampered by soybean's complicated genome structure and large genome size. Progress in molecular identification of legume genes involved in root nodule symbiosis have been mostly achieved by using two model legumes, Lotus japonicus and Medicago truncatula, that have relatively simple and small genomes and are capable of molecular transfection. However, recent development of resources for soybean molecular genetic research, such as genome sequencing, large EST databases, and high-density linkage maps, have enabled us to isolate several Rj genes. This progress has been achieved in connection with systematic utilization of the information obtained from molecular genetics of the model legumes. In this review, we summarize the current status of knowledge of host-controlled nodulation in soybean based on information from recent studies on Rj genes, and discuss the future research prospects.

Evaluation of soybean germplasm conserved in NIAS genebank and development of mini core collections.

Genetic variation and population structure among 1603 soybean accessions, consisted of 832 Japanese landraces, 109 old and 57 recent Japanese varieties, 341 landrace from 16 Asian countries and 264 wild soybean accessions, were characterized using 191 SNP markers. Although gene diversity of Japanese soybean germplasm was slight lower than that of exotic soybean germplasm, population differentiation and clustering analyses indicated clear genetic differentiation among Japanese cultivated soybeans, exotic cultivated soybeans and wild soybeans. Nine hundred ninety eight Japanese accessions were separated to a certain extent into groups corresponding to their agro-morphologic characteristics such as photosensitivity and seed characteristics rather than their geographical origin. Based on the assessment of the SNP markers and several agro-morphologic traits, accessions that retain gene diversity of the whole collection were selected to develop several soybean sets of different sizes using an heuristic approach; a minimum of 12 accessions can represent the observed gene diversity; a mini-core collection of 96 accession can represent a major proportion of both geographic origin and agro-morphologic trait variation. These selected sets of germplasm will provide an effective platform for enhancing soybean diversity studies and assist in finding novel traits for crop improvement.

DaizuBase, an integrated soybean genome database including BAC-based physical maps.

Soybean [Glycine max (L) Merrill] is one of the most important leguminous crops and ranks fourth after to rice, wheat and maize in terms of world crop production. Soybean contains abundant protein and oil, which makes it a major source of nutritious food, livestock feed and industrial products. In Japan, soybean is also an important source of traditional staples such as tofu, natto, miso and soy sauce. The soybean genome was determined in 2010. With its enormous size, physical mapping and genome sequencing are the most effective approaches towards understanding the structure and function of the soybean genome. We constructed bacterial artificial chromosome (BAC) libraries from the Japanese soybean cultivar, Enrei. The end-sequences of approximately 100,000 BAC clones were analyzed and used for construction of a BAC-based physical map of the genome. BLAST analysis between Enrei BAC-end sequences and the Williams82 genome was carried out to increase the saturation of the map. This physical map will be used to characterize the genome structure of Japanese soybean cultivars, to develop methods for the isolation of agronomically important genes and to facilitate comparative soybean genome research. The current status of physical mapping of the soybean genome and construction of database are presented.

Two coordinately regulated homologs of FLOWERING LOCUS T are involved in the control of photoperiodic flowering in soybean.

FLOWERING LOCUS T (FT) is a key flowering integrator in Arabidopsis (Arabidopsis thaliana), with homologs that encode florigens in many plant species regardless of the type of photoperiodic response. We identified 10 FT homologs, which were arranged as five pairs of linked genes in different homoeologous chromosomal regions, in soybean (Glycine max), a paleopolyploid species. Two of the FT homologs, GmFT2a and GmFT5a, were highly up-regulated under short-day (SD) conditions (inductive for flowering in soybean) and had diurnal expression patterns with the highest expression 4 h after dawn. Under long-day (LD) conditions, expression of GmFT2a and GmFT5a was down-regulated and did not follow a diurnal pattern. Flowering took much longer to initiate under LD than under SD, and only the GmFT5a transcript accumulated late in development under LD. Ectopic expression analysis in Arabidopsis confirmed that both GmFT2a and GmFT5a had the same function as Arabidopsis FT, but the effect of GmFT5a was more prominent. A double-mutant soybean line for two PHYTOCHROME A (PHYA) genes expressed high levels of GmFT2a and GmFT5a under LD, and it flowered slightly earlier under LD than the wild type grown under SD. The expression levels of GmFT2a and GmFT5a were regulated by the PHYA-mediated photoperiodic regulation system, and the GmFT5a expression was also regulated by a photoperiod-independent system in LD. Taken together, our results suggest that GmFT2a and GmFT5a coordinately control flowering and enable the adaptation of soybean to a wide range of photoperiodic environments.

The soybean stem growth habit gene Dt1 is an ortholog of Arabidopsis TERMINAL FLOWER1.

Classical genetic analysis has revealed that the determinate habit of soybean (Glycine max) is controlled by a recessive allele at the determinate stem (Dt1) locus. To dissect the molecular basis of the determinate habit, we isolated two orthologs of pea (Pisum sativum) TERMINAL FLOWER1a, GmTFL1a and GmTFL1b, from the soybean genome. Mapping analysis indicated that GmTFL1b is a candidate for Dt1. Despite their high amino acid identity, the two genes had different transcriptional profiles. GmTFL1b was expressed in the root and shoot apical meristems (SAMs), whereas GmTFL1a was mainly expressed in immature seed. The GmTFL1b transcript accumulated in the SAMs during early vegetative growth in both the determinate and indeterminate lines but thereafter was abruptly lost in the determinate line. Introduction of the genomic region of GmTFL1b from the indeterminate line complemented the stem growth habit in the determinate line: more nodes were produced, and flowering in the terminal raceme was delayed. The identity between Dt1 and GmTFL1b was also confirmed with a virus-induced gene silencing experiment. Taken together, our data suggest that Dt1 encodes the GmTFL1b protein and that the stem growth habit is determined by the variation of this gene. The dt1 allele may condition the determinate habit via the earlier loss in GmTFL1b expression concomitant with floral induction, although it functions normally under the noninductive phase of flowering. An association test of DNA polymorphisms with the stem growth habit among 16 cultivars suggested that a single amino acid substitution in exon 4 determines the fate of the SAM after floral induction.

The Synchronized Efforts to Decipher the Molecular Basis for Soybean Maturity Loci E1, E2, and E3 That Regulate Flowering and Maturity.

The general concept of photoperiodism, i.e., the photoperiodic induction of flowering, was established by Garner and Allard (1920). The genetic factor controlling flowering time, maturity, or photoperiodic responses was observed in soybean soon after the discovery of the photoperiodism. E1, E2, and E3 were named in 1971 and, thereafter, genetically characterized. At the centennial celebration of the discovery of photoperiodism in soybean, we recount our endeavors to successfully decipher the molecular bases for the major maturity loci E1, E2, and E3 in soybean. Through systematic efforts, we successfully cloned the E3 gene in 2009, the E2 gene in 2011, and the E1 gene in 2012. Recently, successful identification of several circadian-related genes such as PRR3a, LUX, and J has enriched the known major E1-FTs pathway. Further research progresses on the identification of new flowering and maturity-related genes as well as coordinated regulation between flowering genes will enable us to understand profoundly flowering gene network and determinants of latitudinal adaptation in soybean.

Genetic redundancy in soybean photoresponses associated with duplication of the phytochrome A gene.

Gene and genome duplications underlie the origins of evolutionary novelty in plants. Soybean, Glycine max, is considered to be a paleopolyploid species with a complex genome. We found multiple homologs of the phytochrome A gene (phyA) in the soybean genome and determined the DNA sequences of two paralogs designated GmphyA1 and GmphyA2. Analysis of the GmphyA2 gene from the lines carrying a recessive allele at a photoperiod insensitivity locus, E4, revealed that a Ty1/copia-like retrotransposon was inserted in exon 1 of the gene, which resulted in dysfunction of the gene. Mapping studies suggested that GmphyA2 is encoded by E4. The GmphyA1 gene was mapped to a region of linkage group O, which is homeologous to the region harboring E4 in linkage group I. Plants homozygous for the e4 allele were etiolated under continuous far red light, but the de-etiolation occurred partially, indicating that the mutation alone did not cause a complete loss of phyA function. The genetic redundancy suggests that the presence of duplicated copies of phyA genes accounts for the generation of photoperiod insensitivity, while protecting against the deleterious effects of mutation. Thus, this phenomenon provides a link between gene duplication and establishment of an adaptive response of plants to environments.

Genetic mapping of Cgdef gene controlling accumulation of 7S globulin (beta-conglycinin) subunits in soybean seeds.

Soy protein consists of mainly 7S globulin (beta-conglycinin) and 11S globulin (glycinin). The 7S globulin exerts favorable and unfavorable effects on human nutrition, food processing, and human health. Therefore, it is important for the improvement of the soy protein to control the content of 7S globulin. A mutant line lacking the 7S globulin was induced by gamma-ray irradiation, and the deficiency is controlled by a single recessive gene, cgdef. The Cgdef gene, despite its potential for improvement of the soy protein, has not been assigned to a linkage group of a soybean genetic map. We crossed "Mo-shi-dou Gong 503" with plants homozygous or heterozygous for the Cgdef allele and screened an F2 mapping population that segregated with the cgdef allele to locate the Cgdef gene on a soybean genetic map. By linkage analysis, we assigned the Cgdef gene to chromosome 19 at the position between the Satt523 and Sat_388 simple sequence repeat (SSR) markers. Six SSR markers (Sat_134, Sat_405, Satt143, Satt398, Sat_195, and Satt694) and 2 amplified fragment length polymorphism markers identified previously were mapped at the same position of the Cgdef gene. These markers should enable to conduct map-based cloning of the Cgdef gene.