Genomic Location of a Gene Conditioning a Miniature Phenotype in Soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.]

The potential for global warming and climate change has increased the focus of research on plant genes that respond to high temperatures. Previous research identified a temperature-sensitive miniature soybean mutant that was controlled by a single gene. The objectives of our research were to confirm the single-gene control and to determine the genomic location of this gene. Segregation of the combined progeny of four BC₆F₅ plants heterozygous for the miniature trait in a Tracy-M background confirmed that the trait was conditioned by a single gene (1:2:1, χ ² = 4.38, P = 0.1120). Molecular marker analysis identified three SSR markers and a SNP marker on molecular linkage group B2 (chromosome 14) associated with segregation for the miniature trait. One of these, marker Satt560, co-segregated perfectly with the miniature trait. The data from these four polymorphic markers indicated that the gene conditioning this miniature phenotype is at or near Satt560. Given this newly identified location of the gene and the recently published soybean genomic sequence, it may be feasible to isolate the gene and determine its mechanism of action in responding to temperature. Such knowledge may be of use in understanding how plants respond to increased temperature.     

<Emphasis Type="Italic">Organogenic callus</Emphasis> as the target for plant regeneration and transformation via <Emphasis Type="Italic">Agrobacterium</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.)

A regeneration and transformation system has been developed using organogenic calluses derived from soybean axillary nodes as the starting explants. Leaf-node or cotyledonary-node explants were prepared from 7 to 8-d-old seedlings. Callus was induced on medium containing either Murashige and Skoog (MS) salts or modified Finer and Nagasawa (FNL) salts and B5 vitamins with various concentrations of benzylamino purine (BA) and thidiazuron (TDZ). The combination of BA and TDZ had a synergistic effect on callus induction. Shoot differentiation from the callus occurred once the callus was transferred to medium containing a low concentration of BA. Subsequently, shoots were elongated on medium containing indole-3-acetic acid (IAA), zeatin riboside, and gibberellic acid (GA). Plant regeneration from callus occurred 90 ∼ 120 d after the callus was cultured on shoot induction medium. Both the primary callus and the proliferated callus were used as explants for Agrobacterium-mediated transformation. The calluses were inoculated with A. tumefaciens harboring a binary vector with the bar gene as the selectable marker gene and the gusINT gene for GUS expression. Usually 60–100% of the callus showed transient GUS expression 5 d after inoculation. Infected calluses were then selected on media amended with various concentrations of glufosinate. Transgenic soybean plants have been regenerated and established in the greenhouse. GUS expression was exhibited in various tissues and plant organs, including leaf, stem, and roots. Southern and T₁ plant segregation analysis of transgenic events showed that transgenes were integrated into the soybean genome with a copy number ranging from 1–5 copies.     

Wind tunnel and field assessment of pollen dispersal in Soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.]

Although genetically modified (GM) soybean has never been cultivated commercially in Japan, it is essential to set up the isolation distance required to prevent out-crossing between GM and conventional soybean in preparation for any future possibility of pollen transfer. The airborne soybean pollen was sampled using some Durham pollen samplers located in the range of 20 m from the field edge. In addition, the dispersal distance was assessed in a wind tunnel under constant air flow and then it was compared with the anticipated distances based on the pollen diameter. In the field, the maximum pollen density per day observed was 1.235 grains cm⁻² day⁻¹ at three observation points within 2.5 m from the field and inside the field the mean density did not reach the rate of 1 grain cm⁻² day⁻¹ during 19 flowering days. The results of the wind tunnel experiment also showed that the plants had almost no airborne release of pollen and the dispersal distance was shorter than theoretical value due to clustered dispersal. This study showed little airborne pollen in and around the soybean field and the dispersal is restricted to a small area. Therefore, wind-mediated pollination appears to be negligible.     

Cloning and functional analysis of two <Emphasis Type="Italic">GmDeg</Emphasis> genes in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.]

Although light is the ultimate substrate in photosynthesis, strong light can also be harmful and lead to photoinhibition. The DEG proteases play important roles in the degradation of misfolded and damaged proteins. In this study, two photoinhibition-related genes from soybean [Glycine max (L.) Merr.], GmDeg1 and GmDeg2, were cloned. Bioinformatics analysis indicated that these two proteases both contain a PDZ domain and are serine proteases. The expression levels of GmDeg1 and GmDeg2 increased significantly after 12 h of photooxidation treatment, indicating that GmDeg1 and GmDeg2 might play protective roles under strong light conditions. In in vitro proteolytic degradation assays, recombinant GmDeg1 and GmDeg2 demonstrated biological activities at temperatures ranging from 20°C to 60°C and at pH 5.0 to 8.0. By contrast, the proteases showed no proteolytic effect in the presence of a serine protease inhibitor. Taken together, these results provided strong evidence that GmDeg1 and GmDeg2 are serine proteases that could degrade the model substrate in vitro, indicating that they might degrade damaged D1 protein and other mis-folded proteins in vivo. Furthermore, GmDeg1 and GmDeg2 were transformed into Arabidopsis thaliana to obtain transgenic plants. Leaves from the transgenic and wild-type plants were subjected to strong light conditions in vitro, and the PSII photochemical efficiency (Fv/Fm) was measured. The Fv/Fm of the transgenic plants was significantly higher than that of the wild-type plants at most time points. These results imply that GmDeg1 and GmDeg2 would have similar functions to Arabidopsis AtDeg1, thus accelerating the recovery of PSII photochemical efficiency.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

Polygenic inheritance of canopy wilting in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.]

As water demand for agriculture exceeds water availability, cropping systems need to become more efficient in water usage, such as deployment of cultivars that sustain yield under drought conditions. Soybean cultivars differ in how quickly they wilt during water-deficit stress, and this trait may lead to yield improvement during drought. The objective of this study was to determine the genetic mechanism of canopy wilting in soybean using a mapping population of recombinant inbred lines (RILs) derived from a cross between KS4895 and Jackson. Canopy wilting was rated in three environments using a rating scale of 0 (no wilting) to 100 (severe wilting and plant death). Transgressive segregation was observed for the RIL population with the parents expressing intermediate wilting scores. Using multiple-loci analysis, four quantitative trait loci (QTLs) on molecular linkage groups (MLGs) A2, B2, D2, and F were detected (P ≤ 0.05), which collectively accounted for 47% of the phenotypic variation of genotypic means over all three environments. An analysis of the data by state revealed that 44% of the observed phenotypic variation in the Arkansas environments could be accounted for by these QTLs. Only the QTL on MLG F was detected at North Carolina where it accounted for 16% of the phenotypic variation. These results demonstrate that the genetic mechanism controlling canopy wilting was polygenic and environmentally sensitive and provide a foundation for future research to examine the importance of canopy wilting in drought tolerance of soybean.     

Genome-wide analysis of the PHB gene family in <Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.

Prohibitins (PHBs) have one SPFH domain in common and present in species ranging from prokaryotes to eukaryotes. Although a number of researches on PHBs were performed in different plant species, a systematic analysis of the PHB family in soybean is still remains uncharacterized. In the present study, 24 putative PHB genes have been first systemically identified in soybean. According to phylogenetic analysis, these GmPHBs could be classified into four groups. Gene structures and motif patterns showed high levels of conservation within the phylogenetic subgroups. Several members of this family have undergone purifying selection based on Ka/Ks analysis on duplicated PHB genes in soybean. We performed microsynteny analysis across four legume species based on the comparisons among the specific regions contained in PHB genes. As a result, numerous microsyntenic gene pairs among soybean, Medicago, Lotus and Phaseolus were identified. Most soybean PHB genes exhibited different expression levels in various tissues and developmental stages through expression analysis using publicly available RNA-seq datasets. The 11 GmPHB genes from III_B subgroup were examined by qPCR for their expression in two soybean cultivar after infection by Phytophthora sojae. Besides three GmPHB genes previous reported by us, here other four genes also were rapidly induced by P. sojae infection in the resistant genotype, while induction was very weak in the susceptible genotype. The comprehensive overview of the PHB gene family in soybean genome will provide useful information for further functional analysis of the PHB gene family in soybean.     

Biosynthesis of pectic galactan by membrane-bound galactosyltransferase from soybean (<Emphasis Type="Italic">Glycine max</Emphasis> Merr.) seedlings

We investigated the properties of a galactosyltransferase (GalT) that is involved in the synthesis of -(14)-galactan side chains of pectins. A membrane preparation of etiolated 6-day-old soybean (Glycine max Merr.) hypocotyls transferred [¹⁴C]Gal from UDP-[¹⁴C]Gal into intact and partially hydrolyzed lupin -(14)-galactans of various chain lengths as exogenous acceptors, while activity to endogenous acceptors was negligible. Maximal activity occurred at pH 6.5 and 20–25°C in the presence of 25 mM Mn²⁺ and 0.75% Triton X-100. The transfer reaction onto the unmodified commercial pectic galactan (M _r>150,000) from lupin we used was very low but increased when the M _r of the galactan was reduced by partial acid hydrolysis. Among the partially hydrolyzed galactans, high-M _r (average M _r 60,000) -(14)-galactan was a more efficient acceptor [specific activity 2,000–3,000 pmol min^–1 (mg protein)^–1] than low-M _r (average M _r 10,000 and 5,000) polymers. Digestion of the radiolabeled product from high-M _r galactan with endo--(14)-galactanase released mainly radioactive -(14)-galactobiose and Gal, indicating that the transfer of [¹⁴C]Gal occurred through -(14)-linkages. HPLC analysis showed that the enzyme also catalyzes incorporation of Gal into pyridylaminated (PA) -(14)-galactooligomers with degree of polymerization at least 5. Gal₇-PA chains were elongated by attachment of one, two, or three Gal residues leading to the formation of Gal_8–10-PA.Abbreviations AGP Arabinogalactan-protein - Ara Arabinose - DP Degree of polymerization - GalA Galacturonic acid - Gal _n -PA Pyridylaminated -(14)-galactooligosaccharides - GalT Galactosyltransferase - MALDI–TOF–MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry - Rha Rhamnose Sugars described in this paper belong to the d-series unless otherwise noted     

Fine mapping of a <Emphasis Type="Italic">Phytophthora</Emphasis>-resistance gene <Emphasis Type="Italic">RpsWY</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L.) by high-throughput genome-wide sequencing

Key message

Using a combination of phenotypic screening, genetic and statistical analyses, and high-throughput genome-wide sequencing, we have finely mapped a dominant Phytophthora resistance gene in soybean cultivar Wayao.

Abstract

Phytophthora root rot (PRR) caused by Phytophthora sojae is one of the most important soil-borne diseases in many soybean-production regions in the world. Identification of resistant gene(s) and incorporating them into elite varieties are an effective way for breeding to prevent soybean from being harmed by this disease. Two soybean populations of 191 F₂ individuals and 196 F_7:8 recombinant inbred lines (RILs) were developed to map Rps gene by crossing a susceptible cultivar Huachun 2 with the resistant cultivar Wayao. Genetic analysis of the F₂ population indicated that PRR resistance in Wayao was controlled by a single dominant gene, temporarily named RpsWY, which was mapped on chromosome 3. A high-density genetic linkage bin map was constructed using 3469 recombination bins of the RILs to explore the candidate genes by the high-throughput genome-wide sequencing. The results of genotypic analysis showed that the RpsWY gene was located in bin 401 between 4466230 and 4502773 bp on chromosome 3 through line 71 and 100 of the RILs. Four predicted genes (Glyma03g04350, Glyma03g04360, Glyma03g04370, and Glyma03g04380) were found at the narrowed region of 36.5 kb in bin 401. These results suggest that the high-throughput genome-wide resequencing is an effective method to fine map PRR candidate genes.

     

Mapping quantitative trait loci for seed size traits in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.)

Seed size traits in soybean—length, width and thickness—and their corresponding ratios—length-to-width, length-to-thickness and width-to-thickness—play a crucial role in determining seed appearance, quality and yield. In this study, an attempt was made to detect quantitative trait loci (QTL) for the aforementioned seed size traits in F_2:3, F_2:4 and F_2:5 populations from the direct and reciprocal crosses of Lishuizhongzihuang with Nannong 493-1, using multi-QTL joint analysis (MJA) along with composite interval mapping (CIM). A total of 121 main-effect QTL (M-QTL), six environmental effects, eight environment-by-QTL interactions, five cytoplasmic effects and 92 cytoplasm-by-QTL interactions were detected. Fifty-two common M-QTL across MJA and CIM, 21 common M-QTL in more than two populations and 5 M-QTL in all three populations showed the stability of the results. Five M-QTL had higher heritability, greater than 20%. In addition, 28 cytoplasm-by-QTL and 4 environment-by-QTL interactions were confirmed by CIM. Most M-QTL were clustered in eight chromosomal regions. Our results provide a good foundation for fine mapping, cloning and designed molecular breeding of favorable genes related to soybean seed size traits.     

Improved <Emphasis Type="Italic">Agrobacterium tumefaciens-</Emphasis>mediated transformation of soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.] following optimization of culture conditions and mechanical techniques

In the present study, Agrobacterium tumefaciens-mediated transformation of Glycine max (L.) Merr. (soybean) cv. DS-9712 using half-seed explants was optimized for eight different parameters, including seed imbibition, medium pH, infection mode (sonication and vacuum infiltration), co-cultivation conditions, concentrations of supplementary compounds, and selection. Using this improved protocol, maximum transformation of 14% and regeneration efficiencies of 45% were achieved by using explants prepared from mature seeds imbibed for 36 h, infected with A. tumefaciens strain EHA105 at an optical density (OD₆₀₀) of 0.8, suspended in pH 5.4 medium containing 0.2 mM acetosyringone and 450 mg L^?1 L-cysteine, followed by sonication for 10 s, vacuum infiltration for 2 min, and co-cultivated for 3 d on 35 mg L^?1 kanamycin-containing medium. Independent transgenic lines were confirmed to be transgenic after ß-glucuronidase histochemical assays, polymerase chain reaction, and southern hybridization analysis. The protocol developed in the present study showed high regeneration efficiency within a relatively short time of 76 d. This rapid and efficient protocol might overcome some hurdles associated with the genetic manipulation of soybean.     

Genetic variability in <Emphasis Type="Italic">Bradyrhizobium japonicum</Emphasis> strains nodulating soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merrill]

Brazil has succeeded in sustaining production of soybean [Glycine max (L.) Merrill] by relying mainly on symbiotic N₂ fixation, thanks to the selection and use in inoculants of very effective strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii. It is desirable that rhizobial strains used in inoculants have stable genetic and physiological traits, but experience confirms that rhizobial strains nodulating soybean often lose competitiveness in the field. In this study, soybean cultivar BR 16 was single-inoculated with four B. japonicum strains (CIAT 88, CIAT 89, CIAT 104 and CIAT 105) under aseptic conditions. Forty colonies were isolated from nodules produced by each strain. The progenitor strains, the isolates and four other commercially recommended strains were applied separately to the same cultivar under controlled greenhouse conditions. We observed significant variability in nodulation, shoot dry weight, shoot total N, nodule efficiency (total N mass over nodule mass) and BOX-PCR fingerprinting profiles between variant and progenitor strains. Some variant strains resulted in significantly larger responses in terms of shoot total N, dry weight and nodule efficiency, when compared to their progenitor strain. These results highlight the need for intermittent evaluation of stock bacterial cultures to guarantee effective symbiosis after inoculation. Most importantly, it indicates that it is possible to improve symbiotic effectiveness by screening rhizobial strains for higher N₂ fixation capacity within the natural variability that can be found within each progenitor strain.     

Generation and characterization of two novel low phytate mutations in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> 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 ∼4.63 and ∼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.