Mapping QTLs for tissue culture response in soybean (Glycine max (L.) Merr.)

Hempseed is rich in polyunsaturated fatty acids (PUFAs), which have potential as therapeutic compounds for the treatment of neurodegenerative and cardiovascular disease. However, the effect of hempseed meal (HSM) intake on the animal models of these diseases has yet to be elucidated. In this study, we assessed the effects of the intake of HSM and PUFAs on oxidative stress, cytotoxicity and neurological phenotypes, and cholesterol uptake, using Drosophila models. HSM intake was shown to reduce H₂O₂ toxicity markedly, indicating that HSM exerts a profound antioxidant effect. Meanwhile, intake of HSM, as well as linoleic or linolenic acids (major PUFA components of HSM) was shown to ameliorate Aβ42-induced eye degeneration, thus suggesting that these compounds exert a protective effect against Aβ42 cytotoxicity. On the contrary, locomotion and longevity in the Parkinson’s disease model and eye degeneration in the Huntington’s disease model were unaffected by HSM feeding. Additionally, intake of HSM or linoleic acid was shown to reduce cholesterol uptake significantly. Moreover, linoleic acid intake has been shown to delay pupariation, and cholesterol feeding rescued the linoleic acid-induced larval growth delay, thereby indicating that linoleic acid acts antagonistically with cholesterol during larval growth. In conclusion, our results indicate that HSM and linoleic acid exert inhibitory effects on both Aβ42 cytotoxicity and cholesterol uptake, and are potential candidates for the treatment of Alzheimer’s disease and cardiovascular disease.     

Some factors affecting somatic embryogenesis efficiency in soybean (Glycine max (L.) Merr.)

Selected factors affecting somatic embryogenesis efficiency have been studied, namely genotype, explant type and its orientation in the medium, different basal media, different auxins for somatic embryo induction, and two ways of donor plant cultivation. The key role is played by genotype and auxin used, the minimum effect was observed due to basal media. In the series of subsequent experiments we have found the best combination of individual factors as follows: cv. Altona, 10 uM 2,4-D, L2 basal medium, central part of immature cotyledon as initial expiant oriented by adaxial side down on the agar medium, and field grown donor plants. This combination exhibited 100 % embryogenic explants with 5.43 ± 0.65 somatic embryos per expiant,i.e. somatic embryogenesis efficiency 5.43.     

Regeneration of transformed shoots from electroporated soybean (Glycine max (L.) Merr.) protoplasts

Stable transformation of soybean (Glycine max (L.) Merr.) protoplasts isolated from immature cotyledons was achieved following electroporation with plasmid DNA carrying chimeric genes encoding ß-glucuronidase (GUS) and hygromycin phosphotransferase (HPT) under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Transformed colonies were stringently selected by growing 15-day-old protoplast-derived cells in the presence of 40 g/ml of hygromycin-B for 6 weeks. Over 93% of the resistant cells and colonies exhibited GUS activity, indicating that the two marker genes borne on a single plasmid were co-introduced and co-expressed at a very high freguency. This transformation procedure reproducibly yields transformants at frequencies of 2.9–6.8 × 10^–4 (based on the number of protoplasts electroporated) or 23.0% (based on the number of control microcalli formed) counted after 6 weeks of selection. After repeated subculturing on regeneration medium, shoots were induced from 8.0% of the transformed calli. Southern hybridization confirmed the presence of both the GUS and hygromycin genes in the transformed calli and shoots.     

Mutagenesis of soybean (Glycine max (L.) Merr.) and the isolation of non-nodulating mutants

Soybean seeds (cv. Bragg) were mutagenized with either ethyl methanesulphonate (EMS), gamma-rays or sodium azide (NaN₃). EMS was the most efficient in generating chlorophyll-deficient variants in the M₂. NaN₃ was not an effective mutagen. Approximately 25 200 M₂ plants were screened for absence of nodulation after inoculation with Bradyrhizobium japonicum strain CB1809 (=USDA136). Three stable non-nodulation mutants were recovered and designated nod49, nod772 and nod139. Mutant nod49 was isolated from a mixture of M₂ populations and this mutant also failed to nodulate upon inoculation with Rhizobium fredii strain USDA257, which nodulated the parent cultivar. Mutants nod772 and nod139 came from segregating EMS-derived M₂ families, indicating that these mutations were a result of mutagenesis. Inheritance of the non-nodulation character has been demonstrated through to the M₅, M₄ and M₃ generations for nod49, nod772 and nod139, respectively.     

Intracellular redistribution of phytochrome in etiolated soybean (Glycine max L.) seedlings

The intracellular distribution of phytochrome in hypocotyl hooks of etiolated soybean (Glycine max L.) has been examined by immunofluorescence using a newly produced monoclonal antibody (Soy-1) directed to phytochrome purified from etiolated soybean shoots. Cortical cells in the hook region exhibit the strongest phytochrome-associated fluorescence, which is diffusely distributed throughout the cytosol in unirradiated, etiolated seedlings. A redistribution of immunocytochemically detectable hytochrome to discrete areas (sequestering) following irradiation with red light requires a few minutes at room temperature in soybean, whereas this redistribution is reversed rapidly following irradiation with far-red light. In contrast, sequestering in oat (Avena sativa L.) occurs within a few seconds (D. McCurdy and L. Pratt, 1986, Planta 167, 330–336) while its reversal by far-red light requires hours (J. M. Mackenzie Jr. et al., 1975, Proc. Natl. Acad. Sci. USA 72, 799–803). The time courses, however, of red-light-enhanced phytochrome pelletability and sequestering are similar for soybean as they are for oat. Thus, while these observations made with a dicotyledon are consistent with the previous conclusion derived from work with oat, namely that sequestering and enhanced pelletability are different manifestations of the same intracellular event, they are inconsistent with the hypothesis that either is a primary step in the mode of action of phytochrome.Abbreviations DIC differential interference contrast - FR far-red light - Ig immunoglobulin - Pfr, P far-red- and red-absorbing form of phytochrome, respectively - R red light This work was supported by National Science Foundation grant No. DCB-8703057.     

Cytological evidence of a new male-sterile mutant in soybean (Glycine max (L.) Merr.)

Summary From one plant of soybean (Glycine max (L.) Merr.) with only two one-seeded pods, found in an F₄ population maintained by single-seed descent procedure, two fully fertile plants were obtained which, in turn, produced two progeny segregating for male sterility. Segregation ratios, observed on progeny from fertile plants in three successive generations, indicated that the male-sterility trait was under the control of a single recessive gene. Cytological observations made on malesterile, female-fertile plants showed the occurrence of a complete and properly timed cytokinesis with the formation of tetrad cells whose size was very variable, one of which sometimes had two nuclei. During pollen maturation binucleate microspores and grains with reduced size (micropollens) were frequently observed. Massive pollen degeneration occurred at a rather later stage. Structural evidence points to a normally functioning tapetum.On the basis of these cytological observations we conclude that the abnormalities observed in the mutant we studied have to be considered to be different from those caused by any other known ms allele. Tests of allelism with other sources of male sterility are in progress.     

Genetic structure and diversity of cultivated soybean (Glycine max (L.) Merr.) landraces in China

The Chinese genebank contains 23,587 soybean landraces collected from 29 provinces. In this study, a representative collection of 1,863 landraces were assessed for genetic diversity and genetic differentiation in order to provide useful information for effective management and utilization. A total of 1,160 SSR alleles at 59 SSR loci were detected including 97 unique and 485 low-frequency alleles, which indicated great richness and uniqueness of genetic variation in this core collection. Seven clusters were inferred by STRUCTURE analysis, which is in good agreement with a neighbor-joining tree. The cluster subdivision was also supported by highly significant pairwise F _st values and was generally in accordance with differences in planting area and sowing season. The cluster HSuM, which contains accessions collected from the region between 32.0 and 40.5°N, 105.4 and 122.2°E along the central and downstream parts of the Yellow River, was the most genetically diverse of the seven clusters. This provides the first molecular evidence for the hypotheses that the origin of cultivated soybean is the Yellow River region. A high proportion (95.1%) of pairs of alleles from different loci was in LD in the complete dataset. This was mostly due to overall population structure, since the number of locus pairs in LD was reduced sharply within each of the clusters compared to the complete dataset. This shows that population structure needs to be accounted for in association studies conducted within this collection. The low value of LD within the clusters can be seen as evidence that much of the recombination events in the past have been maintained in soybean, fixed in homozygous self-fertilizing landraces. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Seasonal and diurnal variations in nitrate reductase activity of soybean (Glycine max (L.) Merr.)

Summary The seasonal and diurnal variations in nitrate reductase (NR) activity of field grown Altona soybean, with and without applied nitrogen, were determined. The NR activity in the fortnightly collected leaf samples was, on the average, 20 percent higher throughout the season in N-treated plants, the highest being early in the season and declining gradually in the samples of subsequent dates. Diurnal variations were marked by increase in the NR activity from 7 a.m. to 7 p.m. and then declining gradually to a minimum at 7 a.m. the next morning.     

Responses of photosynthesis to irradiance and temperature in soybean,Glycine max (L.) Merr

The response of photosynthesis to irradiance and temperature during growth was investigated in two soybean genotypes. Soybean is a species that can modify its structure and metabolism so as to adapt to differing light conditions; its responses to rapid changes in irradiance are characterized by their flexibility. However, the temperature during growth can change the response to irradiance: moreover, there may be a marked interaction with genotype.The response of photosynthesis to irradiance consists of changes in leaf thickness, which bring about variations in the mesophyll resistance to CO₂ transfer. The increase in net photosynthesis per unit of leaf area is due to the increase in the amount of assimilating material beneath unit of area, as corroborated by the stability of the net photosynthesis per unit volume. Moreover, the response of photosynthesis to temperature is due to the mesophyll diffusion constant which decreases with the growth temperature.     

Genome-scale identification of MLO domain-containing genes in soybean (Glycine max L. Merr.)

In plants, powdery-mildew-resistance locus o (Mlo) genes encode proteins that are calmodulin-binding proteins involved in a variety of cellular processes. However, systematic characterization of this gene family in soybean (Glycine max L. Merr.) has not been yet reported. In this study, we identified MLO domain-contained members in soybean and examined their expression under phytohormone treatment and abiotic stress conditions. A total of 20 soybean Mlo genes were identified (GmMlo1-20), which are distributed on 13 chromosomes, and display diverse exon-intron structures. Phylogenetic analysis indicated that the Mlo family can be classified into four subfamilies. Sequence comparison was used to reveal the conserved calmodulin-binding domain (CaMBD) in GmMLO proteins. The expression of GmMlo genes was influenced by various phytohormone treatments and abiotic stresses, suggesting that these Mlo genes have various roles in the response of soybean to environmental stimuli. Promoter sequence analysis revealed an overabundance of stress and/or phytohormone-related cis-elements in GmMlo genes. These data provide important clues for elucidating the functions of genes of the Mlo gene family.     

Association mapping for partial resistance to Phytophthora sojae in soybean (Glycine max (L.) Merr.)

Association mapping is a powerful high-resolution mapping tool for complex traits. The objective of this study was to identify QTLs for partial resistance to Phytophthora sojae. In this study, we evaluated a total of 214 soybean accessions by the hypocotyl inoculation method, and 175 were susceptible. The 175 susceptible accessions were then evaluated for P. sojae partial resistance using slant board assays. The 175 accessions were screened with 138 SSR markers that generated 730 SSR alleles. A subset of 495 SSR loci with minor allele frequency (MAF) ≥ 0.05 was used for association mapping by the Tassel general linear model (GLM) and mixed linear model (MLM) program. This soybean population could be divided into two subpopulations and no or weak relatedness was detected between pairwise accessions. Four SSR alleles, Satt634-133, Satt634-149, Sat_222-168 and Satt301-190, associated with partial resistance to P. sojae were detected by both GLM and MLM methods. Of these identified markers, one marker, Satt301, was located in regions where P. sojae resistance QTL have been previously mapped using linkage analysis. The identified markers will help to understand the genetic basis of partial resistance, and facilitate future marker-assistant selection aimed to improve resistance to P. sojae and reduce disease-related mortality in soybean.     

Manganese and iron interactions on their uptake and distribution in soybean (Glycine max (L.) Merr.)

Summary The uptake and distribution of iron and manganese were studied in a manganese-sensitive soybean cultivar (‘Bragg’) grown over a range of supply levels of these nutrients in solution culture. At high (90 and 275 μM) manganese levels, increasing the iron concentration in solution from 2 to 100 μM partially overcame the effects of manganese toxicity. Interactions between manganese and iron occurred for dry matter yields, rate of Mn absorption by the roots, and the proportions of manganese and iron transported to the tops. No interaction was observed for the rate of root absorption of iron. The percentage distribution of manganese in the plant top increased with increasing iron, despite a reduced rate of Mn uptake. On the other hand, iron uptake was independent of solution Mn concentration and increased with increasing solution Fe. Also more iron was retained in the roots at high Mn and/or Fe levels in solution. Concentrations of manganese and iron in roots, stems and individual leaves were affected independently by the manganese and iron supplyi.e. without any interaction occurring between the two elements. In general, the concentration in a plant part was related directly to the solution concentration. Symptoms resembling iron deficiency correlated poorly with leaf Fe concentrations whereas high levels of manganese were found in leaves displaying Mn toxicity symptoms.     

RFLP mapping using near-isogenic lines in the soybean [Glycine max (L.) Merr.]

Summary A molecular marker analysis of a near-isogenic line (NIL), its donor parent (DP), and its recurrent parent (RP) can provide information about linkages between molecular markers and a conventional marker introgressed into the NIL. If the DP and RP possess different alleles for a given molecular marker, and if the NIL possesses the same allele as the DP, then it is reasonable to presume a linkage between that molecular marker and the introgressed marker. In this study, we examined the utility of RFLPs as molecular markers for the NIL genemapping approach. The allelic status of fifteen RFLP loci was determined in 116 soybean RP/NIL/DP line sets; 66 of the Clark RP type and 50 of the Harosoy RP type. Of the 1740 possible allelic comparisons (116 NILs x 15 RFLP loci), 1638 were tested and 462 (33.9%) of those were informative (i.e., the RP and DP had different RFLP alleles). In 15 (3.2%) of these 462 cases the NIL possessed the DP-derived RFLP allele, leading to a presumption of linkage between the RFLP locus and the introgressed conventional marker locus. Two presumptive linkages, pK-3 — and pK-472 — Lf _i, were subsequently confirmed by cosegregation linkage analysis. Although not yet confirmed, two other associations, pk-7 ab and pK-229 — y ₉ seemed to be plausible linkages, primarily because the pk-7 — ab association was detected in two independently derived NILs and both markers of the pK-229 — y ₉ association were known to be linked to Pb. The data obtained in this investigation indicated that RFLP loci were useful molecular markers for the NIL gene-mapping technique.Published as Paper no. 9101, Journal Series, Nebraska Agric. Res. Div. Project no. 12-091. Research partially funded by a grant from the Nebraska Soybean Development, Utilization, and Marketing Board     

Regeneration of soybean (Glycine max L. Merr.) from cultured primary leaf tissue

A reproducible method for regeneration of plants from primary leaf tissue of 27 varieties of soybean (Glycine max), encompassing maturity groups 00 to VIII, has been developed. Progeny from seeds recovered from regenerated plants appear normal. Best regeneration was from leaf explants (2.1–4.0 mm) obtained from 5 day old seedlings. While 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) was demonstrated to be essential for regeneration, addition of benzyladenine (BA) was found to enhance regeneration. Of the 6 other auxins tested, only picloram induced any regenerative response. Using identical volumes of medium and other conditions, regeneration could be obtained in 95 × 25 mm glass culture tubes but not in 60 × 15 mm Petri dishes.The regeneration of soybeans from primary leaf tissue was shown to be greatly enhanced by pyroglutamic acid (5-oxoproline). Stimulatory effects were attained if pyroglutamic acid was added directly to the medium or if it was formed in situ as a result of chemical transformation of glutamine during autoclaving. The active component produced by autoclaving glutamine was not a conjugate of glutamine with inorganic salts or another organic component of the medium. Filter-sterilized glutamine was shown to be inhibitory to regeneration.Murashige and Skoog (MS) and Schenk and Hildebrandt (SH) basal media were compared to Gamborg B5 medium. All contained 0.1 mg/l 2,4,5-T, 40 mg/l adenine sulfate and 10 mM pyroglutamic acid. No regeneration occurred when MS medium was used. Growth and appearance of callus growing on SH and B5 media with the additives were similar. The incidence of regeneration among cultures growing on SH medium was only one third compared to cultures grown on B5 medium.     

Quantitative content of total polar lipids in soybean seeds Glycine max (L.) Merr]

The exhaustive extraction of lipids from soybean seeds in the native state, separation of polar lipids and determination of their functional groups (ester and phosphate) revealed that dry seeds may contain 36,7 mumole (+/- 4,6%) of polar lipids and 25,9 mumole (+/- 1.1%) phospholipids per gramme.     

Phosphorus deficiency increases the acetylene-induced decline in nitrogenase activity in soybean (Glycine max (L.) Merr.)

Open-flow assays of acetylene reduction activity {ARA)and CO₂production in nodulated roots were performed in situ with soybean{Glycine max (L.) Merr.) cv. Kingsoy grown hydroponically withorthophosphate (Pi) nutrition either limiting (low-P) or non-limiting(control) for plant growth. Nodule growth was more limited thanshoot growth by P deficiency. During ARA assays, nitrogenaseactivity declined a few minutes after exposure of the nodulatedroots to C₂H₂, and this acetyleneinduced decline (C₂H₂-ID) wastwice as intense at low-P. Moreover, the minimum ARA after theC₂H₂-ID was reached about 10 min earlier at low-P. The intensityof the C₂H₂-ID was correlated negatively with nodule mass perplant and positively with the ratio of shoot/nodule mass. Afterinitial exposure to C₂H₂, the nodulated-root CO₂ productionwas transiently stimulated and, moreover, this increase was2-fold higher at low-P. Then, the nodulated-root CO₂ productiondecreased with nodule C₂H₄ production. During the C₂H₂-ID, thenodule nitrogenase-linked respiration, which was computed asthe variable component of the linear regression between CO₂and C₂H₄ production, was 2-fold higher at low-P. Furthermore,the microscopic observation of nodule sections revealed thatstarch deposits were decreased at low-P. However, nitrogenaseactivity, i.e. ARA before the C₂H₂-ID, was not affected by Pdeficiency. It is argued that P deficiency increased the C₂H₂-IDbecause it increased nodule permeability to O₂ diffusion. Key words: Acetylene reduction, nitrogen fixation, phosphorus, respiration, soybean, Glycine max (L.) Merr     

Molecular cloning and ethylene-inducible expression of Chib1 chitinase from soybean (Glycine max (L.) Merr.)

A soybean seed-specific PR-8 chitinase, named Chib2, has a markedly extended C-terminal segment compared to other plant Chib1 homologues of the PR-8 chitinase family known to date. To further characterize the molecular structure and the expression pattern of this chitinase family, we cloned two typical Chib1-similar cDNAs (Chib1-1 and Chib1-2) from soybeans by PCR-cloning techniques. The deduced primary sequence of Chib1-1 chitinase is composed of a signal peptide segment (26 amino acid residues) and a mature 273 amino acid sequence (calculated molecular mass 28,794, calculated pI 3.7). This Chib1-1 enzyme is more than 90% identical to Chib1-2 chitinase but is below 50% identical to Chib2 enzyme. Thus, we confirmed the occurrence of two distinct classes, Chib1 and Chib2 in the plant PR-8 chitinase family. The Chib1 genes, interrupted by one intron, were found to be up-regulated in response to ethylene in stems and leaves, but scarcely expressed in developing soybean seeds. Chib1 chitinases may be responsible for protecting the plant body from various pathogenic attacks.     

Molecular characterization and genetic diversity analysis of soybean (Glycine max (L.) Merr.) germplasm accessions in India

Molecular characterization and genetic diversity among 82 soybean accessions was carried out by using 44 simple sequence repeat (SSR) markers. Of the 44 SSR markers used, 40 markers were found polymorphic among 82 soybean accessions. These 40 polymorphic markers produced a total of 119 alleles, of which five were unique alleles and four alleles were rare. The allele number for each SSR locus varied between two to four with an average of 2.97 alleles per marker. Polymorphic information content values of SSRs ranged from 0.101 to 0.742 with an average of 0.477. Jaccard’s similarity coefficient was employed to study the molecular diversity of 82 soybean accessions. The pairwise genetic similarity among 82 soybean accessions varied from 0.28 to 0.90. The dendrogram constructed based on genetic similarities among 82 soybean accessions identified three major clusters. The majority of genotypes including four improved cultivars were grouped in a single subcluster IIIa of cluster III, indicating high genetic resemblance among soybean germplasm collection in India.

Electronic supplementary material

The online version of this article (doi:10.1007/s12298-014-0266-y) contains supplementary material, which is available to authorized users.     

Genetic correlations derived from Full-sib relationships in soybean (Glycine max Merr.)

Summary Spaced plants of a segregating soybean hybrid population in the F₆ generation were scored for fourteen quantitative traits related to yield, foliage development and growth duration. Full-sib relationships were used to estimate the genetic additive components of variation and covariation. All genetic correlations between traits, as well as phenotypic and environmental correlations, were estimated separately. A principal component analysis was further performed in all three cases. Genetic correlations identified four different groups of traits comprised of: (I) seed number per pod; (II) mean seed weight; (III) dry weight and chlorophyll content per unit leaf area; (IV) all the other characters, including seed yield and total plant weight at maturity. Among these traits, stem diameter at ground level appeared to be a good indicator of yield. This distribution remained about the same for the environmental correlations, except that growth duration traits and foliage development traits became independent of yield. The implications of these results are discussed in relation to soybean breeding for climatic adaptation.