Identification of genomic regions determining flower and pod numbers development in soybean （Glycine max L.）

Flower and pod numbers per plant are important agronomic traits underlying soybean yield.So far quantitative trait loci (QTL) detected for flower and pod-related traits have mainly focused on the final stage,and might therefore have ignored genetic effects expressed during a specific developmental stage.Here,dynamic expressions of QTL for flower and pod numbers were identified using 152 recombinant inbred lines (RILs) and a linkage map of 306 markers.Wide genetic variation was found among RILs;17 unconditional and 18 conditional QTL were detected for the two traits at different developmental stages over two years.Some QTL were detected only at one stage and others across two or more stages,indicating that soybean flower and pod numbers development may be governed by time-dependent gene expression.Three main QTL (qfn-Chr18-2,qfn-Chr20-1,and qfn-Chr19) were detected for flower number,and two main QTL (qpn-Chr11 and qpn-Chr20) were detected for pod number.The phenotypic variation explained by them ranged from 6.1% to 34.7%.The markers linked to these QTL could be used in marker-assisted selection for increasing soybean flower and pod numbers,with the ultimate aim of increasing soybean yield.Comparison of the QTL regions for flower and pod numbers traits with the related genes reported previously showed that seven and four related genes were located in the QTL regions of qfn-Chr11 and qfn-Chr19,respectively.Tbese results provide a basis for fine mapping and cloning of flower and pod development-related genes.     

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.     

Mapping QTLs for seed yield and drought susceptibility index in soy-bean（Glycine max L.） across different environments

Drought stress has long been a major constraint in maintaining yield stability of soybean （Glycine max （L.） Merr.） in rainfed ecosystems. The identification of consistent quantitative trait loci （QTL） involving seed yield per plant （YP） and drought susceptibility index （DSI） in a population across different environments would therefore be important in molecular marker-assisted breeding of soybean cultivars suitable for rainfed regions. The YP of a recombinant line population of 184 F2：7：11 lines from a cross of Kefengl and Nannong1138-2 was studied under water-stressed （WS） and well-watered （WW） conditions in field （F） and greenhouse （G） trials, and DSI for yield was calculated in two trials. Nineteen QTLs associated with YP-WS and YP-WW, and 10 QTLs associated with DSI, were identi- fied. Comparison of these QTL locations with previous findings showed that the majority of these regions control one or more traits re- lated to yield and other agronomic traits. One QTL on molecular linkage group （MLG） K for YP-F, and two QTLs on MLG C2 for YP-G, remained constant across different water regimes. The regions on MLG C2 for YP-WW-F and MLG H for YP-WS-F had a pleiotropic effect on DSI-F, and MLG A1 for YP-WS-G had a pleiotropic effect on DSI-G. The identification of consistent QTLs for YP and DSI across different environments will significantly improve the efficiency of selecting for drought tolerance in soybean.     

Mapping and validation of simple sequence repeat markers linked to a major gene controlling seed cadmium accumulation in soybean [Glycine max (L.) Merr]

Daily consumption of cadmium (Cd) contaminated foods poses a risk to human health. Cultivar selection is an important method to limit Cd uptake and accumulation, however, analyzing grain Cd concentration is costly and time-consuming. Developing markers for low Cd accumulation will facilitate marker assisted selection (MAS). Inheritance studies using a threshold value of 0.2 mg kg^?1 for low and high and an F_2:3 population showed that low Cd accumulation in soybean seed is under the control of a major gene (Cda1, proposed name) with the allele for low accumulation being dominant. A recombinant inbred line (RIL) population (F_6:8) derived from the cross AC Hime (high Cd accumulation) and Westag-97 (low Cd accumulation) was used to identify the DNA markers linked to Cda gene(s) or quantitative trait loci (QTLs) controlling low Cd accumulation. We screened 171 simple sequence repeat (SSR) primers that showed polymorphism between parents on the 166 RILs. Of these, 40 primers were newly developed from the soybean genomic DNA sequence. Seven SSR markers, SatK138, SatK139, SatK140 (0.5 cM), SatK147, SacK149, SaatK150 and SattK152 (0.3 cM), were linked to Cda1 in soybean seed. All the linked markers were mapped to the same linkage group (LG) K. The closest flanking SSR markers linked to Cda1 were validated using a parallel population (RILs) involving Leo × Westag-97. Linked markers were also validated with diverse soybean genotypes differing in their seed Cd concentration and showed that SSR markers SatK147, SacK149, and SattK152 clearly differentiated the high and low Cd accumulating genotypes tested. To treat Cd uptake as a quantitative trait, QTL analysis using a linkage map constructed with 161 markers identified a major QTL associated with low Cd concentration in the seeds. The QTL was also mapped to the same location as Cda1 on LG-K. This QTL accounted for 57.3% of the phenotypic variation. Potential candidate genes (genes with known or predicted function that could influence the seed Cd concentration) like protein kinase, putative Adagio-like protein, and plasma membrane H⁺-ATPase were found to be located in the locus of interest. Of the four SSR markers located in the region, SattK152 was localized in the plasma membrane H⁺-ATPase gene. SSR markers closely linked to Cda1 in seeds of soybean were identified and have potential to be used for MAS to develop low Cd accumulating cultivars in a breeding program.     

QTL analyses of seed weight during the development of soybean (Glycine max L. Merr.)

At harvest traits such as seed weight are the sum of development and responses to stresses over the growing season and particularly during the reproductive phase of growth. The aim here was to measure quantitative trait loci (QTL) underlying the seed weight from early development to drying post harvest. One hundred forty-three F(5) derived recombinant inbred lines (RILs) developed from the cross of soybean cultivars 'Charleston' and 'Dongnong 594' were used for the analysis of QTL underlying mean 100-seed weight at six different developmental stages. QTL x Environment interactions (QE) were analyzed by a mixed genetic mode based on 3 years' data. At an experiment-wise threshold of a=0.05 and by single-point analysis 94 QTL unaffected by QE underlay the mean seed weight at different developmental stages. Sixty-eight QTL affected by QE that also underlay mean seed weight were identified. From the 162 QTL 42 could be located on 12 linkage groups by composite interval mapping (LOD>2.0). The numbers, locations and types of the QTL and the genetic effects were different at each developmental stage. On linkage group C2 the distantly linked QTL swC2-1, swC2-2 and swC2-3 each affected mean seed weight throughout the different developmental stages. The DNA markers linked to the QTL possessed potential for use in marker-assisted selection for soybean seed size. The identification of QTL with genetic main effects and QE interaction effects suggested that such interactions might significantly alter seed weight during seed development.     

Plant regeneration from protoplasts of soybean (Glycine max L.)

Protoplasts were isolated from immature cotyledons of six cultivars of Glycine max L. and cultured in the KP8 liquid medium supplemented with 0.2 mg/L 2,4-D, 1 mg/L NAA and 0.5 mg/L ZT. The protoplasts started to divide after 3–5 days of culture. Sustained divisions resulted in mass production of cell colonies and small calli in 6 weeks. The calli further grew to 2–3 mm on the gelritesolidified K8 medium and were transferred onto the MSB medium with 1 mg/L 2,4-D and 0.25 mg/L BA, to obtain compact and nodular calli. Shoot formation was initiated on MSB medium with 0.15 mg/L NAA, and BA, KT and ZT, 0.5 mg/L of each, with or without 500 mg/L CH. It was followed by plant regeneration. So far, 87 plants have been regenerated from 4 cultivars, and normal seeds were obtained from them after transplanting into pots.Abbreviation IAA indol-3-acetic acid - NAA naphthalene acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid - KT kinetin - BA 6-benzyladenine - ZT zeatin - CH casein hydrolysate     

Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.     

Host genetic control of symbiosis in soybean (Glycine max L.)

Genes controlling nitrogen-fixing symbioses of legumes with specialized bacteria known as rhizobia are presumably the products of many millions of years of evolution. Different adaptative solutions evolved in response to the challenge of survival in highly divergent complexes of symbionts. Whereas efficiency of nitrogen fixation appears to be controlled by quantitative inheritance, genes controlling nodulation are qualitatively inherited. Genes controlling nodulation include those for non-nodulation, those that restrict certain microsymbionts, and those conditioning hypernodulation, or supernodulation. Some genes are naturally occurring polymorphisms, while others were induced or were the result of spontaneous mutations. The geographic patterns of particular alleles indicate the role of coevolution in determining symbiont specificites and compatibilities. For example, the Rj4 allele occurs with higher frequency (over 50%) among the soybean (G. max) from Southeast Asia. DNA homology studies of strains of Bradyrhizobium that nodulate soybean indicated two groups so distinct as to warrant classification as two species. Strains producing rhizobitoxine-induced chlorosis occur only in Group II, now classified as B. elkanii. Unlike B. japonicum, B. elkanii strains are characterized by (1) the ability to nodulate the rj1 genotype, (2) the formation of nodule-like structures on peanut, (3) a relatively high degree of ex planta nitrogenase activity, (4) distinct extracellular polysaccharide composition, (5) distinct fatty acid composition, (6) distinct antibiotic resistance profiles, and (7) low DNA homology with B. japonicum. Analysis with soybean lines near isogenic for the Rj4 versus rj4 alleles indicated that the Rj4 allele excludes a high proportion of B. elkanii strains and certain strains of B. japonicum such as strain USDA62 and three serogroup 123 strains. These groups, relatively inefficient in nitrogen fixation with soybean, tend to predominate in soybean nodules from many US soils. The Rj4 allele, the most common allelic form in the wild species, has a positive value for the host plants in protecting them from nodulation by rhizobia poorly adapted for symbiosis.     

Cadmium-induced senescence in nodules of soybean (Glycine max L.) plants

The relationship between cadmium-induced oxidative stress and nodule senescence in soybean was investigated at two different concentrations of cadmium ions (50 and 200 μM), in solution culture. High cadmium concentration (200 μM) resulted in oxidative stress, which was indicated by an increase in thiobarbituric acid reactive substances content and a decrease in leghemoglobin levels. Consequently, nitrogenase activity was decreased, and increases in iron and ferritin levels were obtained. Senescent parameters such as ethylene production, increased levels of ammonium and an increase in protease activity were simultaneously observed. Glutamate dehydrogenase activity was also increased. Peroxidase activity decreased at the higher cadmium concentration while the lower cadmium treatment produced changes in peroxidase isoforms, compared to control nodules. Ultrastructural investigation of the nodules showed alterations with a reduction of both bacteroids number per symbiosome and the effective area for N₂-fixation. These results strongly suggest that, at least at the higher concentration, cadmium induces nodule senescence in soybean plants.     

Mapping quantitative trait loci associated with chlorophyll a fluorescence parameters in soybean (Glycine max (L.) Merr.)

Chlorophyll a fluorescence parameters can provide qualitative and quantitative information about photosynthetic processes in chloroplasts. JIP-test and modulated fluorescence (MF) parameters are commonly used chlorophyll a fluorescence parameters. This study was conducted to identify quantitative trait loci (QTLs) associated with JIP-test parameters, MF parameters, and photosynthetic rate (P_N), and to examine the relationships among them in soybean (Glycine max (L.) Merr.). Pot and field experiments were performed to evaluate 184 recombinant inbred lines (RILs) for five JIP-test parameters (ABS/RC, TR_o/ABS, ET_o/TR_o, RE_o/ET_o, and PI_ABS), four MF parameters (Fv/Fm, Fv′/Fm′, ΦPSII, and qP), and P_N. Significant correlations were commonly observed among JIP-test parameters, MF parameters, and P_N. QTL mapping analysis identified 13, 9, and 4 QTLs for JIP-test parameters, MF parameters, and P_N, respectively, of which 13 were stable. Four major genomic regions were detected: LG A2 (19.81 cM) for JIP-test parameters, LG C1 (94.31 and 97.61 cM) for P_N and MF parameters, LG M (100.51 cM) for JIP-test and MF parameters, and LG O (30.61–49.91 cM) for P_N, JIP-test, and MF parameters. These results indicate that chlorophyll fluorescence parameters, especially ΦPSII and qP, could play an important role in regulating P_N, and that JIP-test and MF parameters could be controlled by the same or different genes. The QTLs identified in this study will help in the understanding of the genetic basis of photosynthetic processes in plants. They will also contribute to the development of marker-assisted selection breeding programs for photosynthetic capacity in soybean.     

Sowing seasons and drying methods during post-harvest influence the seed vigour of soybean (Glycine max (L.) Merr.)

Experiments were conducted to study the influence of sowing seasons and drying methods on the seed vigour of two spring soybean (Glycine max (L.) Merr.) cultivars. Two cultivars, ‘Huachun18’ and ‘Huachun 14’, were sown in three seasons viz., spring, summer and autumn and the harvested seeds were dried using three different methods. The results showed that soybean sown in spring had a higher number of branches per plant, pods per branch and seed weight, and consequently resulted in higher seed yields than that of soybean sown in autumn or summer seasons. Seeds sown in the autumn season had the lowest values of electrical conductivity during seed imbibitions, higher peroxidase (POD) activity in germinated seedlings and lower contamination by the seed-borne fungi on the MS medium, which indirectly improved the seed vigour, which was followed by summer sown seeds. Seeds sown during the spring season resulted in poor seed vigour. In addition, the effect of drying methods on the seed vigour was also clarified. Seeds that hung for four days before threshing and then air-dried had the poorest seed vigour which was determined by germination, electrical conductivity, POD activity and seed borne fungal growth. There was no difference in seed vigour between other methods, i.e. seeds threshed directly at harvest and then air-dried on a bamboo sifter or concrete floor. These results indicated that autumn sowing soybean and the drying method in which seeds were threshed directly at harvest and then air-dried on a bamboo sifter resulted in higher seed vigour.     

The translocation of Zn65 and Cs134 between seed generations in soybean (Glycine max (L.) Merr.)

Summary In this study 23.9 per cent of the Zn⁶⁵ and 3.4 per cent of the Cs¹³⁴ contained in the first generation seeds was translocated to the second generation. The advisability of using Zn⁶⁵ or Cs¹³⁴ (and presumably Cs¹³⁷) contaminated soybean seed for crop production, even in uncontaminated soil, is questioned.Paper number 7851 Scientific Journal Series, University of Minnesota Agricultural Experiment Station, St. Paul, Minnesota.     

Molecular-genetic identification and certification of soybean (Glycine max L.) cultivars

An approach to certification of soybean genotypes has been developed. The procedure employs three methods of DNA analysis based on polymerase chain reaction (PCR): PCR with arbitrary primers (AP PCR), simple sequence repeat polymorphism (SSRP) analysis, and inter-simple sequence repeat (ISSR) analysis. The approach to certification proposed may be used in both genetic and breeding research and seed production. A "certificate" form that reflects the unique characteristics of each cultivar studied is proposed. The results of molecular genetic analysis of allele distribution in genotypes of soybean from different ecological geographic zones permit estimation of the adaptive significance of individual alleles.     

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.     

Fatty acid composition of soybean (Glycine max (L.) merr.) somatic embryos

Summary Somatic embryos from four soybean cultivars were matured for 30 and 45 d. Success of embryo germination was evaluated for each length of maturation. The percentage of somatic embryos undergoing successful germination, as defined by rooting and shoot emergence, was greater for embryos matured 45 d than for embryos matured 30 d. Therefore, embryos matured for 45 d are probably physiologically more mature than embryos matured for 30 d. Relative percentages of fatty acids comprising oils and lipids of somatic embryos were determined for each length of maturation and for each cultivar. Variation in relative percentages of palmitic acid, oleic acid, and linoleic acid was affected by length of maturation. However, these changes were genotype dependent. A significant interaction between the cultivars Clark and Maple Arrow and stage of maturation was observed for levels of oleic acid. No other interactions were observed. These data suggest that if changes in relative percentages of certain fatty acids are associated with soybean somatic embryo maturation the changes are genotype dependent. This is journal paper No. J-12870 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project No. 2763. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the United States Department of Agriculture and does not imply its approval to the exclusion of other products that also may be suitable. This research was supported in part by grants from the American Soybean Association Development Foundation and the Iowa Soybean Promotion Board.     

Morpho-physiological parameters affecting iron deficiency chlorosis in soybean (Glycine max L.)

Background and aims

Iron deficiency chlorosis (IDC) leads to severe leaf chlorosis, low photosynthetic rates, and yield reductions of several million metric tonnes each year. In order to devise breeding and genetic transformation programs that aim at generating high-yielding and IDC-tolerant soybean lines, it is necessary to better understand the mechanisms that enable tolerant plants to survive under Fe-limiting conditions.

Methods

An in silico analysis in the USDA soybean collection allowed the identification of a set of novel efficient and inefficient soybean cultivars which can be used in future studies concerning IDC response. Plants were grown in iron deficient and iron sufficient conditions using a bicarbonate system and several IDC-related aspects were studied.

Results

A new set of efficient and inefficient soybean lines were identified in silico, and their tolerance to IDC was confirmed under laboratorial conditions. New plant traits that are highly correlated to IDC scoring were identified: a negative correlation was found between SPAD values and stem weight, weight of the unifoliolates and iron concentration of the first unifoliolates was found; higher SPAD values were correlated with the amount of iron in the first trifoliate leaves. Our data also show that having higher concentrations of iron in the seeds provides increased resistance to IDC. No correlation was found between root iron reductase activity and chlorosis.

Conclusions

Soybean differential chlorosis susceptibility between different accessions is linked to specific morpho-physiological parameters such as unifoliolate leaf size, stem weigh, concentration of iron in the seeds, and tissue iron partitioning.     

Antioxidant enzymes and isoflavonoids in chilled soybean (Glycine max (L.) Merr.) seedlings

Changes of activity antioxidant enzymes and of levels of isoflavonoids were studied in the roots and hypocotyls of the etiolated soybean (Glycine max (L.) Merr. var. Essor) seedlings, submitted to cold. Prolonged exposure to 1 degrees C inhibited hypocotyl and root elongation and limited their growth after seedlings were transferred to 25 degrees C. Roots were more sensitive to chilling than hypocotyls. At 1 degrees C a gradual increase in MDA concentration in roots but not in hypocotyls was observed. An increase in catalase (CAT, EC 1.11.1.6) and superoxide dismutase (SOD, EC 1.15.1.1) activity in hypocotyls was observed both at 1 degrees C and after transfer of plants to 25 degrees C. In roots, CAT activity increased after 4 days of chilling, while SOD activity only after rewarming. L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) activity decreased in roots of chilled seedlings, but did not change in hypocotyls until activity increased after transfer to 25 degrees C. The content of genistein and daidzein increased after 24 h of treatment by low temperature and then decreased with prolonged chilling in hypocotyls and remained high in roots. However, it should be noted that genistin level (genistein glucoside) in chilled hypocotyls is 10 times higher than in roots, despite falling tendency. The role of antioxidant enzymes and isoflavonoids in preventing chilling injury in hypocotyls and roots of soybean seedlings is discussed.     

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.