Factors affecting the survival of Bradyrhizobium applied in liquid cultures to soya bean [Glycine max (L.) Merr.] seeds

AIMS: To determine the impact of medium composition, bacterial strain, trehalose accumulation, and relative humidity during seed storage on the survival of Bradyrhizobium japonicum on soya bean [Glycine max (L.) Merr.] seeds. METHODS AND RESULTS: Bacteria in liquid cultures were applied to seeds, and the number of survivors was quantified after 2, 24, 48, or 96 h. Addition of yeast extract to a defined medium increased on-seed survival 50- to 80-fold. Addition of 40 mmol l(-1) of NaCl to the medium doubled or tripled the accumulation of trehalose in cells and increased survival several fold, and the addition of both salt and trehalose had an additive effect. There was a threefold difference among strains in survival, and survival of the various strains was significantly correlated with differences in the accumulation of trehalose. The correlation between trehalose accumulation by bacteria and survival was also highly significant in other experiments. Studies in controlled humidity environments showed 100-fold or more differences in survival. CONCLUSIONS: The consistently significant correlation of trehalose content of cells with survival on seed suggests that trehalose is an important component of the survival mechanisms. When some of the factors (salt and trehalose in the medium plus humidity control) were studied in combination, several 100-fold increases in survival of bacteria on seeds were recorded. SIGNIFICANCE AND IMPACT OF THE STUDY: It is possible by manipulation of several parameters--strain selection, salt and trehalose content of the medium, control of relative humidity--to achieve substantial improvements in survival of Bradyrhizobium on soya bean seeds.     

Factors underlying genotypic differences in the induction of photosynthesis in soybean [Glycine max (L.) Merr.]

Crop leaves are subject to continually changing light levels in the field. Photosynthetic efficiency of a crop canopy and productivity will depend significantly on how quickly a leaf can acclimate to a change. One measure of speed of response is the rate of photosynthesis increase toward its steady state on transition from low to high light. This rate was measured for seven genotypes of soybean [Glycine max (L.) Merr.]. After 10 min of illumination, cultivar ‘UA4805’ (UA) had achieved a leaf photosynthetic rate (P_n) of 23.2 μmol · m^?2 · s^?1, close to its steady‐state rate, while the slowest cultivar ‘Tachinagaha’ (Tc) had only reached 13.0 μmol · m^?2 · s^?1 and was still many minutes from obtaining steady state. This difference was further investigated by examining induction at a range of carbon dioxide concentrations. Applying a biochemical model of limitations to photosynthesis to the responses of P_n to intercellular CO₂ concentration (C_i), it was found that the speed of apparent in vivo activation of ribulose‐1:5‐bisphosphate carboxylase/oxygenase (Rubisco) was responsible for this difference. Sequence analysis of the Rubisco activase gene revealed single nucleotide polymorphisms that could relate to this difference. The results show a potential route for selection of cultivars with increased photosynthetic efficiency in fluctuating light.     

Genetics and cytology of a new genic male-sterile soybean [Glycine max (L.) Merr.]

 Genetic and cytological studies were conducted with a new male-sterile, female-fertile soybean [Glycine max (L.) Merr.] mutant. This mutant was completely male sterile and was inherited as a single-recessive gene. No differences in female or male gamete transmission of the recessive allele were observed between reciprocal cross-pollinations in the F₁ or F₂ generations. This mutant was not allelic to any previously identified soybean genic male-sterile mutants: ms1, ms2, ms3, ms4, ms5, or ms6. No linkage was detected between sterility and flower color (W1 locus), or between sterility and pubescence color (T1 locus). Light microscopic and cytological observations of microsporogenesis in fertile and sterile anthers were conducted. The structure of microspore mother cells (MMC) in male-sterile plants was identical to the MMCs in male-fertile plants. Enzyme extraction analyses showed that there was no callase activity in male-sterile anthers, and this suggests that sterility was caused by retention of the callose walls, which normally are degraded around tetrads at the late tetrad stage. The tapetum from male-sterile anthers also showed abnormalities at the tetrad stage and later stages, which were expressed by an unusual formation of vacuoles, and by accumulation of densely staining material. At maturity, anthers from sterile plants were devoid of pollen grains. Received: 13 May 1996 / Revision accepted: 19 August 1996     

Sequence Level Analysis of Recently Duplicated Regions in Soybean [Glycine max (L.) Merr.] Genome

A single recessive gene, rxp, on linkage group (LG) D2 controlsbacterial leaf-pustule resistance in soybean. We identifiedtwo homoeologous contigs (GmA and GmA') composed of five bacterialartificial chromosomes (BACs) during the selection of BAC clonesaround Rxp region. With the recombinant inbred line populationfrom the cross of Pureunkong and Jinpumkong 2, single-nucleotidepolymorphism and simple sequence repeat marker genotyping wereable to locate GmA' on LG A1. On the basis of information inthe Soybean Breeders Toolbox and our results, parts of LG A1and LG D2 share duplicated regions. Alignment and annotationrevealed that many homoeologous regions contained kinases andproteins related to signal transduction pathway. Interestingly,inserted sequences from GmA and GmA' had homology with transposaseand integrase. Estimation of evolutionary events revealed thatspeciation of soybean from Medicago and the recent divergenceof two soybean homoeologous regions occurred at 60 and 12 millionyears ago, respectively. Distribution of synonymous substitutionpatterns, K_s, yielded a first secondary peak (mode K_s = 0.10–0.15)followed by two smaller bulges were displayed between soybeanhomologous regions. Thus, diploidized paleopolyploidy of soybeangenome was again supported by our study.     

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     

Cryopreservation of the SB-M photosynthetic soybean [Glycine max (L.) Merr.] suspension culture

Summary The photosynthetic cell suspension culture of soybean [Glycine max (L.) Merr. cv. Corsoy] (SB-M) was successfully cryopreserved in liquid nitrogen using a preculture and controlled freezing to −40° C (two-step) freezing method. The effective method included a preculture treatment with gradually increasing levels of sorbitol added to the 3% sucrose already present in the medium. The cells were then placed in a cryoprotectant solution [10% DMSO (dimethylsulfoxide) and 9.1% sorbitol, or 10% DMSO and 8% sucrose], incubated for 30 min at 0° C, cooled at a rate of 1° C/min to −40° C, held at −40° C for 1 h, and then immersed directly into liquid nitrogen. The cells were thawed at 40° C and then immediately placed in liquid culture medium. The cell viabilities immediately after thawing were 75% or higher in all cases where cell growth resumed. The original growth rate and chlorophyll level of the cells was recovered within 40 to 47 d. If the sorbitol level was not high enough or the preculture period too short, growing cultures could not be recovered. Likewise, survival was not attained with cryoprotectant mixtures consisting of 15% DMSO, 15% glycerol, and 9.1% sucrose or 15% glycerol and 8% sucrose. The successful method was reproducible, thus allowing long-term storage of this and certain other unique photosynthetic suspension cultures in liquid nitrogen.     

Multiple origins of the determinate growth habit in domesticated common bean (Phaseolus vulgaris)

Background and Aims

The actual number of domestications of a crop is one of the key questions in domestication studies. Answers to this question have generally been based on relationships between wild progenitors and domesticated descendants determined with anonymous molecular markers. In this study, this question was investigated by determining the number of instances a domestication phenotype had been selected in a crop species. One of the traits that appeared during domestication of common bean (Phaseolus vulgaris) is determinacy, in which stems end with a terminal inflorescence. It has been shown earlier that a homologue of the arabidopsis TFL1 gene – PvTFL1y – controls determinacy in a naturally occurring variation of common bean.

Methods

Sequence variation was analysed for PvTFL1y in a sample of 46 wild and domesticated accessions that included determinate and indeterminate accessions.

Key Results

Indeterminate types – wild and domesticated – showed only synonymous nucleotide substitutions. Determinate types – observed only among domesticated accessions – showed, in addition to synonymous substitutions, non-synonymous substitutions, indels, a putative intron-splicing failure, a retrotransposon insertion and a deletion of the entire locus. The retrotransposon insertion was observed in 70 % of determinate cultivars, in the Americas and elsewhere. Other determinate mutants had a more restricted distribution in the Americas only, either in the Andean or in the Mesoamerican gene pool of common bean.

Conclusions

Although each of the determinacy haplotypes probably does not represent distinct domestication events, they are consistent with the multiple (seven) domestication pattern in the genus Phaseolus. The predominance of determinacy in the Andean gene pool may reflect domestication of common bean prior to maize introduction in the Andes.     

The genome of cowpea (Vigna unguiculata [L.] Walp.)

Cowpea (Vigna unguiculata [L.] Walp.) is a major crop for worldwide food and nutritional security, especially in sub‐Saharan Africa, that is resilient to hot and drought‐prone environments. An assembly of the single‐haplotype inbred genome of cowpea IT97K‐499‐35 was developed by exploiting the synergies between single‐molecule real‐time sequencing, optical and genetic mapping, and an assembly reconciliation algorithm. A total of 519 Mb is included in the assembled sequences. Nearly half of the assembled sequence is composed of repetitive elements, which are enriched within recombination‐poor pericentromeric regions. A comparative analysis of these elements suggests that genome size differences between Vigna species are mainly attributable to changes in the amount of Gypsy retrotransposons. Conversely, genes are more abundant in more distal, high‐recombination regions of the chromosomes; there appears to be more duplication of genes within the NBS‐LRR and the SAUR‐like auxin superfamilies compared with other warm‐season legumes that have been sequenced. A surprising outcome is the identification of an inversion of 4.2 Mb among landraces and cultivars, which includes a gene that has been associated in other plants with interactions with the parasitic weed Striga gesnerioides. The genome sequence facilitated the identification of a putative syntelog for multiple organ gigantism in legumes. A revised numbering system has been adopted for cowpea chromosomes based on synteny with common bean (Phaseolus vulgaris). An estimate of nuclear genome size of 640.6 Mbp based on cytometry is presented.     

Correlation between AS1 gene expression and seed protein contents in different soybean (Glycine max [L.] Merr.) cultivars

In higher plants, asparagine synthetase (AS) plays an important role in regulating the nitrogen sink-source relationship. We studied the expression of AS genes in five Chinese soybean cultivars exhibiting contrasting seed protein contents. We found that only the AS2 but not the AS1 gene was induced by dark treatment. On the other hand, the expression of AS1 in leaves (especially in trifoliate leaves of young seedlings) showed a positive correlation with seed protein contents in the soybean cultivars tested. Therefore, in spite of the fact that the principle transporting compounds in soybean plants for nitrogen acquired via symbiotic fixation are ureides, AS may still play an important role in the process of nitrogen assimilation.     

Low concentrations of ammonium inhibit specific nodulation (nodule number g-1 root DW) in soybean (Glycine max [L.] Merr.)

Experiments on peas (Gulden and Vessey, 1997) have indicated that NH ₄ ⁺ stimulates both whole plant (nodules plant^-1) and specific nodulation (nodules g^-1 root DW). The effect of low concentrations of NH ₄ ⁺ on the soybean/Bradyrhizobium symbiosis is unknown. The objectives of the current study were to determine the immediate and residual effects of NH ₄ ⁺ on nodulation and N₂ fixation in soybean (Glycine max [L.] Merr.) in sand culture. Soybean (cv. Maple Ridge) were exposed to 0.0, 0.5, 1.0 and 2.0 mM of ¹⁵N-labelled (NH₄)₂SO₄ for 28 days after inoculation (DAI). From 29 to 56 DAI the plants were grown on NH ₄ ⁺ -free nutrient solution. Plants were harvested at 7, 14, 21, 28 and 56 DAI for root, shoot and nodule dry weight (DW), total N content, nodule counts and ¹⁵N enrichment of plant composites. Nitrogenase activity was measured by gas exchange at 28 DAI. The plants in the control (0.0 mM NH ₄ ⁺ ) treatment had consistently lower relative growth rates than the plants in the NH ₄ ⁺ treatments during the first 28 DAI. Plant growth was also less at 2.0 mM NH ₄ ⁺ compared to growth at 0.5 and 1.0 mM NH ₄ ⁺ . At 28 DAI, plants exposed to 0.5 and 1.0 mM NH ₄ ⁺ had significantly more nodules per plant and larger individual nodules than either the NH ₄ ⁺ -free controls or the 2.0 mM NH ₄ ⁺ -supplied plants. However, specific nodulation (nodule number g^-1 root DW) and specific nitrogenase activity (nitrogenase activity g^-1 nodule DW) were on average approximately 286% and 60% higher in the control plants, respectively, than for plants in the NH ₄ ⁺ treatments at 28 DAI. Also at 28 DAI, specific nodule DW (nodule DW g^-1 root DW) were 17, 44 and 53% higher in control plants than plants that had been exposed to 0.5, 1.0 and 2.0 mM NH ₄ ⁺ . At 56 DAI, after an additional 4 weeks of NH ₄ ⁺ -free nutrition, the plants which had previously received 0.5 and 1.0 mM NH ₄ ⁺ still maintained the highest plant DW and N contents, however, specific nodule DW had become similar at 600 mg nodule DW g^-1 root DW among all treatments. It is concluded that NH ₄ ⁺ has a negative effect on the nodulation process in the soybean/Bradyrhizobium symbiosis (as best indicated by the negative effect of NH ₄ ⁺ on specific nodulation). Despite this negative effect on specific nodulation, 0.5 and 1.0 mM NH ₄ ⁺ resulted in higher whole plant nodulation and N₂ fixation due to a compensating, positive effect on overall plant growth (i.e. fewer nodules g^-1 root DW, but much larger roots). Once NH ₄ ⁺ was removed from all treatments, the soybean plants appeared to move toward a consistent level of nodule DW relative to root DW.     

Loci and candidate gene identification for resistance to Sclerotinia sclerotiorum in soybean (Glycine max L. Merr.) via association and linkage maps

Soybean white mold (SWM), caused by Sclerotinia sclerotiorum ((Lib.) W. Phillips), is currently considered to be the second most important cause of soybean yield loss due to disease. Research is needed to identify SWM‐resistant germplasm and gain a better understanding of the genetic and molecular basis of SWM resistance in soybean. Stem pigmentation after treatment with oxaloacetic acid is an effective indicator of resistance to SWM. A total of 128 recombinant inbred lines (RILs) derived from a cross of ‘Maple Arrow’ (partial resistant to SWM) and ‘Hefeng 25’ (susceptible) and 330 diverse soybean cultivars were screened for the soluble pigment concentration of their stems, which were treated with oxalic acid. Four quantitative trait loci (QTLs) underlying soluble pigment concentration were detected by linkage mapping of the RILs. Three hundred and thirty soybean cultivars were sequenced using the whole‐genome encompassing approach and 25 179 single‐nucleotide polymorphisms (SNPs) were detected for the fine mapping of SWM resistance genes by genome‐wide association studies. Three out of five SNP markers representing a linkage disequilibrium (LD) block and a single locus on chromosome 13 (Gm13) were significantly associated with the soluble pigment content of stems. Three more SNPs that represented three minor QTLs for the soluble pigment content of stems were identified on another three chromosomes by association mapping. A major locus with the largest effect on Gm13 was found both by linkage and association mapping. Four potential candidate genes involved in disease response or the anthocyanin biosynthesis pathway were identified at the locus near the significant SNPs (<60 kbp). The beneficial allele and candidate genes should be useful in soybean breeding for improving resistance to SWM.     

Cloning, mapping, and analyses of expression of the Em-like gene family in soybean [Glycine max (L). Merr.]

 The entire Em-like Group-1 late embryogenesis abundant (Lea) gene family from soybean was cloned and characterized. The five Group-1 Lea genes (Sle1-5) were divided into two classes based on sequence identity. Sle1-4 were genetically mapped to four different linkage groups. Nucleotide sequencing indicated that Sle1, Sle2, Sle3, and Sle5 encode polypeptides differing primarily by the presence of a repeated 20-amino acid motif. Sle1 and Sle5 were shown by Northern analysis to be expressed in developing embryos weeks earlier than Sle2 and Sle3. Sle4 was shown to be a pseudogene. Maximal levels of mRNA for all functional Sle genes accumulated in maturation-phase seeds, before significant desiccation had occurred, and declined rapidly upon seed imbibition. Desiccation did not induce Sle expression in seeds or vegetative tissue. Sle expression was confined to embryo tissues and Sle mRNA accumulated at similar levels in both the embryo axis and in the cotyledons. Received: 25 September 1996 / Accepted: 27 September 1996     

Molecular mapping of k2 Mdh1-n y20, an unstable chromosomal region in soybean [Glycine max (L.) Merr.]

In the soybean genome, a chromosomal region covering three tightly linked genes, k2, Mdh1-n, and y20, was found very unstable. It was suspected that the instability of the k2 Mdh1-n y20 chromosomal region was caused by a non-autonomous transposable element residing adjacent to or in this region. In this study, we located and mapped this region with simple sequence repeat (SSR) markers on the soybean integrated map using five mapping populations. The k2 Mdh1-n y20 chromosomal region was located on molecular linkage group H. The integrated map from five mapping populations consisted of 13 loci in the order Satt541, Satt469, Sat_122, Satt279, Satt253, Satt314, Mdh1-n,y20, k2, Satt302, Satt142, Satt181, and Satt434. The k2 Mdh1-n y20 chromosomal region was very close to Satt314, Satt253, and Satt279. The genetic distance between the Mdh1-n gene and Satt314 was less than 1 cM. The results of the mapping study were consistent with the results from previous studies that the Mdh1-n mutation in T261 (k2 Mdh1-n) and the Mdh1-n y20 mutation in T317 (Mdh1-n y20) were caused by deletions. In addition, another putative deletion was found in the genome of T261 which covered three SSR markers (Satt314, Satt253, and Satt279). This is a joint contribution of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 3769, and from the USDA, Agricultural Research Service, Corn Insects and Crop Genetics Research Unit, and supported by the Hatch Act and the State of Iowa. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by Iowa State University or the USDA, and the use of the name by Iowa State University or the USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Effects of norflurazon (San 9789) on light-increased extractable nitrate reductase activity in soybean [Glycine max (L.) Merr.]seedlings

Abstract. The effects of norflurazon (San 9789) on light-increased extractable NADH nitrate reductase activity (NRA) in soybean seedlings were studied. Continuous white light (W) increased NRA steadily in root and cotyledonary tissues over a 5 d period. Morflurazon, a pyridazinone herbicide which causes chlorophyll bleaching in W, reduced the initial NRA induction rate in roots and cotyledons. However, in cotyledons of norfiurazon-treated plants NRA increased at a more rapid rate than in the control after 24 h of W, with activity levels reaching three times those of control seedlings after 5 d. NRA induced by W in control and norflurazon-treated cotyledons was fluence-rate dependent. Continuous FR induced equal amounts of NRA in control and norflurazontreated tissues, suggesting that the superinduceable NRA of norflurazon-treated plants under W is not phytochrome induced. The FR-induced NRA of control and norflurazon-treated cotyledons had pH optima of 6.6, but during development under W the pH optimum of control cotyledons changed from 6.3 to between 6.6 and 7.1. The pH optimum of the norflurazon-induced NRA of the cotyledon under W was about 7.5. The NADH/NADPH NRA ratio after 4 d of W was 1.3 in control and 2.5 in norflurazontreated cotyledons. These data indicate that photosynthelic pigments are involved only secondarily in light-induction of NRA in this system.