Utility of DNA amplified by degenerate oligonucleotide-primed PCR (DOP-PCR) from the total genome and defined chromosomal regions of field bean

Degenerate oligonucleotide primed (DOP)-PCR has emerged as a simple and rapid method for representative amplification of highly complex genomic DNA from humans, mice and Drosophila. The present paper describes the adaptation of this method for use on a plant species, Vicia faba, with a large genome (2C = 30 pg). Specific low-copy-number sequences as well as highly repeated sequences were detectable among DOP-PCR products obtained from small samples of purified genomic DNA (100 pg), DNA from 10 prophase nuclei, 10 flow-sorted chromosomes or 15 microdissected chromosome segments (satellites) following reamplification with sequence-specific primers and/or Southern hybridization. Biotinylated chromosome-specific DOP-PCR products were used for fluorescent in situ hybridization. All chromosomes showed hybridization signals, with the exception of regions containing Fok elements which are not present in the chromosomal DNA targeted by DOP-PCR.     

Fluorescentin situ hybridization to soybean metaphase chromosomes

Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescentin situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosome [27] showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescentin situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.     

Characterization of soybean vegetative storage proteins and genes

Summary Soybean vegetative storage proteins (VSPs) were purified and characterized. Anion exchange HPLC resolved partially purified VSPs into fractions containing 27-kD/27-kD and 29-kD/29-kD homodimers and 27-kD/29-kD heterodimers. Reversed-phase HPLC resolved partially purified VSPs into three fractions. One fraction contained only 27-kD VSP and the other two contained 29-kD VSP. The two 29-kD VSP fractions differed with respect to their cyanogen bromide cleavage patterns, an observation that indicated the 29-kD VSPs were heterogeneous. Genomic clones that contained 29-kD VSP genes were also isolated and characterized. One genomic clone contained a complete 29-kD VSP gene and was sequenced. The coding region in the clone contained two introns whose borders had regulatory sequences typical of other eukaryotic genes. Putative polyadenlyation signals were present in the 3-flanking region of the gene, while putative TATA, CAAT, and enhancer core sequences were found in the 5-flanking regions. A second genomic clone that was studied contained the 5 regions of two partial 29-kD VSP genes in an inverted linkage. Genomic DNA gel blots showed that the two genes were organized in the same arrangement in the soybean genome.Cooperative research between USDA/Agricultural Research Service and the Indiana Agricultural Experiment Station. Journal Paper No. 12,192 from the Indiana Agricultural Experiment Station     

Lectin in five soybean cultivars previously considered to be lectin-negative

Hemagglutinating proteins were isolated by affinity chromatography from seeds of each of five cultivars of soybeans (Clycine max (L.) Merr.) previously reported to lack detectable lectin (S.P. Pull et al., 1978; Science 200, 1277). Quantities were between 1,000 and 10,000 times less than that found in the seeds of the reference cultivar, Chippewa. The sensitivity of the hemagglutinating assay was 0.05 g ml^-1. Hemagglutinating activity was demonstrated in affinity-purified fractions from bulk seeds and seeds from individual plants in two cultivars, 30–70% ammonium-sulfate-precipitable fractions of seeds from individual plants of all five cultivars, and in whole crude extracts of individual seeds from each cultivar. In all instances, hemagglutinating activity was inhibited by galactose, anti-soybean agglutinin (SBA), and lectin-binding polysaccharide produced by Rhizobium japonicum. Affinity-purified lectin from seeds of a single Columbia plant was labeled with fluorescein isothiocyanate (FITC) and observed by fluorescence microscopy to bind to R. japonicum cells with specificity, intensity and localization indistinguishable from FITC-SBA. Lectins from distinguishable from FITC-SBA. Lectins from three cultivars in sufficiently high concentration for study had molecular properties very similar to Chippewa SBA.Abbreviations FITC fluorescein isothiocyanate - IgG immunoglobulin G - SBA soybean agglutinin     

Ribosomal RNA genes in soybean and common bean: chromosomal organization, expression, and evolution

Ribosomal RNA (5S and 45S) genes were investigated by FISH in two related legumes: soybean [Glycine max (L.) Merr.] and common bean (Phaseolis vulgaris L.). These species are both members of the same tribe (Phaseoleae), but common bean is diploid while soybean is a tetraploid which has undergone diploidization. In contrast to ploidy expectations, soybean had only one 5S and one 45S rDNA locus whereas common bean had more than two 5S rDNA loci and two 45S rDNA loci. Double hybridization experiments with differentially labelled probes indicated that the soybean 45S and 5S rDNA loci are located on different chromosomes and in their distal regions. Likewise, the common bean 45S and 5S rDNA loci were on unique chromosomes, though two of the 5S rDNA loci were on the same chromosome. FISH analysis of interphase nuclei revealed the spatial arrangement of rDNA loci and suggested expression patterns. In both species, we observed one or more 5S rDNA hybridization sites and two 45S rDNA hybridization sites associated with the nucleolar periphery. The 45S rDNA hybridization patterns frequently exhibited gene puffs as de-condensed chromatin strings within the nucleoli. The other condensed rDNA sites (both 5S and 45S) were spatially distant from the nucleolus in nucleoplasmic regions containing heterochromatin. The distribution of rDNA between the nucleoplasm and the nucleoli is consistent with differential gene expression between homologous alleles and among homoeologous loci.     

Identification of molecular markers in soybean comparing RFLP,RAPD and AFLP DNA mapping techniques

Three different DNA mapping techniques—RFLP, RAPD and AFLP—were used on identical soybean germplasm to compare their ability to identify markers in the development of a genetic linkage map. Polymorphisms present in fourteen different soybean cultivars were demonstrated using all three techniques. AFLP, a novel PCR-based technique, was able to identify multiple polymorphic bands in a denaturing gel using 60 of 64 primer pairs tested. AFLP relies on primers designed in part on sequences for endonuclease restriction sites and on three selective nucleotides. The 60 diagnostic primer pairs tested for AFLP analysis each distinguished on average six polymorphic bands. Using specific primers designed for soybean fromEco RI andMse I restriction site sequences and three selective nucleotides, as many as 12 polymorphic bands per primer could be obtained with AFLP techniques. Only 35% of the RAPD reactions identified a polymorphic band using the same soybean cultivars, and in those positive reactions, typically only one or two polymorphic bands per gel were found. Identification of polymorphic bands using RFLP techniques was the most cumbersome, because Southern blotting and probe hybridization were required. Over 50% of the soybean RFLP probes examined failed to distinguish even a single polymorphic band, and the RFLP probes that did distinguish polymorphic bands seldom identified more than one polymorphic band. We conclude that, among the three techniques tested, AFLP is the most useful.     

Genetic characteristics of two genes for resistance to soybean mosaic virus in PI486355 soybean

Soybean [Glycine max (L.) Merr.] PI486355 is resistant to all the identified strains of soybean mosaic virus (SMV) and possesses two independently inherited resistance genes. To characterize the two genes, PI486355 was crossed with the susceptible cultivars Lee 68 and Essex and with cultivars Ogden and Marshall, which are resistant to SMV-G1 but systemically necrotic to SMV-G7. The F₂ populations and F₂₃ progenies from these crosses were inoculated with SMV-G7 in the greenhouse. The two resistance genes were separated in two F₃₄ lines, LR1 and LR2, derived from Essex x PI486355. F₁ individuals from the crosses of LR1 and LR2 with Lee 68, Ogden, and York were tested with SMV-G7 in the greenhouse; the F₂ populations were tested with SMV-G1 and G7. The results revealed that expression of the gene in LR1 is gene-dosage dependent, with the homozygotes conferring resistance but the heterozygotes showing systemic necrosis to SMV-G7. This gene was shown to be an allele of the Rsv1 locus and was designated as Rsv1-s. It is the only allele identified so far at the Rsv1 locus which confers resistance to SMV-G7. Rsv1-s also confers resistance to SMV-G1 through G4, but results in systemic necrosis with SMV-G5 and G6. The gene in LR2 confers resistance to strains SMV-G1 through G7 and exhibits complete dominance. It appears to be epistatic to genes at the Rsv1 locus, inhibiting the expression of the systemic necrosis conditioned by the Rsv1 alleles. SMV-G7 induced a pin-point necrotic reaction on the inoculated primary leaves in LR1 but not in LR2. The unique genetic features of the two resistance genes from PI486355 will facilitate their proper use and identification in breeding and contribute to a better understanding of the interaction of SMV strains with soybean resistance genes.     

用万能引物PCR法从YAC中分离制备荧光原位杂交探针的研究

报道了一种从微量且未知碱基顺序的YAC DNA中制备FISH探针的新方法——万能引物PCR(UP PCR),即把复性后的UP-Linker连接于PFGE分离后经Alu Ⅰ酶切的YAC DNA上,再用UP对其进行PCR扩增和标记。由于Alu Ⅰ的广谱性和UP与Linker长度不同等,使该法能根据PCR效率调节扩增产物的广泛性向特异性的转变,且产物能覆盖YAC嵌入DNA的全长。此法制备的人21号染色体FISH探针,特异性强,杂交信号明亮,21号染色体的检出率高。该法稳定简便。     

Transformation of 12 different plasmids into soybean via particle bombardment

Particle bombardment offers a simple method for the introduction of DNA into plant cells. Multiple DNA fragments may be introduced on a single plasmid or on separate plasmids (co-transformation). To investigate some of the properties and limits of co-transformation, 12 different plasmids were introduced into embryogenic suspension culture tissue of soybean [Glycine max (L.) Merrill] via particle bombardment. The DNAs used for co-transformation included 10 plasmids containing KFLP markers for maize and 2 plasmids separately encoding hygromycin-resistance and ß-glucuronidase. Two weeks following bombardment with the 12 different plasmids, suspension culture tissue was placed under hygromycin selection. Hygromycin-resistant clones were isolated after an additional 5 to 6 weeks. Southern hybridization analysis of 26 hygromycin-resistant embryogenic clones verified the presence of introduced plasmid DNAs. All of the co-transforming plasmids were present in most of the transgenic soybean clones and there was no preferential uptake and integration of any of the plasmids. The copy number of individual plasmids was approximately equal within clones but highly variable between clones. While some clones contained as few as zero to three copies of each plasmid, others clones contained as many as 10 to 15 copies of each of the 12 different plasmids.     

Tissue-specific and developmental regulation of a gene encoding a low molecular weight sulfur-rich protein in soybean seeds

A gene corresponding to a cDNA clone, SE60, encoding a low molecular weight sulfur-rich protein in soybean seeds was isolated from a soybean genomic library and characterized at the nucleotide level. The SE60 gene is interrupted by an intervening sequence of 694 by in size. The 5 flanking region of the gene contained various regulatory sequences such as the RY repeat and CACA elements found in other seed protein genes of legumes. The SE60 gene encoded a pre-protein of 75 amino acids, having a signal sequence of 28 amino acids at the N-terminus. The mature protein of 47 amino acids was basic and cysteine-rich. Northern blot analysis suggested that the SE60 gene is expressed in a tissue-specific and developmentally regulated manner during soybean seed development. The SE60 genes form a small multigene family composed of about four members in the soybean genome.     

Effects of quercetin and enhanced UV-B radiation on the soybean (Glycine max) leaves

The possible ameliorative effects of quercetin on soybean [Glycine max (L.) Merr.] leaves exposed to UV-B radiation were conducted in greenhouse. The symmetrical leaves supplied with quercetin solution (0.2%, 1%) were exposed to UV-B radiation (0, 3.5, 6.5 kJ m⁻² d⁻¹). 0.2% quercetin ameliorated leaf photosynthesis, improved leaf water content (LWC), and decreased lipid oxidation. The unfavorable effect on photosynthetic parameter was displayed in 1% quercetin treatment. The effect of quercetin on phenylalanine ammonia lyase (PAL) activity varied with the quercetin concentration, UV-B radiation intensity and leaf development. In the later development polyphenol oxidase (PPO) activity was increased significantly by quercetin treatments. We suggested that quercetin with suitable concentration could serve as UV-B protective agent partly due to its antioxidant capacity.     

1. Effects of Embedding 2. Experiments with Modifications

Paraffin embedding was found to be satisfactory for brain stained by a modification of the Golgi dichromate-silver method. Nitrocellulose embedding caused fading in a few specimens. Several modifications in which the tissue was impregnated with silver nitrate before treating it with potassium dichromate were investigated. The following one is recommended. Fix pieces of brain 5-6 mm. thick for 2 days in: silver nitrate;0.5%, 90 ml.; formalin, comml. unneutralized (37-40% gas), 10 ml.; pyridine, pure, 0.05-0.1 ml. Mix in the order given and test for pH with brom cresol purple. A pH of 5.5-6.0 is about optimum and the amount of pyridine added can be varied to adjust it. A slight turbidity of the fixing fluid may be disregarded, but precipitation indicates too much alkalinity. Rinse the tissues with distilled water and place them in a mixture of potassium dichromate, 2.5%, 100 ml. and osmic acid, 1%, 1 ml., for 3-5 days. Wash in water, dehydrate with alcohol and embed in soft paraffin for thick sectioning. Greater intensity of staining (but with an increase in precipitate) can be secured by rinsing the blocks after the dichromate treatment and resilvering in a 0.5% solution of silver nitrate for a day or two, then washing, dehydrating and embedding. This modification of the Golgi method was worked out on brain of adult rat, guinea pig, cat and monkey. Results with fetal material were not good. All solutions used were aqueous, and staining was done at room temperature.     

Identification of quantitative trait loci for plant height, lodging, and maturity in a soybean population segregating for growth habit

The use of molecular markers to identify quantitative trait loci (QTLs) has the potential to enhance the efficiency of trait selection in plant breeding. The purpose of the present study was to identify additional QTLs for plant height, lodging, and maturity in a soybean, Glycine max (L.) Merr., population segregating for growth habit. In this study, 153 restriction fragment length polymorphisms (RFLP) and one morphological marker (Dt1) were used to identify QTLs associated with plant height, lodging, and maturity in 111 F₂-derived lines from a cross of PI 97100 and Coker 237. The F₂-derived lines and two parents were grown at Athens, Ga., and Blackville, S.C., in 1994 and evaluated for phenotypic traits. The genetic linkage map of these 143 loci covered about 1600 cM and converged into 23 linkage groups. Eleven markers remained unlinked. Using interval-mapping analysis for linked markers and single-factor analysis of variance (ANOVA), loci were tested for association with phenotypic data taken at each location as well as mean values over the two locations. In the combined analysis over locations, the major locus associated with plant height was identified as Dt1 on linkage group (LG) L. The Dt1 locus was also associated with lodging. This locus explained 67.7% of the total variation for plant height, and 56.4% for lodging. In addition, two QTLs for plant height (K007 on LG H and A516b on LG N) and one QTL for lodging (cr517 on LG J) were identified. For maturity, two independent QTLs were identified in intervals between R051 and N100, and between B032 and CpTI, on LG K. These QTLs explained 31.2% and 26.2% of the total variation for maturity, respectively. The same QTLs were identified for all traits at each location. This consistency of QTLs may be related to a few QTLs with large effects conditioning plant height, lodging, and maturity in this population.     

5S ribosomal gene variation in the soybean and its progenitor

Summary The soybean, Glycine max and its wild progenitor, Glycine soja, have been surveyed for repeat length variation for the nuclearly encoded 5S ribosomal RNA genes. There is little variation among the 33 accessions assayed, with a common repeat length of 345 bases being typical of both taxa. A 334 base size variant was encountered in individuals from two populations of G. soja from China. The low level of variability is in marked contrast to the variation observed within and between the species of the perennial subgenus Glycine.     

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.     

Characterization of a class of small auxin-inducible soybean polyadenylated RNAs

Four new auxin-responsive RNAs from soybean (Glycine max (L.) Merr., var. Wayne) are described. The RNAs were identified by hybridization to three cDNA probes obtained from a library enriched for sequences which increase in abundance within 60 min after 2,4-D (2,4-dichlorophenoxyacetic acid) treatment. These RNAs appear to define a new class of small (i.e. approximately 550 nucleotides) RNAs that respond extremely rapidly to application of exogenous auxin. In excised elongating hypocotyl sections, an increase in the abundance of these RNAs can be detected 2 to 5 min after treatment with 50 M 2,4-D. This response is half maximal after 10 min and reaches steady state in 60 min. RNA blot analysis shows that these RNAs are expressed differentially in various parts of the seedling. The degree of inducibility by auxin is also organ-specific, with the elongating hypocotyl being the most responsive of the organs tested. The RNAs display identical response specificities with one exception. Accumulation of one RNA, designated 10A, is completely abolished by simultaneous addition of cycloheximide and 2,4-D. This RNA also displays a different 2,4-D dose response than other RNAs examined. These results suggest that more than one mechanism is involved in rapid modulation of gene expression by auxin.     

The frequency of polyembryonic seedlings and polyploids from ms1 soybean

Summary Seed from homozygous recessivems ₁ genetic male-sterile soybean (Glycine max (L.) Merr.) plants was studied for frequencies of polyembryonic seedlings and different levels of polyploidy among abnormal seedlings from six different source populations: Amesms ₁ (Ams), North Carolinams ₁ (NCms), Tonicams ₁ (Tms), Urbanams ₁ (Ums), and F₄ generation seed obtained from crosses ofms ₁ to two chromosome interchange lines (Ams x Clark T/T and Ums x KS-172-11-3). Frequencies of polyembryony observed in Tms, Ums, Ams, NCms, F₄ seed from Ams x Clark T/T, and F₄ seed from Ums x KS-172-11-3 were 3.6%, 2.4%, 3.1%, 2.5%, 2.2% and 0.1%, respectively. Frequencies of abnormal seedlings from these six sources varied from 1.7% (Ums X KS-172-11-3) to 16.8% (Ams X Clark T/T). Frequencies of polyploids among the abnormal seedlings ranged from 6.8% in Ums x Ks-172-11-3 to 66.7% in Tms. On average, the frequency of polyploid individuals from monoembryonic seedlings was 1.22%. Chromosome number of these seedlings varied from 20 to 200. Variation of the frequencies of polyembryonic seedlings and polyploid progeny among abnormal seedlings suggested that the mechanism(s) controlling the characters of polyembryony and formation of polyploids was associated with thems ₁ gene and was affected by other gene(s) or environmental factors.Joint contribution: Agricultural Research Service, US Department of Agriculture, and Journal Paper No. J-11255 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA. Project 2471     

Development of a highly efficient, repetitive system of organogenesis in soybean (Glycine max (L.) Merr).

A highly efficient, repetitive system of organogenesis was developed in soybean. Seeds of soybean cv. White hilum pretreated with TDZ formed multiple bud tissue(s) (MBT) at the cotyledonary nodes. MBT initiation occurred only if the axillary buds were not removed from the cotyledonary node. The best MBT formation was achieved by pretreating the seeds for 1 week on medium supplemented with 0.1 mg/l TDZ, followed by culture of the cotyledonary node on medium supplemented with 0.5 mg/l BA for 4 weeks. Culture of the MBT on medium supplemented with 0.1 mg/l TDZ resulted in the proliferation of MBT. MBT was maintained in this way for 12 months. Three hundred thirty six shoots were obtained when 1 g of MBT was subcultured on medium supplemented with 0.5 mg/l BA. Plants were rooted on medium without growth regulators. The regenerated plants grew normally in the greenhouse. Unfortunately, they did not set seeds because of the long-day conditions during growth. This system was successfully applied in three other genotypes.     

Hypomethylated sequences: Characterization of the duplicate soybean genome

Soybean is believed to be a diploidized tetraploid generated from an allotetraploid ancestor. In this study, we used hypomethylated genomic DNA as a source of probes to investigate the genomic structure and methylation patterns of duplicated sequences. Forty-five genomic clones from Phaseolus vulgaris and 664 genomic clones from Glycine max were used to examine the duplicated regions in the soybean genome. Southern analysis of genomic DNA using probes from both sources revealed that greater than 15% of the hypomethylated genomic regions were only present once in the soybean genome. The remaining ca. 85% of the hypomethylated regions comprise duplicated or middle repetitive DNA sequences. If only the ratio of single to duplicate probe patterns is considered, it appears that 25% of the single-copy sequences have been lost. By using a subset of probes that only detected duplicated sequences, we examined the methylation status of the homeologous genomes with the restriction enzymes MspI and HpaII. We found that in all cases both copies of these regions were hypomethylated, although there were examples of low-level methylation. It appears that duplicate sequences are being eliminated in the diploidization process. Our data reveal no evidence that duplicated sequences are being silenced by inactivation correlated with methylation patterns.     

Cotransformation frequencies of foreign genes in soybean cell cultures

Summary Through the use of electroporation and a soybean (Glycine max L.) protoplast system, we generated stably transformed cell lines expressing a number of foreign genes (neomycin phosphotransferase,-glucuronidase, chloramphenicol acetyl transferase, and phosphinothricin acetyl transferase). Selected and unselected marker genes were cointroduced either linked on a single plasmid or as separate plasmids. Calli expressing multiple genes were recovered, and Cotransformation frequencies were established for both cases. Our results show a 50% cotransformation frequency in the case of linked genes. In situations in which two genes are introduced on independent plasmids, cotransformation frequencies are 18%–27%. Similar rates of cotransformation were observed among various marker pairs.