We sought to determine the genetic basis of expression of the ubiquitous (metabolic) urease of soybean. This isozyme is termed the metabolic urease because its loss, in eu4/eu4 mutants, leads to accumulation of urea, whereas loss of the embryo-specific urease isozyme does not. The eu4 lesion eliminated the expression of the ubiquitous urease in vegetative and embryonic tissues. RFLP analysis placed urease clone LC4 near, or within, the Eu4 locus. Sequence comparison of urease proteins (ubiquitous and embryo-specific) and clones (LC4 and LS1) indicated that LC4 and LS1 encode ubiquitous and embryo-specific ureases, respectively. That LC4 is transcribed into poly(A)+ RNA in all tissues was indicated by the amplification of its transcript by an LC4-specific PCR primer. (The LS1-specific primer, on the other hand, amplified poly(A)+ RNA only from developing embryos expressing the embryo-specific urease.) These observations are consistent with Eu4 being the ubiquitous urease structural gene contained in the LC4 clone. In agreement with this notion, the mutant phenotype of eu4/eu4 callus was partially corrected by the LC4 urease gene introduced by particle bombardment.
Surveys of variability at loci that condition simply inherited, phenotypically neutral characters can be used to investigate relationships among accessions in germplasm collections. We performed a survey of isozyme variability among 1005 domesticated soybean [Glycine max (L.) Merr.] and 258 wild soybean [G. soja (L.) Siebold & Zucc.] accessions from the USDA soybean germplasm collection. By using eight activity stains, we detected polymorphisms conditioned by 13 isozyme loci. The average gene diversity of the Asian G. max accessions in this study was 0.198, while that of the G. soja accessions was 0.235. The relatively high variability of this set of loci made it possible to investigate relationships among groups of accessions from six diverse geographic regions in Asia: China, India and South‐Central Asia, Japan, Korea, Manchuria‐Siberia, and Southeast Asia. The G. max and G. soja accessions fell into two separate groups, based both on genetic distance and canonical discriminant analysis. Publicly developed soybean cultivars released for production in North America clustered with the G. max accessions from northeast Asia. The group of accessions from India and South‐Central Asia was the G. max group most closely related to the G. soja accessions and may include some primitive agronomic types. The group of G. max accessions from Southeast Asia stood out from the other G. max groups. This difference was due to high frequencies of the Aco3‐b, Dial, and Enp‐a alleles among accessions in Maturity Groups VII through X. The accessions in the later maturity groups may represent a distinct population, relative to the accessions in the earlier maturity groups.
Horizontal starch gel electrophoresis, followed by enzyme-specific staining, allows us to separate and visualize several soybean [Glycine max [L.] Merr.] isozymes. Using extracts prepared from the cotyledons of seeds germinated 3 to 4 days, we detect five bands (zones) of aconitase [aconitate hydratase, enzyme commission (EC) 4.2.1.3] activity and one band of endopeptidase activity. We have observed mobility variants of all of these isozymes. An understanding of the genetic basis of the variants would facilitate their use as markers in soybean genetics or breeding studies. One objective of this research was to investigate the inheritance of mobility variants for four of the five aconitase isozymes, as well as a mobility variant for the endopeptidase band. Segregation data (either individual F2 zymotypes or F2 genotypes determined by progeny testing) were collected on F2 populations derived from Fj individuals whose parents had different zymotypes. Mobility variants of the four aconitase isozymes are conditioned by four independent loci with codominant alleles. The aconitase loci and alleles are: Acol (a, b), Aco2 (a, b), Aco3 (a, and Aco4 (a, b, c). The endopeptidase mobility variants are conditioned by the codominant alleles Enp-a and Enp-b. A second objective was to determine if these five isozyme loci are linked to one another, or to other known loci. Data were collected on F2 populations segregating for several loci conditioning isozyme and morpholigical traits. Deviations from independent assortment were determined by ×2 analysis. Locus Aco3 is linked to the Spl locus; the frequency of recombination varied from 4.6 _+ 0.9% in the cross 'A1937')< P1342662A (G. soja Sieb. et Zucc.) to 30.6 + 3.0% in the cross PI 437728 (G. max) × 'Evans'. Locus Aco3 also is linked to the T locus, with a recombination frequency of 28.8 _+ 4.3%, as determined in the cross 'Amsoy 71' × PI 342622A. Both Spl and T are on linkage group 1. The present data allow us to assign Aco3 to linkage group 1, but do not allow us to unambiguously establish its position relative to other markers. The other four loci also were tested for linkage with several loci; Acol (11 loci), Aco2 (10 loci), .4co4 (13 loci) and Enp (8 loci) segregated independent of all loci tested. These five loci conditioning isozyme mobility variants can be useful tools in soybean genetic research.
Shoot regeneration from seed-derived callus cultures of Kentucky bluegrass (Poa pratensis L.) was tested on MS basal medium supplemented with four different growth regulators. Regeneration frequencies for medium supplemented with 10 μM 2,4-dichlorophenoxyacetic acid (2,4-D), 60 μM 4amino-3, 5,6-picolinic acid (picloram), or 30 μM 3,6dichloro-o-anisic acid (dicamba) ranged from 0.4 to 4%. Medium supplemented with 30 μM dicamba plus 10 μM 6-benzylaminopurine (BA) resulted in regeneration of shoots from 20% of the calli tested. Higher rates of growth regulators (60 or 90 μM dicamba, 20 μM BA) resulted in regeneration of shoots from 45% of calli of the cultivar 'Baron'. In a subsequent study, the response of 12 North American cultivars grown on these media was cultivar-specific, with mean frequencies of regeneration ranging from 4% to 40%.
The soybean (Glycine max [L.] Merr.) contains two urease isozymes whose expression is regulated in a tissue-specific and temporal manner. The ubiquitous urease is expressed in all tissues examined (leaf, embryo, seed coat, cell culture); the embryo-specific urease is synthesized exclusively in the developing embryo. The embryo-specific urease accumulates during seed development while the ubiquitous urease is found in highest levels during early development of both leaves and seeds. We have isolated mutants which fall in three phenotypic classes lacking one or both urease isozyme activities. Genetic analysis has thus far identified three unlinked loci which control the expression of urease(s). Genomic and cDNA clones of urease structural genes have also been recovered and we are working to assign these to genetic loci by sequence and RFLP analyses. That the ubiquitous urease isozyme is expressed in cell culture makes it possible to include cell culture in physiological and developmental studies. Additionally, we have developed direct selections for ureasenegative mutants, and their revertants, in cell culture. These selections will facilitate the study of the expression of cloned urease genes in genetically transformed tissue.
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