Fatty acid profiling of soybean cotyledons by near-infrared spectroscopy.

Genetically improved soybean grain often contains altered fatty acid profiles. Such alterations can have deleterious effects on seed germination and seedling development, making it necessary to monitor fatty acid profiles in follow-up physiological studies. The objective of this research was to quantify the five fatty acids in soybean (Glycine max) cotyledons using near-infrared (NIR) spectroscopy. Soybean cotyledon samples were dried, ground, and scanned with visible and NIR radiation from 400 to 2500 nm, and reflectance was recorded. Samples were also analyzed by gas chromatography (GC) for palmitic, stearic, oleic, linoleic, and linolenic acids and total oil; GC data, expressed as actual concentration and proportion of total oil, were regressed against spectral data to develop calibration equations. Equation statistics indicated that four of the five fatty acids could be predicted accurately by NIR spectroscopy; the fifth fatty acid could be determined by subtraction. Principal component analysis revealed that most of the spectral variation in this population was due to chlorophyll absorbance in the visible region. Therefore, the spectra were trimmed to include the NIR region only (1100-2500 nm), and a second set of equations was developed. Equations based exclusively on NIR spectra had equal or greater precision than equations based on visible and NIR spectra. Principal component analysis and partial least squares analysis revealed that even after trimming, at least 90% of the spectral variation was unrelated to fatty acid, though variation from fatty acid was identified in the second and third principal components. This research provides an NIR method for complete fatty acid profiling of soybean cotyledons. Equations were achieved with NIR spectra only, so spectrophotometers that analyze both the visible and NIR regions are not needed for this analysis. In addition, equations were possible with a 250 mg sample, which is one-tenth the normal sample size for this analysis.

Genetic divergence between North American ancestral soybean lines and introductions with resistance to soybean cyst nematode revealed by chloroplast haplotype.

Domesticated soybean [Glycine max (L.) Merr.] is a major crop with an established ancestral relationship to wild soybean (Glycine soja Sieb. & Zucc.) native to Asia. Soybean genetic diversity can be assessed at different levels by identification of polymorphic alleles at genetic loci, in either the plastid or nuclear genomes. The objective of this study was to evaluate genetic diversity based on chloroplast haplotypes for soybean genotypes present in the USDA germplasm resource collection. Shared chloroplast haplotypes represent broad groups of genetic relatedness. Previous work categorized three-quarters of the cultivated soybeans from Asia into a single haplotype group. Our results confirmed the close relationship of North American soybean ancestors and G. max plant introductions previously identified as representing potential sources of soybean genetic variation with the finding that these genotypes belonged to a single chloroplast haplotype group. Genetic diversity was identified in soybean genotypes determined to have a high density of single nucleotide polymorphisms and in a screen of accessions with resistance to soybean cyst nematode. Characterization of soybean plant introduction lines into chloroplast haplotype group may be an important initial step in evaluating the appropriate use of particular soybean genotypes.

Molecular and biochemical characterization of a cytokinin oxidase from maize.

It is generally accepted that cytokinin oxidases, which oxidatively remove cytokinin side chains to produce adenine and the corresponding isopentenyl aldehyde, play a major role in regulating cytokinin levels in planta. Partially purified fractions of cytokinin oxidase from various species have been studied for many years, but have yet to clearly reveal the properties of the enzyme or to define its biological significance. Details of the genomic organization of the recently isolated maize (Zea mays) cytokinin oxidase gene (ckx1) and some of its Arabidopsis homologs are now presented. Expression of an intronless ckx1 in Pichia pastoris allowed production of large amounts of recombinant cytokinin oxidase and facilitated detailed kinetic and cofactor analysis and comparison with the native enzyme. The enzyme is a flavoprotein containing covalently bound flavin adenine dinucleotide, but no detectable heavy metals. Expression of the oxidase in maize tissues is described.

Isolation of a gene encoding a glycosylated cytokinin oxidase from maize.

The major cytokinin oxidase in immature maize kernels was purified to homogeneity. Selected tryptic peptides were used to design degenerate oligonucleotide primers for PCR isolation of a fragment of the oxidase gene. Hybridization of the PCR fragment to a maize genomic library allowed isolation of a full-length cytokinin oxidase gene (ckx1). The gene encodes a protein of approximately 57 kDa that possesses a signal peptide, eight consensus N-glycosylation sequences and a consensus FAD binding sequence. Expression of ckx1 in Pichia caused secretion of active glycosylated cytokinin oxidase that contains a substrate-reducible FAD. The gene displays sequence homology with a putative oxidoreductase from Arabidopsis thaliana and with the fas5 gene from Rhodococcus fascians.

Replication timing properties across the pseudoautosomal region boundary and cytogenetic band boundaries on human distal Xp.

The establishment of human chromosomal regions as distinct and characteristic domains has been demonstrated by the reproducible banding patterns observed on metaphase chromosomes as a result of various staining techniques. Although the exact molecular properties responsible for the patterns are not well understood, a general correlation has been established between the time of replication of a particular region of DNA and its banding characteristics. Using a replication timing assay based on fluorescence in situ hybridization patterns, we investigated replication timing properties across chromosomal regions with potentially distinct chromatin properties. Relative replication timing values were determined using cosmid DNA probes around the pseudoautosomal region boundary in Xp22.3 and the cytogenetic band boundary regions surrounding Xp22.2. Although we observed replication timing domains that were generally consistent with cytogenetic banding patterns, we did not find sharp replication timing boundaries at either the pseudoautosomal region boundary or at the cytogenetic band boundaries.

Genome rearrangements by residual IS10 elements in strains of Escherichia coli K-12 which had undergone Tn10 mutagenesis and fusaric acid selection.

Mutant strains selected as survivors of the lethal overexpression of a plasmid-encoded bovine somatotropin-beta-galactosidase fusion protein were found to include instances where an IS10 element had transposed from the chromosome into the fusion protein structural gene on the plasmid. Two distinct types of IS10 elements were found in these mutants, the well-known IS10R and a novel hybrid element composed of portions of both IS10R and IS10L. The strain in which the selection scheme was carried out had been constructed in a series of steps, including alteration of two loci by Tn10-mediated intramolecular transposition involving fusaric acid (FA) selection for loss of tetracycline resistance. Genetic dissection of this strain revealed that one of these altered loci was an origin for both types of IS10 elements, while the other locus was an origin for only IS10R elements. The finding that residual IS10 elements, left after FA selection for Tcs derivatives of Tn10-containing strains, can be a significant source of spontaneous mutation should be of interest to workers using strains that have been 'cured' of Tn10 in this way.