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In order to implement reliable marker-assisted selection systems for the restorer-of-fertility locus ( Ms) in onions ( Allium cepa L.), simple PCR-based codominant markers linked to the Ms locus were developed. Based on the EST probe sequences of previously reported RFLP markers, full-length genomic sequences of the gene encoding putative oligopeptide transporter (OPT) was obtained by RACE. The first intron contained two 108 and 439-bp indel polymorphisms between the two Ms allele-linked OPT alleles. A simple PCR marker for OPT was developed by designing a primer pair on the flanking regions of the 108-bp indel which is created by two tandem repeats. The second simple PCR marker was developed from the EST probe encoding photosystem I subunit O (PsaO). Two 14 and 39-bp tandem repeats were identified from the 5′ upstream sequences of the PsaO-coding gene, which were isolated by genome walking. Three different compositions of these tandem repeats were identified from diverse onion germplasm. A primer set binding to the flanking sequence of these polymorphic repeats was used to amplify three different marker haplotypes. The OPT marker was tightly linked to the Ms locus at a distance of 1.5 cM, but the analysis of the linkage relationship showed little linkage disequilibrium between the marker and the Ms locus. Even so, these simple PCR markers are valuable tools for the marker-assisted selection of segregating individuals in onion F hybrid breeding programs. [ABSTRACT FROM AUTHOR]

Onion pungency is commonly measured on absorbency of the wine pink color that results from adding NaOH to the heated mixture of dinitrophenylhydrazine (DNPH) and onion juice. However, significant variation exists among several modifications of the original Schwimmer and Weston (SW) method. We observed differences in pyruvic acid concentrations of 20%-30% between our automated method and a batch method with manual absorbency readings. To determine the source of the differences, we examined the heating time and waiting time of the sample-DNPH mixtures and found no differences. The differences were caused by differential color degradation between the pyruvic acid standards and onion juice samples. These differences could be minimized by reading the absorbency within 1 min of NaOH addition. Using this information, we devised the one-by-one method to control the reading time at 30 s. We compared 5 different analysis methods of 40 onion samples representing 4 onion colors. The automated, high-performance liquid chromatography, and SW methods had similar results, with only about a 5% difference. However, the batch method resulted in differences of approximately 24% as compared to the automated method. The one-by-one method produced very comparable results, within 5%, to the automated method. By modifying the procedure to ensure a uniform and fast reading time, we increased the consistency between the pungency analysis methods. Therefore, fast and uniform absorbency reading time is essential for an accurate measurement of pungency in undiluted onion juice. [ABSTRACT FROM AUTHOR]

Asparagus ( Asparagus officinalis L.) spears are rich in bioactive compounds such as protodioscin, a saponin, and rutin, a flavonoid. Protodioscin and rutin are routinely quantified separately, and an approach permitting simultaneous measurement would significantly improve speed of analysis. We have optimized an extraction procedure and modified a method of high-performance liquid chromatography by coupling to an ultraviolet detector to simultaneously analyze protodioscin and rutin in asparagus extracts. An acidic ethanol solvent was more efficient than methanol, acetonitrile, or water in coextraction of protodioscin and rutin. Protodioscin and rutin were detected at 210 nm, with retention times of 12.6 min and 7.9 min, respectively. The method was validated by high linear correlations between 3.13 and 1000.0 μg/mL for protodioscin ( r= 0.9999), and between 0.3 and 1087.5 μg/mL for rutin ( r= 0.9997). The limit(s) of detection and quantification for protodioscin were 1.6 μg/mL and 3.13 μg/mL, respectively, and for rutin 0.2 μg/mL and 0.3 μg/mL, respectively. White asparagus spears and the crown of the plants were revealed to be rich sources of protodioscin and contained 2.59 to 10.4 mg/g dry weight. Green asparagus spears, particularly the upper portion, were rich in rutin and contained between 1.51 and 7.29 mg/g dry weight. [ABSTRACT FROM AUTHOR]

The formation of pink-red pigments ('pinking') by various amino acids was investigated by reacting amino acids with compounds present in onion juice. The unknown pink-red pigments were generated and separated using high-performance liquid chromatography (HPLC) and a diode array detector (DAD) in the range of 200 to 700 nm. To generate pink-red pigments, we developed several reaction systems using garlic alliinase, purified 1-propenyl-L-cysteine sulfoxide (1-PeCSO), onion thiosulfinate, natural onion juice, and 21 free amino acids. The compound 1-PeCSO was a key compound associated with pinking in the presence of both the alliinase and amino acids. Numerous naturally occurring pink-red pigments were detected and separated from pink onion juice using the HPLC-DAD system at 515 nm. Most free amino acids, with the exceptions of histidine, serine, and cysteine, formed various pink-red pigments when reacted with onion thiosulfinate. This observation indicated that onion pinking is caused not by a single pigment, but by many. Furthermore, more than one color compound could be produced from a single amino acid; this explains, in part, why there were many pink-red compound peaks in the chromatogram of discolored natural onion juice. We presumed that the complexity of the pink-red pigments was due to the involvement of more than 21 natural amino acids as well as several derivatives of the color products produced from each amino acid. We observed that the pinking process in onion juice is very similar to that of the greening process in crushed garlic, emphasizing that both thiosulfinate from flavor precursors and free amino acids are absolutely required for the discoloration. [ABSTRACT FROM AUTHOR]

This study was performed to purify and quantify quercetin glycosides (QG) and aglycone (free) quercetin (Q) in 6 selected onion cultivars and to compare analytical approaches based on high-performance liquid chromatography (HPLC) and spectrophotometry for the quantification of total quercetin (TQ) concentrations. Individual mono- and di-glycoside Q compounds were purified using a semipreparative HPLC and identified by comparing spectral data and by confirming corresponding peaks of QG and Q after incomplete enzyme-hydrolysis. Purified QG were quantified as Q by enzyme-hydrolysis/HPLC. TQ concentrations obtained from 20 onion bulbs with enzyme-hydrolysis/HPLC, no-hydrolysis/HPLC, and a spectrophotometric method without prior hydrolysis were significantly correlated ( r2= 0.99) and were about 15% higher, identical, or 10% less than those concentrations by a standard acid-hydrolysis/HPLC method, respectively. During enzyme-hydrolysis of onion extracts, progressive reduction of the QG and formation of the corresponding mono-glycosides and Q were monitored using an analytical HPLC. TQ ranged from 83 to 330 μg/g F.W. in 6 selected cultivars of long-day or short-day onions. Q3,4′G and Q4′G were the 2 major compounds and comprised approximately between 94% and 97% of TQ in onions. [ABSTRACT FROM AUTHOR]

The color-forming ability of amino acids with thiosulfinate in crushed garlic was investigated. We developed reaction systems for generating pure blue pigments using extracted thiosulfinate from crushed garlic and onion and all 22 amino acids. Each amino acid was reacted with thiosulfinate solution and was then incubated at 60 °C for 3 h to generate pigments. Unknown blue pigments, responsible for discoloration in crushed garlic cloves ( Allium sativum L.), were separated and tentatively characterized using high-performance liquid chromatography (HPLC) and a diode array detector ranging between 200 and 700 nm. Blue pigment solutions exhibited 2 maximal absorbance peaks at 440 nm and 580 nm, corresponding to yellow and blue, respectively, with different retention times. Our findings indicated that green discoloration is created by the combination of yellow and blue pigments. Eight naturally occurring blue pigments were separated from discolored garlic extracts using HPLC at 580 nm. This suggests that garlic discoloration is not caused by only 1 blue pigment, as reported earlier, but by as many as 8 pigments. Overall, free amino acids that formed blue pigment when reacted with thiosulfinate were glycine, arginine, lysine, serine, alanine, aspartic acid, asparagine, glutamic acid, and tyrosine. Arginine, asparagine, and glutamine had spectra that were more similar to naturally greened garlic extract. [ABSTRACT FROM AUTHOR]

Bulb color in onions ( Allium cepa) is an important trait, and homogenous red coloration is desirable in red onion cultivars. The gene encoding anthocyanin synthase (ANS) is required for anthocyanin biosynthesis in onions. We have previously described three different alleles of the ANS gene. Here we report identification of the fourth allele of ANS, ANS-h1, found in a dark red doubled haploid line. ANS-h1 is similar to a non-functional allele found in Brazilian yellow cultivars except that it has several point mutations and indels throughout the promoter and coding regions, none of which are predicted to inactivate enzymatic activity. F2 and backcross populations originating from the crosses between wild-type ( ANS-L) allele-containing red and pink ( ANS-p) allele-containing white or yellow parents show a discrete segregation ratio of 3 red to 1 light pink, indicating that the wild-type allele is completely dominant over the pink allele. In contrast, segregating populations derived from the crosses between ANS-h1 allele-containing red and the same white or yellow parents show a gradient of red intensity from light pink to dark red, suggesting that other genetic factors may affect expression of ANS-h1. A newly developed PCR-based marker and two previously developed markers for allelic selection of the ANS gene were used to examine allele composition in fifty-six breeding lines and commercial cultivars. Most lines are heterogeneous for the ANS gene with two or three alleles detected. The frequency of the pink allele is low in red breeding lines, but it is predominant in white and yellow lines. [ABSTRACT FROM AUTHOR]

Here we demonstrate that fruit from tomato (Lycopersicon esculentum) plants expressing Arabidopsis (Arabidopsis thaliana) H+/cation exchangers (CAX) have more calcium (Ca2+) and prolonged shelf life when compared to controls. Previously, using the prototypical CAX1, it has been demonstrated that, in yeast (Saccharomyces cerevisiae) cells, CAX transporters are activated when the N-terminal autoinhibitory region is deleted, to give an N-terminally truncated CAX (sCAX), or altered through specific manipulations. To continue to understand the diversity of CAX function, we used yeast assays to characterize the putative transport properties of CAX4 and N-terminal variants of CAX4. CAX4 variants can suppress the Ca2+ hypersensitive yeast phenotypes and also appear to be more specific Ca2+ transporters than sCAX1. We then compared the phenotypes of sCAX1- and CAX4-expressing tomato lines. The sCAX1-expressing tomato lines demonstrate increased vacuolar H+/Ca2+ transport, when measured in root tissue, elevated fruit Ca2+ level, and prolonged shelf life but have severe alterations m plant development and morphology, including increased incidence of blossom-end rot. The CAX4-expressing plants demonstrate more modest increases in Ca2+ levels and shelf life but no deleterious effects on plant growth. These findings suggest that CAX expression may fortify plants with Ca2+ and may serve as an alternative to the application of CaCl2 used to extend the shelf life of numerous agriculturally important commodities. However, judicious regulation of CAX transport is required to assure optimal plant growth. [ABSTRACT FROM AUTHOR]

Bulb color in onions ( Allium cepa) is an important trait, but its complex, unclear mechanism of inheritance has been a limiting factor in onion cultivar improvement. The identity of the L locus, which is involved in the color difference between Brazilian yellow and red onions, is revealed in this study. A cross was made between a US-type yellow breeding line and a Brazilian yellow cultivar. The segregation ratio of nine red to seven yellow onions in the F2 population supports the involvement of two complementary genes in anthocyanin production in the F1 hybrids. The high-performance liquid chromatography (HPLC) and reverse-transcriptase (RT)-PCR analysis of the Brazilian yellow onions indicated that the genes are involved late in the anthocyanin synthesis pathway. The genomic sequence of the anthocyanidin synthase (ANS) gene in Brazilian yellow onions showed a point mutation, which results in an amino acid change of a glycine to an arginine at residue 229. Because this residue is located adjacent to a highly conserved iron-binding active site, this mutation is likely responsible for the inactivation of the ANS gene in Brazilian yellow onions. Following the isolation of the promoter sequence of the mutant allele, a PCR-based marker for allelic selection of the ANS gene was designed. This assay is based on an insertion (larger than 3 kb) mutation. The marker perfectly co-segregated with the color phenotypes in the F2 populations, thereby indicating that the L locus encodes ANS. [ABSTRACT FROM AUTHOR]

Bulb color in onions (Allium cepa) is an important trait and is inherited in a complex manner. However, the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that pink bulb color in onions is inherited as a single recessive trait. This trait is attributable to a significantly reduced transcription of the anthocyanidin synthase (ANS) gene. In this study, we developed a PCR-based marker for an allelic selection of theANSgene to avoid the laborious progeny tests traditionally employed. To identify polymorphisms between pink and red alleles of theANSgene, promoter sequences of both alleles were isolated. There was 97% nucleotide sequence identity between the promoter sequences of the two alleles. A 390-bp insertion was identified 632 bp upstream from the putative transcription start site in the pink allele. A pair of primers was designed on the flanking sequences of the inserted region and utilized as a PCR-based marker for allelic selection of theANSgene. The reliability of the marker was tested using parents, F1 hybrids, and F3 lines whose genotypes had been identified by progeny tests. The marker was also used to evaluate the distribution of the pink allele in white and yellow breeding lines. The results indicated that a majority of the breeding lines tested were homozygous recessive. [ABSTRACT FROM AUTHOR]

Bulb color in onions (Allium cepa) is an important trait, but the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that inactivation of the dihydroflavonol 4-reductase (DFR) gene at the transcriptional level resulted in a lack of anthocyanin production in yellow onions. The objectives of the present study were the identification of the critical mutations in the DFR gene (DFR-A) and the development of a PCR-based marker for allelic selection. We report the isolation of two additional DFR homologs (DFR-B and DFR-C). No unique sequences were identified in either DFR homolog, even in the untranslated region (UTR). Both genes shared more than 95% nucleotide sequence identity with the DFR-A gene. To obtain a unique sequence from each gene, we isolated the promoter regions. Sequences of the DFR-A and DFR-B promoters differed completely from one another, except for an approximately 100-bp sequence adjacent to the 5'UTR. It was possible to specifically amplify only the DFR-A gene using primers designed to anneal to the unique promoter region. The sequences of yellow and red DFR-A alleles were the same except for a single base-pair change in the promoter and an approximately 800-bp deletion within the 3' region of the yellow DFR-A allele. This deletion was used to develop a co-dominant PCR-based marker that segregated perfectly with color phenotypes in the F2 population. These results indicate that a deletion mutation in the yellow DFR-A gene results in the lack of anthocyanin production in yellow onions. [ABSTRACT FROM AUTHOR]

Bulb color in onions (Allium cepa) is an important trait and is inherited in a complex manner. However, the mechanism of color inheritance is poorly understood at the molecular level. A previous study showed that pink bulb color in onions is inherited as a single recessive trait. This trait is attributable to a significantly reduced transcription of the anthocyanidin synthase (ANS) gene. In this study, we developed a PCR-based marker for an allelic selection of theANSgene to avoid the laborious progeny tests traditionally employed. To identify polymorphisms between pink and red alleles of theANSgene, promoter sequences of both alleles were isolated. There was 97% nucleotide sequence identity between the promoter sequences of the two alleles. A 390-bp insertion was identified 632 bp upstream from the putative transcription start site in the pink allele. A pair of primers was designed on the flanking sequences of the inserted region and utilized as a PCR-based marker for allelic selection of theANSgene. The reliability of the marker was tested using parents, F1 hybrids, and F3 lines whose genotypes had been identified by progeny tests. The marker was also used to evaluate the distribution of the pink allele in white and yellow breeding lines. The results indicated that a majority of the breeding lines tested were homozygous recessive. [ABSTRACT FROM AUTHOR]