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  • Author or Editor: John McCallum x
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Genetic and environmental factors affect onion (Allium cepa L.) pungency but the molecular basis for this variation is not understood. To initiate molecular analysis of onion sulfur metabolism we isolated cDNAs from onion associated with sulfur assimilation and compared gene expression and sulfur metabolism of mild and pungent onion cultivars. We isolated cDNAs encoding homologues of 5'adenosine-phosphosulfate (APS) reductase, γ-glutamylcysteine synthetase and serine acetyl transferase using a homology-based RT-PCR approach. Homologues of high-affinity sulfate transporters and sulfite reductase were isolated from an onion root differential cDNA library enriched for genes up-regulated by 48 hours sulfur deprivation. The influence of genotype and sulfur nutrition on root expression of selected genes was measured in an experiment in which a low pungency onion cultivar (`Houston Grano') and a high pungency cultivar (`Canterbury Longkeeper') were grown hydroponically in low (0.1 meq·L-1) or high (4.0 meq·L-1) sulfate medium and harvested before bulbing. `Canterbury Longkeeper' contained higher concentrations of (+)-S-methyl-L-cysteine sulfoxide in leaf and root than `Houston Grano' but cultivars did not differ in leaf trans-(+)-S-(1-propenyl)-L-cysteine sulfoxide concentrations. `Houston Grano' accumulated significantly higher concentrations of total N, nitrate, and basic amino acids in leaves and roots, suggesting these cultivars differ markedly in maintenance of S/N homeostasis. Steady-state transcript levels of APS reductase and high-affinity sulfate transporter in roots were significantly higher (2- to 3-fold) at low sulfate. By contrast, steady state levels of ATP sulfurylase transcript were significantly higher at high sulfate levels and in `Canterbury Longkeeper'. We conclude that differences in regulation of the sulfur assimilation pathway may underlie genetic differences in pungency.

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The commercial production of onion (Allium cepa L.) inbreds, hybrids, and open-pollinated (OP) cultivars would benefit from a robust set of molecular markers that confidently distinguish among elite germplasms. Large-scale DNA sequencing has revealed that single nucleotide polymorphisms (SNPs), short insertion-deletion (indel) events, and simple sequence repeats (SSRs) are relatively abundant classes of codominant DNA markers. We identified 398 SNPs, indels, and SSRs among 35 elite onion ulations and observed that all populations could be distinguished. Phylogenetic analyses of simple-matching and Jaccard's coefficients for SSRs produced essentially identical trees and relationships were consistent with known pedigrees and previous marker evaluations. The SSRs revealed that elite germplasms from specific companies or breeding programs were often closely related. In contrast, phylogenetic analyses of SNPs and indels did not reveal clear relationships among elite onion populations and there was no agreement among trees generated using SNPs and indels vs. SSRs. This discrepancy was likely due to SNPs and indels occurring among amplicons from duplicated regions (paralogs) of the onion genome. Nevertheless, these PCR-based markers will be useful in the quality control of inbred, hybrid, and OP onion seed lots.

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Cytoplasmic-genic male sterility (CMS) is used to produce hybrid onion (Allium cepa L.) seed. For the most widely used source of onion CMS, male sterility is conditioned by the interaction of the male-sterile (S) cytoplasm and the homozygous recessive genotype at a nuclear male-fertility restoration locus (Ms). Maintainer lines are used to seed propagate male-sterile lines, possess normal (N) male-fertile cytoplasm, and are homozyous recessive at the Ms locus. Due to the biennial nature of onion, it takes 4 to 8 years of crossing and scoring of progeny phenotypes to establish if maintainer lines can be extracted from an uncharacterized population or family. Identification of nuclear markers tightly linked to the Ms locus would allow for molecular-facilitated selection of maintainer lines. We evaluated testcross progenies from a segregating family for nuclear restoration of male fertility over at least three environments. Although segregations in the F2 family fit the expected 1:2:1 ratio (P = 0.973), the proportion of male-sterile testcross progenies showed significant (P < 0.01) year effects and it is therefore imperative to score male-fertility restoration over environments. Too many male-sterile testcross progenies were often observed, indicating that the dominant allele conditioning male-fertility restoration for S cytoplasm may not show complete penetrance. Segregations of amplified fragment length polymorphisms and restriction fragment length polymorphisms (RFLPs) revealed RFLPs flanking the Ms locus at 0.9 and 8.6 cM. An onion cDNA showing highly significant homology to the aldehyde dehydrogenase conditioned by the rf2 locus of maize was identified and mapped to linkage group I, independent of the Ms locus. A sample of commercial onion germplasm was evaluated for putative allelic diversity at the RFLP loci linked to Ms. The genomic region corresponding to the cDNA (AOB272) revealing the closest RFLP to Ms was sequenced to reveal numerous single nucleotide polymorphisms. Single-stranded conformational polymorphisms and single nucleotide extensions were developed that revealed genomic variation at AOB272-EcoRI. The use of these molecular markers to select maintainer lines in onion is discussed.

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Developmental, environmental, and genetic factors affecting seed color were studied in the progeny of a cross between two white-flowered (aa) green cotyledon (ii) field peas (Pisum sativum L.): the pale large-seeded Marrowfat cultivar Primo and the greener small-seeded Prussian Blue OSU442-15. Changes in chlorophyll and carotenoid content during seed development of the parental genotypes were determined by high performance liquid chromatography analysis. Both cultivars accumulated similar pigment quantities per seed, but pigment loss was greater during maturation of `Primo'. Bleached and unbleached mature seed tissues also were compared for pigment composition. Lutein was the predominant pigment in bleached cotyledons of both cultivars. Only trace amounts of pheophytins were detected in unbleached seed. In both genotypes, chlorophyll A : B ratios were ≈1:1 in seed coats compared to 3:1 in cotyledons. Objective measurements of seed color in terms of luminance (lightness) and chrominance (hue and saturation) were made in YUV color space by video image analysis. Inheritance of seed color was studied in an F2 population derived from the `Primo' × `OSU442-15' cross and inbred descendants. Quantitative trait loci (QTL) for seed color were localized by interval mapping using a linkage map of 199 molecular markers spanning most of the genome and by bulked segregant analysis and selective genotyping. Four genomic regions affecting seed color were detected. A major gene accounting for 61% of the phenotypic variance in seed lightness (Y luminance component) was identified on linkage group V linked to r locus. Another major gene, which accounted for 56% of the phenotypic variance in seed hue (U chrominance component), was mapped to a linkage group containing group III and IV markers. A QTL with smaller effect on seed hue (U and V chrominance components) was detected on linkage group VII. Support for overdominant allelic interaction for a QTL on linkage group I, adjacent to the legumin locus Lg-J, was obtained by selective genotyping of the seed lightness distributional extremes.

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Bulb onion (Allium cepa L.) is a globally significant crop, but the structure of genetic variation within and among populations is poorly understood. We broadly surveyed genetic variation in a cultivated onion germplasm using simple sequence repeat (SSR) markers and sequenced regions flanking expressed sequence tag (EST)-SSRs to develop single-nucleotide polymorphism (SNP) markers. Samples from 89 inbred and open-pollinated (OP) bulb onion populations of wide geographical adaptation and four related Allium L. accessions were genotyped with 56 EST-SSR and four genomic SSR markers. Multivariate analysis of genetic distances among populations resolved long-day, short-day, and Indian populations. EST-SSR markers frequently revealed two major alleles at high frequency in OP populations. The median proportion of single-locus polymorphic loci was 0.70 in OP and landrace populations compared with 0.43 in inbred lines. Resequencing of 24 marker amplicons revealed additional SNPs in 17 (68%) and five SNP assays were developed from these, suggesting that resequencing of EST markers can readily provide SNP markers for purity testing of inbreds and other applications in Allium genetics.

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Bulb onion (Allium cepa L.) is a challenging subject for experimental studies because of its slow growth, genetic heterogeneity, and sensitivity to environmental and biotic stresses. Sharing of common germplasm and controlled propagation practices has underpinned research on model plants, such as Arabidopsis and tomato, but not in onion. To encourage wider evaluation of onion for physiological and molecular studies in controlled environments, we describe the growing practices we have developed over two decades of research on adaptive and nutrient assimilation traits. Key aspects covered include choice of germplasm, propagation media, nutrition, and environmental control. Adopting common onion genetics and cultivation techniques across laboratories will allow researchers to answer deeper research questions and increase the reproducibility of the research.

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