Variation in Key Flavonoid Biosynthetic Enzymes and Phytochemicals in 'Rio Red' Grapefruit (Citrus paradisi Macf.) during Fruit Development.

In the current study, the phytochemical contents and expression of genes involved in flavonoid biosynthesis in Rio Red grapefruit were studied at different developmental and maturity stages for the first time. Grapefruit were harvested in June, August, November, January, and April and analyzed for the levels of carotenoids, vitamin C, limonoids, flavonoids, and furocoumarins by HPLC. In addition, genes encoding for phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), chalcone isomerase (CHI), and 1,2-rhamnosyltransferase (2RT) were isolated, and their expression in grapefruit juice vesicles was studied. Fruit maturity had significant influence on the expression of the genes, with PAL, CHS, and CHI having higher expression in immature fruits (June), whereas 2RT expression was higher in mature fruits (November and January). The levels of flavonoids (except naringin and poncirin), vitamin C, and furocoumarins gradually decreased from June to April. Furthermore, limonin levels sharply decreased in January. Lycopene decreased whereas ß-carotene gradually increased with fruit maturity. Naringin did not exactly follow the pattern of 2RT or of PAL, CHS, and CHI expression, indicating that the four genes may have complementary effects on the level of naringin. Nevertheless, of the marketable fruit stages, early-season grapefruits harvested in November contained more beneficial phytochemicals as compared to mid- and late-season fruits harvested in January and April, respectively.

Identification of a novel chimeric gene, orf725, and its use in development of a molecular marker for distinguishing among three cytoplasm types in onion (Allium cepa L.).

A novel chimeric gene with a 5' end containing the nearly complete sequence of the coxI gene and a 3' end showing homology with chive orfA501 was isolated by genome walking from two cytoplasm types: CMS-S and CMS-T, both of which induce male-sterility in onion (Allium cepa L.). In addition, the normal active and variant inactive coxI genes were also isolated from onions containing the normal and CMS-S cytoplasms, respectively. The chimeric gene, designated as orf725, was nearly undetectable in normal cytoplasm, and the copy number of the normal coxI gene was significantly reduced in CMS-S cytoplasm. RT-PCR results showed that orf725 was not transcribed in normal cytoplasm. Meanwhile, the normal coxI gene, which is essential for normal mitochondrial function, was not expressed in CMS-S cytoplasm. However, both orf725 and coxI were transcribed in CMS-T cytoplasm. The expression of orf725, a putative male-sterility-inducing gene, was not affected by the presence of nuclear restorer-of-fertility gene(s) in male-fertility segregating populations originating from the cross between a male-sterile plant containing either CMS-T or CMS-S and a male-fertile plant whose genotypes of nuclear restorer gene(s) might be heterozygous. The specific stoichiometry of orf725 and coxI in the mtDNA of the three cytoplasm types was consistent among diverse germplasm. Therefore, a molecular marker based on the relative copy numbers of orf725 and coxI was designed for distinguishing among the three cytoplasm types by one simple PCR. The reliability and applicability of the molecular marker was shown by testing diverse onion germplasm.

Marker-assisted genotype analysis of bulb colors in segregating populations of onions (Allium cepa).

Bulb color in onions (Allium cepa) is an important trait whose complex inheritance mechanism involves epistatic interactions among major color-related loci. Recent studies revealed that inactivation of dihydroflavonol 4-reductase (DFR) in the anthocyanin synthesis pathway was responsible for the color differences between yellow and red onions, and two recessive alleles of the anthocyanidin synthase (ANS) gene were responsible for a pink bulb color. Based on mutations in the recessive alleles of these two genes, PCR-based markers for allelic selection were developed. In this study, genotype analysis of onions from segregating populations was carried out using these PCR-based markers. Segregating populations were derived from the cross between yellow and red onions. Five yellow and thirteen pink bulbs from one segregating breeding line were genotyped for the two genes. Four pink bulbs were heterozygous for the DFR gene, which explains the continuous segregation of yellow and pink colors in this line. Most pink onions were homozygous recessive for the ANS gene, except for two heterozygotes. This finding indicated that the homozygous recessive ANS gene was primarily responsible for the pink color in this line. The two pink onions, heterozygous for the ANS gene, were also heterozygous for the DFR gene, which indicated that the pink color was produced by incomplete dominance of a red color gene over that of yellow. One pink line and six other segregating breeding lines were also analyzed. The genotyping results matched perfectly with phenotypic color segregation.