Haejeen Bang

Development of a codominant CAPS marker for allelic selection between canary yellow and red watermelon based on SNP in lycopene β-cyclase (LCYB) gene

Flesh color of watermelon is an agronomically important trait that is predominantly determined by a network of the carotenoid biosynthetic pathway, which also contributes to the nutritional value of the fruit through the health-promoting function of carotenoids. We have identified a key gene, lycopene β-cyclase (LCYB) that may determine canary yellow and red flesh color of watermelon and developed a zero-distance molecular marker that identifies a critical single nucleotide polymorphism (SNP) that distinguishes different alleles of the LCYB gene. Analysis of the flesh color inheritance in segregating populations indicated that a single gene determines the color difference between canary yellow and red flesh in watermelon. The sequence comparison of full-length cDNA of LCYB, which was isolated using degenerate PCR and RACE, identified three SNPs in the coding region of LCYB between canary yellow and red. These SNPs showed perfect co-segregation with flesh color phenotypes. One of the SNPs introduces an amino acid replacement of evolutionarily conserved Phe226 to Val, which may impair the catalytic function of LCYB. This SNP was used to develop a cleaved amplified polymorphic sequence (CAPS) marker, which perfectly cosegregated with flesh color phenotype. Our results strongly suggest that LCYB may be the genetic determinant for canary yellow or red flesh color and our CAPS marker will allow breeders to economically distinguish between canary yellow and red watermelon fruit color at the seedling stage.

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.

Deficit irrigation impact on lycopene, soluble solids, firmness and yield of diploid and triploid watermelon in three distinct environments

Summary Water conservation practices are critical for production and quality of watermelon [Citrullus lanatus (Thunb.) Matsum & Nakai] in southern regions of the USA. The objective of this experiment was to determine the effects of deficit irrigation and environment on lycopene content, total soluble solids, firmness, and yield of diploid and triploid watermelon. Irrigation rates were 1.0 evapotranspiration (ET), 0.75 ET, and 0.5 ET, which were calculated based on climatic parameters applied to the Penman-Monteith equation. Diploid cultivars were ‘Summer Flavor 710’ and ‘Summer Flavor 800’, and triploid cultivars were ‘Summer Sweet 5244’ and ‘Super Seedless 7187’. To investigate if there was a consistent response in cultivars across diverse environments, three studies were conducted in remote Texas locations: Uvalde in the Wintergarden, Weslaco in the Rio Grande Valley, and Lubbock in the High Plains. To isolate the environmental effects due to weather and soil type, all experiments used similar cultural strategies (plant spacing, subsurface drip irrigation, black plastic mulch, and containerized transplants), except for transplanting and harvesting dates. Deficit irrigation reduced total marketable yield by 36% at Uvalde, 30% at Lubbock, and 15% in Weslaco, increasing the yield of small fruits (<5 kg). Location and irrigation rates had major influences on yield of diploid and triploid watermelon. Soluble solids content increased with deficit irrigation at 0.5 ET rate in triploids, but not in diploids. Flesh firmness also increased in triploids compared to diploids. Fruit lycopene content increased with maturity (7 and 22 d after ripening) at all irrigation rates and cultivars. This work across three diverse Texas locations confirms that deficit irrigation directly reduced yield, but does not reduce lycopene development and fruit quality of triploid watermelon.

Construction of high-resolution linkage map of the Ms locus, a restorer-of-fertility gene in onion (Allium cepa L.)

For the purpose of developing closely-linked molecular markers to the Ms locus, a restorer-of-fertility gene in onions (Allium cepa L.), bulked segregant analysis and randomly amplified polymorphic DNA (RAPD) analyses were utilized. Five RAPD markers polymorphic between male-fertile and male-sterile bulks were identified. These RAPD markers were converted into a simple PCR marker or cleaved amplified polymorphic sequence (CAPS) markers after sequencing the RAPD products and obtaining flanking sequences of the RAPD markers by genome walking. A linkage map was constructed with the Ms locus and flanking markers using a F2 population. There was no recombinant between the Ms locus and two CAPS markers, jnurf05 and jnurf17. To increase resolution among these closely linked molecular markers and the Ms locus, a total of 1,346 F2:3 and 2,927 F2:4 plants were analyzed with two flanking markers for detection of recombinants. Segregation of male-fertility phenotypes in large-sized populations confirmed allelic segregation distortion in favor of the recessive Ms allele. Analysis of the recombinants with closely linked markers revealed only two recombinants between the Ms locus and the jnurf05 markers among 4,273 segregating plants, showing very tight linkage between the two loci. However, linkage disequilibrium between the two loci was not too strong among the breeding lines. Despite weak linkage disequilibrium, these tightly linked markers are useful in accurate marker-assisted selection of the Ms alleles and ultimate isolation of the Ms gene by map-based cloning approach.

Variation of Carotenoid, Sugar, and Ascorbic Acid Concentrations in Watermelon Genotypes and Genetic Analysis

The effects of flesh color, season, and test cross on carotenoid, sugar, and ascorbic acid contents of 20 watermelon genotypes with red, pink, orange, and yellow flesh were examined. Red- and pink-fleshed watermelons contained lycopene (4.8–47.8 μg·g−1 fresh weight), β-carotene (< 3.7 μg·g−1), and lutein (< 0.9 μg·g−1) as the major carotenoids. The orange and yellow watermelons contained complex mixtures of prolycopene, lycopene, and β-carotene with levels less than 7.4, 1.5, and 8.5 μg·g−1, respectively. There were also minor carotenoids, such as violaxanthin, lutein, neurosporene, and zeacarotene in the range of 0–1.4 μg·g−1. Total sugar contents ranged from 24 to 91 mg·g−1, with sucrose, glucose, and fructose as the major sugars. The sugar profiles were divided into sucrose-, fructose-, and non-dominant groups. Higher percentages of glucose and fructose were negatively correlated with the total sugar content. Fairly-low levels of ascorbic acid, less than 28.2 μg·g−1, were measured, and some genotypes had nearly no ascorbic acid. There were only slight differences in ascorbic acid, sugar, soluble-solid, and total-carotenoid contents among 11 genotypes harvested in June and November. Five sets of test crosses showed various trends of heritability of carotenoids, sugars, and ascorbic acid in F1 hybrids. In general, carotenoids and total sugar content tended to be inherited from the parent with lower carotenoid and sugar contents, respectively. Paralleled chemical analyses will be beneficial for efficient progress in breeding efforts and genetic research.

Shoot regeneration and ploidy variation in tissue culture of honeydew melon (Cucumis melo L. inodorus)

The Cucumis melo L. inodorus honeydew melon variant is one of the most consumed melons in the USA, and has important commercial and nutritional value. There is a need for improvement of several genetic traits in the US honeydew melon, such as nutrition, drought tolerance, and disease resistance. We investigated the existing regeneration media and optimized the medium composition for an elite honeydew diploid breeding line, “150”, using cotyledonary explants. Four combinations of three different plant growth regulators, 6-benzyladenine, abscisic acid, and indole-3-acetic acid (IAA), were tested in the shoot regeneration media. The presence of IAA in the medium caused the cotyledon explants to curl away from the medium, which made antibiotic selection problematic in our previous study. Omission of IAA from the culture media eliminated this problem and did not impact the shoot regeneration capacity of the cotyledonary explants. We also estimated the ploidy of regenerated plants using flow cytometry, and 50–60% were found to be polyploid (tetraploid or mixoploid). However, contrary to other studies, these polyploid plants did not show major morphological differences compared to the diploid plants.

Watermelon lycopene β-cyclase: promoter characterization leads to the development of a PCR marker for allelic selection

In the carotenoid biosynthetic pathway, lycopene β-cyclase (LCYB) catalyzes the cyclization that converts lycopene into β-carotene. Only a single copy of LCYB was identified and was suggested to encode a chromoplast-specific LCYB (CYCB type) in watermelon [Citrullus lanatus (Thunb.), Matsum & Nakai]. Splicing variants in the 5′-untranslated region were identified, but alternative splicing did not provide an explanation of the regulation of carotenoid accumulation in watermelon flesh. A quantitative assay using real time-PCR showed that differential expression was not detected between red- and canary yellow-fleshed watermelon cultivars. LCYB promoter regions were isolated and characterized, and a sequence difference was identified in the promoter region between red and canary yellow LCYB alleles. This polymorphism did not change the expression of LCYB, but does provide a reliable marker for discriminating LCYB alleles for red and canary yellow flesh. To develop a PCR-based marker to distinguish between the two LCYB alleles, we designed primers flanking the polymorphic region. The newly developed marker, designated Clcyb.600, co-segregated perfectly with flesh color phenotypes and single nucleotide polymorphism (SNP) markers developed in our previous study. Moreover, the Clcyb.600 marker offers easier discrimination of LCYB alleles than SNP or cleaved amplified polymorphic sequence markers, as it does not require restriction enzyme digestion for genotyping. Genotyping of LCYB promoter alleles in various commercial cultivars and plant introductions indicated that watermelon cultivars can be classified into two groups, those carrying a red LCYB allele or a canary yellow LCYB allele.