Identification of a novel locus C2 controlling canary yellow flesh color in watermelons.

The flesh color of watermelon is an important trait that is determined by carotenoid composition and affects consumers' fruit desirability. Although a complete dominant control by C locus (Cllcyb) for canary yellow flesh (CY) over red flesh has been reported, red and CY colors frequently appear as a mixed pattern in the same flesh (incomplete canary yellow, ICY) in F1 and inbred lines carrying dominant C alleles. Therefore, we examined the genetic control of the mixed color pattern in ICY using whole-genome resequencing of three ICY (ICY group) and three CY inbred lines (CY group), as well as genetic linkage mapping of an F2 population. The segregation pattern in 135 F2 plants indicated that CY is controlled by a single locus (named C 2) dominant over ICY. The whole-genome resequencing of ICY and CY inbred lines revealed an ICY/CY-specific region of approximately 27.60-27.88 Mb on Chr. 2 that was polymorphic between the ICY and CY groups. Our genetic map, using nine cleaved amplified polymorphic sequence markers developed based on the single-nucleotide polymorphisms from the ICY/CY-specific region, confirmed that C 2 is located on Chr. 2 and cosegregated with the marker (M7) derived from a non-synonymous single-nucleotide polymorphism of the pentatricopeptide repeat (PPR) gene (ClPPR, Cla97C02G039880). Additionally, 27 watermelon inbred lines of ICY, CY, and red flesh were evaluated using previously reported Cllcyb (C locus)-based markers and our C 2 locus-linked ClPPR-based marker (M7). As a result, dominant alleles at the C 2 locus were required to produce CY, in addition to dominant alleles at the C locus, while a recessive homozygous genotype at the C locus gave the red flesh irrespective of the genotype at the C 2 locus. Using a ClPPR-based cleaved amplified polymorphic sequence developed in this study and Cllcyb-based markers, watermelon cultivars with CY, ICY, and red flesh could be successfully discerned, implying that the combined use of these markers will be efficient for marker-assisted selection of flesh color in watermelon breeding.

First Report of Cercospora citrullina Causing Spot Disease on Watermelon in China.

Watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai], Cucurbitaceae family, is an important vegetable crop. It is believed to be native to Africa and is cultivated in the temperate regions of Africa, central Asia, Americas and the Mediterranean (Chomicki & Renner 2015). China is the largest producer and consumer of watermelon, with an annual production of about 60.25 million tons in 2020 (https://www.fao.org/faostat/en/#home). In May 2022, a new fungal disease was observed on the leaves, vines and fruits of watermelon (cv. Heimeiren, 8424, Qilin) with an incidence of up to 75% in greenhouses, in Gudi Industrial Park, Hanting district, Weifang City, Shandong Province, China. The symptomatic leaves, vines and fruits showed small circular black spots. The disease caused the leaves and vines to desiccate rapidly, and severely affected the fruit quality. Symptomatic leaves, vines and fruits were randomly collected, and isolations were performed from infected tissues. The edges of necrotic lesions were cut into small pieces (about 5 mm), surface sterilized with 2% NaClO for 2 min, followed by 75% ethanol for 30 s, rinsed three times in sterile distilled water and placed in Petri dishes on potato dextrose agar (PDA). The same fungus was isolated from all tissue pieces and formed colonies white fluffy on the surface, and dark gray on the reverse side after 7 days incubation at 25oC. Colonies were subcultured on PDA and Corn Meal Agar (CMA), respectively, and grew slowly (the diameter was approximately 2 cm in 10 days) on PDA showing a white edge, but they grew more rapidly on CMA (approximately 3.5 cm in diameter after 10 days incubation) showing an orange halo. Hyphae were branched, brown and smooth. Conidiophores were fasciculate, brown, straight, unbranched and measured 20.03 to 304.08 × 3.41 to 6.41 µm. Conidia were needle-shaped to clavate, colorless, erect or curved and measured 22.53 to 243.97 × 3.16 to 7.02 µm. According to these morphological characteristics, the fungus was tentatively identified as Cercospora spp. (Chupp 1954). To determine the species of the fungus, three representative isolates, UNL090101, UNL090102 and UNL090103 obtained from symptomatic leaves, vines and fruits, respectively, were characterized. The genomic DNA was extracted to amplify the nuclear ribosomal internal transcribed spacer (ITS) region, translation elongation factor 1-α (TEF-1), histone H3 (HIS), and actin (ACT) genes, using the following primer pairs ITS1/ITS4 (White et al. 1990), EF1-728F/EF1-986R (Carbone & Kohn 1999), CYLH3F/CYLH3R (Crous et al. 2004), ACT-512F/ACT-783R (Carbone & Kohn 1999), respectively. The ITS, EF-1, HIS, and ACT gene sequences were blasted and deposited in GenBank (accession numbers ON849061/OQ102622/OQ102623, ON890306/OQ108278/OQ108281, ON890307/OQ108279/OQ108282 and ON890308/OQ108280/OQ108283, respectively). A phylogenetic tree based on concatenated sequences of ITS-CAT-TEF-H3 from the genus Cerospora was constructed using the maximum likelihood method. Isolates from watermelon and C. citrullina formed a monophyletic group with 100% bootstrap support, which was in accordance with BLAST results. Therefore, the fungus associated to watermelon spot disease was identified as C. citrullina. To fulfill the Koch's postulates, each of the three isolates was artificially inoculated onto watermelon (cv. Qilin) detached expanded leaves, vines and fruits. Three wounds were made with sterilized entomological needles on each leaf, vine and fruit, and each wound was inoculated with 6 mm CMA medium with the fungus, and without fungus as control. All the experiments were conducted for three times. All the inoculated and control leaves were placed in an incubator and incubated at 28oC and 85% relative humidity, with a 12 h photoperiod. Three days after inoculation, the inoculated leaves showed similar symptoms to those observed on naturally infected plants, while the control leaves remained asymptomatic. C. citrullina was re-isolated from symptomatic artificially inoculated leaves and identified by microscopy and re-sequencing, thus fulfilling Koch's postulates. C. citrullina has been reported on several Cucurbitaceae plants worldwide, eg. on watermelon in South Carolina (Rennberger et al. 2019) and on Burcucumber (Sicyos angulatus L.) in Korea (Hong et al. 2014), but, to our knowledge, this is the first report of C. citrullina causing spot disease on watermelon in China.

Development of a Gene-Based Marker Set for Orange-Colored Watermelon Flesh with a High ß-Carotene Content.

The fruit flesh of watermelons differs depending on the distinct carotenoid composition. Orange-colored flesh relates to the accumulation of ß-carotene, which is beneficial to human health. Canary-yellow-fleshed OTO-DAH and orange-ß-fleshed (orange-fleshed with high ß-carotene) NB-DAH near-isogenic lines (NILs) were used to determine the genetic mechanism attributed to orange watermelon flesh. For genetic mapping, an F2 population was developed by crossing the two NILs. The segregation ratio of flesh color in the F2 population indicated that the orange-ß flesh of the NB-DAH NIL was controlled by a single incompletely dominant gene. Through a comparative analysis of the whole-genome sequences of the parent lines and NILs, a major introgression region unique to the NB-DAH NIL was detected on Chr. 1; this was considered a candidate region for harboring genes that distinguish orange from canary-yellow and red flesh. Among the 13 genes involved in the carotenoid metabolic pathway in watermelons, only ClPSY1 (ClCG01G008470), which encodes phytoene synthase 1, was located within the introgression region. The genotyping of F2 plants using a cleaved amplified polymorphic sequence marker developed from a non-synonymous SNP in ClPSY1 revealed its relationship with orange-ß flesh. The insights gained in this study can be applied to marker-assisted breeding for this desirable trait.

Development of DNA markers for Slmlo1.1, a new mutant allele of the powdery mildew resistance gene SlMlo1 in tomato (Solanum lycopersicum).

Reductions in growth and quality due to powdery mildew (PM) disease cause significant economic losses in tomato production. Oidium neolycopersici was identified as the fungal species responsible for tomato PM disease in South Korea in the present study, based on morphological and internal transcribed spacer DNA sequence analyses of PM samples collected from two remote regions (Muju and Miryang). The genes involved in resistance to this pathogen in the tomato accession 'KNU-12' (Solanum lycopersicum var. cerasiforme) were evaluated, and the inheritance of PM resistance in 'KNU-12' was found to be conferred via simple Mendelian inheritance of a mutant allele of the PM susceptibility locus Ol-2 (SlMlo1). Full-length cDNA analysis of this newly identified mutant allele (Slmlo1.1) showed that a 1-bp deletion in its coding region led to a frameshift mutation possibly resulting in SlMlo1 loss-of-function. An alternatively spliced transcript of Slmlo1.1 was observed in the cDNA sequences of 'KNU-12', but its direct influence on PM resistance is unclear. A derived cleaved amplified polymorphic sequence (dCAPS) and a high-resolution melting (HRM) marker were developed based on the 1-bp deletion in Slmlo1.1, and could be used for efficient marker-assisted selection (MAS) using 'KNU-12' as the source for durable and broad-spectrum resistance to PM.