Development of Pollen Parent Cultivar-Specific SCAR Markers and a Multiplex SCAR-PCR System for Discrimination between Pollen Parent and Seed Parent in Citrus.

This study discusses the challenge of distinguishing between two high-quality mandarin cultivars, 'Asumi' and 'Asuki', which have been introduced and cultivated in Korea after being developed through crossbreeding in Japan. Owing to genetic similarities resulting from crossbreeding between the same parent cultivars, it is challenging to differentiate them morphologically at the seedling stage. This difficulty poses challenges for cultivation and harvesting on farms. To address this issue, we developed a method using sequence characteristic amplification region (SCAR) markers for rapid and accurate differentiation between the two cultivars. We selected specific primer sets from random amplified polymorphic DNA-SCAR combinations and sequence-related amplified polymorphism contrast markers. The multiplex PCR system using these molecular markers was able to identify 16 mandarin cultivars, including 'Asumi' and 'Asuki', among 30 cultivars. The use of these SCAR markers is expected to enhance citrus cultivation by accurately identifying mixed cultivars and facilitating proper harvest timing for citrus distribution. Additionally, the markers can help identify the genetic traits of hybrid varieties at the seedling stage.

Comparative Analysis of Physicochemical Properties and Storability of a New Citrus Variety, Yellowball, and Its Parent.

Although numerous citrus varieties have recently been developed to enhance their quality, information on their quality characteristics is limited. We assessed the quality characteristics of Yellowball, a novel citrus variety, by evaluating its appearance, storability, sensory properties, functionality, and metabolite profiles and then comparing these characteristics with those of its parent varieties, Haruka and Kiyomi. The metabolite profiles between the citrus varieties differed significantly, resulting in distinct physicochemical and functional qualities. The storability of Yellowball was significantly increased compared with that of its parent varieties owing to its strong antifungal activity and unique peel morphology, including the stoma and albedo layers. While we did not investigate the volatile compounds, overall functional activities, and detailed characteristics of each metabolite, our data provide valuable insights into the relationship between citrus metabolites, peel morphology, physicochemical properties, and storability, and demonstrate the potential of Yellowball as a promising variety in the citrus industry.

Anther Culture-Derived Haploids of Citrus aurantium L. (Sour Orange) and Genetic Verification of Haploid-Derived Regenerated Plants.

Citrus plants are important fruit tree species; however, the breeding of high-quality varieties of citrus species is a time-consuming process. Using haploid-derived plants from anther culture may reduce the time required for obtaining purebred lines. This study aimed to genetically verify whether anther culture-derived sour orange (Citrus aurantium L.) plants developed from somatic embryos or haploid tissues. Sour orange anthers were cultured in N6 and MS media to induce calli and somatic embryos. N6 liquid medium supplemented with 1 mg·L-1 gibberellic acid and 200 µM spermidine resulted in a 10% increase in callus and embryo induction rates. Regenerated plants were validated using simple sequence repeat markers. Out of the 109 regenerated plants, ploidy analysis identified 99 diploids, two haploids, and eight putative aneuploids; out of the 99 diploid plants, 33 were haploid-derived homozygous diploids. The chromosomal analysis confirmed most plants as diploids, whereas some were identified as aneuploids (19-21 chromosomes). Furthermore, phylogenetic analysis confirmed that the resultant homozygous or heterozygous plants were haploid-derived. This is the first report of haploid-derived homozygous diploid and aneuploid sour orange plants obtained through anther culture. Moreover, the anther cultivation technique described herein can be applied to other citrus varieties.

Comparative Metabolomics Analysis of Citrus Varieties.

Many citrus varieties are hybridized to improve their quality and to overcome the effects of climate change. However, there is limited information on the effect of the chemical profiles of hybrid varieties on their quality. In this study, we analyzed 10 citrus varieties and evaluated the correlation with their general characteristics and antioxidant activities. Chemical profiles, including the contents of sugars, organic acid compounds, flavonoids, limonoids, and carotenoids, which are related to taste, color, and health benefits, were significantly different depending on the citrus varieties, leading to different antioxidant capacities and general quality parameters. Based on these data, the correlations were investigated, and 10 citrus varieties were clustered into four groups-Changshou kumquat and Jeramon (cluster I); Setoka (cluster II-1); Natsumi, Satsuma mandarin, and Navel orange (cluster II-2); Kanpei, Tamnaneunbong, Saybyeolbong, and Shiranui (cluster II-3). Moreover, a metabolomic pathway was proposed. Although citrus peels were not analyzed and the sensory and functional qualities of the citrus varieties were not investigated in this study, our results are useful to better understand the relationship between citrus quality and metabolite profiles, which can provide basic information for the development and improvement of new citrus varieties.

Complete chloroplast genome sequence of a medicinal landrace citrus Jinkyool (Citrus sunki Hort. ex Tanaka) in Jeju Island, Korea.

Citrus sunki (Jinkyool) is a medicinal landrace citrus belonging to the Rutaceae family. We determined the complete chloroplast genome (160,699 bp) of C. sunki CRS0085 in Jeju Island, Korea. The genome is composed of four distinct parts; a large single copy of 87,918 bp, a small single copy of 21,355 bp, and a pair of inverted repeat regions of 25,713 bp. A total of 134 genes including 89 protein-coding genes, 37 tRNA genes, and eight rRNA genes were identified. The phylogenetic tree showed that C. sunki CRS0085 has the closest relationship with C. reticulata within genus Citrus.

Identification of the 'Haryejosaeng' mandarin cultivar by multiplex PCR-based SNP genotyping.

Most satsuma mandarin (Citrus unshiu Marc.) cultivars are difficult to identify in the seedling stage based only on morphological traits. Therefore, simple polymerase chain reaction (PCR)-based single-nucleotide polymorphism (SNP) markers were developed to specifically and rapidly distinguish the 'Haryejosaeng' cultivar, which is generally supplied to breeders of other satsuma mandarin cultivars. SNP markers were verified using high-resolution melt (HRM)-specific primers. PCR was performed to distinguish 'Haryejosaeng' from eight other satsuma mandarin cultivars using six SNP markers (P1-P6) specific for 'Haryejosaeng', with one negative control SNP primer pair. The best results were obtained using three SNP markers (P1, P2, and P5). In the multiplex PCR, markers P1, P2, and P5 yielded 165-, 150-, and 526-base pair amplicons, respectively, in 'Haryejosaeng', distinguishing it from other satsuma mandarin cultivars. The selected SNP markers were validated by HRM with HRM-specific primers. The multiplex PCR with P1/P5 and P2/P5 also identified 'Haryejosaeng' obtained from a farm growing 17 different cultivars of satsuma mandarin. Specific SNP molecular markers were determined for accurately identifying the 'Haryejosaeng' cultivar by multiplex PCR to save the time and costs associated with its supply to breeders of satsuma mandarin.

Extensive chromosomal polymorphism revealed by ribosomal DNA and satellite DNA loci in 13 Citrus species.

Little is known about the chromosomal variability and polymorphism existing in mitotic chromosomes of Citrus, mainly due to lack of reliable chromosomal markers and small chromosome size. To test the hypothesis of chromosomal polymorphism and provide the foundation of the genome organization in the Citrus cultivars, we have developed molecular cytogenetic markers for 13 Citrus species collected from Jeju island, Korea. In this study, we demonstrated that the chromosomal locations of cytogenetic markers are quite variable and extremely polymorphic, in contrast to the previous studies. The data obtained in this study will be of utmost importance in cytological systematics and karyotyping of the Citrus species.

Correlation between flavonoid content and the NO production inhibitory activity of peel extracts from various citrus fruits.

We investigated the correlation between the flavonoid content and NO production inhibitory activity of fruit peel extracts using 20 citrus plants. The contents of seven flavonoids (naringin, naringenin, hesperidin, hesperetin, rutin, nobiletin, and tangeretin) were determined by HPLC analysis. Each citrus peel extract varied in flavonoid content, but the contents of nobiletin and tangeretin, which were contained in all 20 fruit peels, showed a positive and significant correlation with each other (r=0.879, p<0.0005 for immature fruit peels; r=0.858, p<0.0005 for mature fruit peels). All citrus peel extracts dose-dependently inhibited LPS-induced NO production in RAW 264.7 cells. This inhibitory effect was significantly and positively correlated with the content of nobiletin and tangeretin. Nobiletin showed a more potent NO production inhibitory activity (IC50=26.5 microM) compared to tangeretin (IC50=136.6 microM). This result supports the premise that nobiletin-rich citrus may provide protection against disease resulting from excessive NO production.