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.

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.

Development of Molecular Marker to Detect Citrus Melanose Caused by Diaporthe citri from Citrus Melanose-like Symptoms.

It is difficult to distinguish melanose and melanoses-like symptoms with the naked eye because they appear similar. To accurately detect melanose symptoms caused by Diaporthe citri from melanose-like symptoms, we developed PCR-based specific primers Dcitri by aligning the internal transcribed spacer (ITS) region of D. citri with the ITS of Diaporthe cytosporella, Diaporthe foeniculina, Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria citri, and Fusarium oxysporum found on citrus peel. PCR results showed that the specific product was amplified in D. citri but not in other isolates including, C. gloeosporioides, B. cinerea, A. citri, F. oxysporum. In addition, specific products were observed in melanose symptoms caused by D. citri but not in melanose-like symptoms, such as copper-injury, sunscald, damages by yellow tea thrips, and pink citrus rust mite. Using the Dcitri primers developed in this study, it is expected that melanose caused by D. citri could be accurately distinguished from melanose-like symptoms.

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.