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Citrus is one of the most cultivated fruits in the world, and satsuma mandarin (Citrus unshiu Marc.) is a major cultivated citrus in Japan. Many excellent cultivars derived from satsuma mandarin have been released through the improvement of mandarins using a conventional breeding method. The citrus breeding program is a lengthy process owing to the long juvenility, and it is predicted that marker-assisted selection (MAS) will overcome the obstacle and improve the efficiency of conventional breeding methods. To promote citrus molecular breeding in Japan, a genetic mapping was initiated in 1987, and the experimental tools and resources necessary for citrus functional genomics have been developed in relation to the physiological analysis of satsuma mandarin. In this paper, we review the progress of citrus breeding and genome researches in Japan and report the studies on genetic mapping, expression sequence tag cataloguing, and molecular characterization of breeding characteristics, mainly in terms of the metabolism of bio-functional substances as well as factors relating to, for example, fruit quality, disease resistance, polyembryony, and flowering. [ABSTRACT FROM AUTHOR]

Citrus is a major cultivated crop in Japan, and new cultivars are of great interest in the Japanese and global market. Recently, the infringement of breeders' rights to citrus cultivars bred in Japan has become a problem related to the agricultural product export strategy promoted by the Japanese government. Cultivar identification systems using DNA markers are an effective tool for protecting breeders' rights. Here, a novel target cultivar-specific identification system using the chromatographic printed array strip method was developed for eight prominent Japanese citrus cultivars. A polymorphic InDel fragment specific to each cultivar was explored through the screening of published citrus InDel markers and next-generation sequencing of retrotransposon libraries. The cultivar-specific DNA marker set for each cultivar comprised 1-3 polymorphic InDel fragments in combination with a PCR-positive DNA marker for the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene. The DNA markers were detected within 3 hours from DNA extraction to the detection by the C-PAS4 membrane stick following multiplex PCR. The developed system is superior as a convenient, rapid, and cost-effective DNA diagnostic method during inspection. The proposed target cultivarspecific identification system is expected to serve as an efficient tool for the injunction of suspicious registered cultivars, contributing to the protection of breeders' rights. [ABSTRACT FROM AUTHOR]

A citrus cultivar identification system using CAPS marker has been developed on nursery trees, but this needs to be extended to include various product types, such as imported fruits and processed products. Here, we developed a new cultivar identification system using TaqMan-MGB SNP genotyping assay. Eight probe and primer sets were designed to amplify PCR fragments <100 bp to enable the genotyping of fresh and processed fruits in which predicted that insufficient quantities of DNA and residual impurities in the DNA extracts. The TaqMan-MGB SNP genotyping assay was stable and reproducible, and were confirmed to apply various sample sources, including leaves, fresh fruit, juice, canned fruit, and dry fruit. They could provide at least a single differentiating SNP to discriminate any paired combination among 48 citrus cultivars. Minimal marker subsets to identify the target cultivar were listed for each of 18 registered cultivars with valid patent. The allelic SNP genotypes of 48 citrus cultivars, which cover more than 98% of all citrus fruit shipment produced in Japan, is valuable for the referencing information in the DNA-based identification for fresh and processed fruits. This identification system will help protect registered cultivars and facilitate food fraud inspections. [ABSTRACT FROM AUTHOR]

Citrus species are some of the most valuable and widely consumed fruits globally. The genome sequences of representative citrus (e.g., Citrus clementina, C. sinensis, C. grandis) species have been released but the research base for mandarin molecular breeding is still poor. We assembled the genomes of Citrus unshiu and Poncirus trifoliata, two important species for citrus industry in Japan, using hybrid de novo assembly of Illumina and PacBio sequence data, and developed the Mikan Genome Database (MiGD). The assembled genome sizes of C. unshiu and P. trifoliata are 346 and 292 Mb, respectively, similar to those of citrus species in public databases; they are predicted to possess 41,489 and 34,333 protein-coding genes in their draft genome sequences, with 9,642 and 8,377 specific genes when compared to C. clementina, respectively. MiGD is an integrated database of genome annotation, genetic diversity, and Cleaved Amplified Polymorphic Sequence (CAPS) marker information, with these contents being mutually linked by genes. MiGD facilitates access to genome sequences of interest from previously reported linkage maps through CAPS markers and obtains polymorphism information through the multiple genome browser TASUKE. The genomic resources in MiGD (https://mikan.dna.affrc.go.jp) could provide valuable information for mandarin molecular breeding in Japan. [ABSTRACT FROM AUTHOR]

Phytoene synthase (PSY) is one of the key regulatory enzyme on the biosynthesis and accumulation of carotenoid in citrus fruits. The transcriptional diversity of PSY is mainly attributed to the structural variation in promoter region among PSY alleles. In aim to clarify how this transcriptional diversity is regulated among them, PSY alleles responsible for carotenoid biosynthesis in the fruits are characterized and their promoter sequences were compared. Based on gene structure and expression pattern of PSY homologues on the clementine mandarin genome sequence, PSY alleles responsible for carotenoid biosynthesis are derived from a single locus in the scaffold 6. AG mapping population possessed four PSY alleles derived from parent lines of A255 and G434, and their F1 individuals with PSY-g2 allele tended to have low transcription level. From sequence comparison of their promoter regions, the czs-motif alternation from MYBPZM to RAV1AAT might be a candidate to influence the transcription level. Among the ancestral pedigree varieties of AG mapping population, the transcription level of PSY correlated with genotypes of MYBPZM and RAVI AAT motifs in the promoter region of PSY alleles, so that homozygous genotype of MYBPZM showed higher transcription level while heterozygous genotype of MYBPZM and RAVI AAT showed lower transcription level. [ABSTRACT FROM AUTHOR]

Satsuma mandarins (Citrus unshiu Marc.) are the predominant cultivated citrus variety in Japan. Clarification of its origin would prove valuable for citrus taxonomy and mandarin breeding programs; however, current information is limited. We applied genome-wide genotyping using a 384 citrus single nucleotide polymorphism (SNP) array and MARCO computer software to investigate the satsuma mandarin parentage. Genotyping data from 206 validated SNPs were obtained to evaluate 67 citrus varieties and lines. A total of five parent-offspring relationships were newly found by MARCO based on the 206 SNP genotypes, indicating that 'Kishuu mikan' type mandarins (Citrus kinokuni hort. ex Tanaka accession 'Kishuu mikan' and 'Nanfengmiju') and 'Kunenbo' type mandarins (Citrus nobilis Lour. var. kunip Tanaka accession 'Kunenbo' and 'Bendiguangju') are possible parents of the satsuma mandarin. Moreover, cleaved amplified polymorphic sequences analysis showed that the genotypes of four regions in chloroplast DNA of 'Kishuu mikan' type mandarins were identical to that of the satsuma mandarin. Considering the historical background, satsuma mandarins may therefore derive from an occasional cross between a 'Kishuu mikan' type mandarin seed parent (derivative or synonym of 'Nanfengmiju') and a 'Kunenbo' type mandarin pollen parent (derivative or synonym of 'Bendiguangju'). [ABSTRACT FROM AUTHOR]