Japanese Diagnostic Method Targets Breeding Rights Violations

Josh McGillBreeding, International

Citrus is the third most important agricultural product in Japan. The success of the Japanese citrus industry is attributed to the development of new cultivars that are free of pests and diseases, climate-resilient and exhibit superior fruit quality.

To develop this system, first, genomic DNA was extracted from plant tissues using a commercially available kit. Specific DNA markers for each target cultivar were amplified with PCR-positive DNA markers using multiplex PCR. The PCR products were detected within 15 minutes on the C-PAS membrane stick. (Image by Yuki Monden, Okayama University)

There has, however, been a growing concern over the infringement and violation of breeding rights of citrus cultivars developed in Japan. This issue stems from the unauthorized export and cultivation of those cultivars overseas, followed by the importation of infringing fruit back into the country. This practice results in a loss of business in overseas markets, infringing the rights and severely jeopardizing the livelihoods of Japanese farmers.

To prevent overseas outflow of registered cultivars and to safeguard the rights of citrus breeders, the Japanese government in 2021 revised the Plant Variety Protection and Seed Act.

Additionally, researchers developed cultivar-specific DNA identification systems such as cleaved amplified polymorphic site (CAPS) markers and TaqMan-MGB single-nucleotide polymorphism genotyping assays, which could discriminate the target registered cultivar from others. However, these identification methods are time consuming. There is a growing need for faster and more user-friendly systems that can effectively identify Japanese cultivars, particularly at on-field inspection sites, so that the import of counterfeit fruit is curbed.

To this end, a group of Japanese researchers has developed a new DNA marker-based cultivar identification system for eight prominent Japanese citrus cultivars. The group is led by Yuki Monden from Okayama University and Mitsutoshi Okamoto of Ehime Research Institute of Citrus Fruits. Also in the research group are Takehiko Shimada of the National Agriculture and Food Research Organization, and Kazuto Takasaki and Tomoyuki Takeuchi of FASMAC Co. Ltd. A paper detailing their study was published in Breeding Science in June.

“This system accurately identifies citrus cultivars by detecting DNA polymorphisms — fragments of DNA that are unique to each of the target cultivars,” Monden said. The study employed genetic sequencing techniques to identify three retrotransposon families that are transcriptionally active in citrus plants. Retrotransposons are genetic elements that exhibit high copy numbers which can be used to detect cultivar-specific DNA polymorphisms. By analyzing their insertion sites through sequencing, unique DNA markers for specific cultivars were identified. These cultivar-identifying DNA markers were successfully detected within 15 minutes using a small membrane stick, the chromatographic printed array strip.

This straightforward process can be accomplished within one hour of DNA extraction and can clearly determine whether the tested fruit sample belongs to a registered cultivar. The system offers significant advantages as a convenient, rapid and cost-effective DNA diagnostic method to inspect fruit imports. The proposed target cultivar-specific identification system is expected to function as an efficient tool for promptly identifying and acting against suspicious, unlicensed cultivars.

Source: Okayama University