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Outline
Citrus plants are economically and medicinally important crops worldwide. However, due to the highly complex genetic background of the genus, the accurate identification of medicinal species within this genus remains a major challenge. In this study, we employed the analysis of whole-GEnome (AGE), also known as the Shizhen Method, to conduct bioinformatics analysis on 49 genomes from 15 Citrus species, successfully constructing a candidate species-specific target sequence library that covers these species. The number of specific target sequences per species predominantly ranges from 10 000 to 100 000. From this candidate library, species-specific target sequences were selected for the corresponding species. Their high specificity was subsequently verified using Sanger sequencing and CRISPR-Cas12a technology, which confirmed their efficacy in identifying medicinal Citrus species. Based on this foundation, we established an AGE-based identification system for closely related Citrus species, which enabled the precise authentication of 30 commercial samples of Citri Reticulatae Semen and its decoction pieces. This study demonstrates that the AGE can be used for the accurate identification of medicinal Citrus species, which breaks through the bottlenecks of traditional methods and effectively resolves the identification challenges. It thereby provides technical support for quality control of Chinese herbal medicine and clinical medication safety.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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