In total, 35 batches of Juhe samples were collected in this study, including 12 TMV, C. ‘Dahongpao’, and 23 N-TMV. The results showed that among 35 samples, C. sinensis, C. (L.) Osbeck, C. ichangensis Swingle, C. reticulata and Fortunella margarita (Lour.) Swingle significantly differe from the traditional medicinal variety C. ‘Dahongpao’ based on relationship,seed shape and size, and flavonoids content. C. ‘Ponkan’ and C. ‘Dahongpao’ exhibit the highest similarity based on these three aspects, thus, suggesting the need for further researched on this variety as an emerging variety for medical use.
RFLP, RAPD, SSR and SCAR are molecular marker methods usually used to study citrus kinship, but the research found that these research methods have limitations, while the SRAP (sequence-related amplified polymorphism) is Stable, repeatable, short cycle, good polymorphism, which are widely used in the study of genetic diversity and genetic relationship of plant germplasm resources. Therefore, the SRAP molecular marker method was used in this study. Research shows that different varieties of citrus have distant genetic relationships with C. ‘Dahongpao’. This finding may be attributed to the fact that Fortunella margarita (Lour.) Swingle belongs to Fortunella Swingle family, whereas the remaining samples belong to citrus L.. C. reticulata is an artificial hybrids that is gererated from Valencia orange as the female parent and Jiangnan orange or zhusha orange as the male parent. C. sinensis is a type of C. (L.) Osbeck, that was originally produced in the United States and were introduced from Morocco and other countries to China in 1965 and later. C. ichangensis Swingle is a primitive variety in Sichuan, that potantially originated in the Quaternary Ice Age and is distantly related other citrus varieties[28, 29]. The r close relationship between C. ‘Ponkan’ and C. ‘Dahongpao’ is potentially explained by the fact that both belong to the C. reticulata Blanco. Furthermore, C. ‘Ponkan’ was grafted on C. ‘Dahongpao’, and genetic material exchange potentially occurred. The genetic relationship between different variety samples from the same place of origin is closer, thus, the same genetic mutations may occur among different varieties due to similar environmental factors. In addition, citrus varieties easily hybridize, thus increasing the similarity in genetic material. Given the long planting history and different genetic mutations, the relationship between C. ‘Dahongpao’ samples is not similar. Natural environmental factors, such as the climate, soil, and altitude of Nanchong, are particularly suitable for the growth of citrus plants. Therefore, samples of different varieties in Nanchong are less related to the other samples, and were divided into a separate branch.
Since ancient times, Chinese medicine scholars believe that the characteristics of Chinese material medica, such as shape, size and odor, were used as identification characteristics and quality standards. However, human-dependent detection is strongly dependent on personal experience and subjectivie. Therefore, stereomicroscopes are often used to observe the morphological structure of some traditional Chinese medicines. For example, Jie Zhang used stereomicroscopes to identify the seeds of Atractylodes macrocephala and Atractylodes lancea; Xiaolin Li studied the morphological structure of Salvia miltiorrhiza seeds using a stereomicroscopes. Each version of the Chinese Pharmacopoeia has records regulations on the size and shape of Juhe. Therefore, in this study we use a stereomicroscope to measure the size and shape. This technique presents Juhe’s appearance characteristics in a digital form, and avoids subjective error. The study found that Juhe collected from different regions, even in the same variety, exhibit different sizes and shapes, which may be due to different natural factors, such as the sea level, climate conditions, differences in cultivation techniques, cross pollination with other varieties, or different growth periods. Discrepancies in different varieties may be caused by genetic differences.
The main active components of tangerine kernels are limonoids and flavonoids. information on limonoids can be found in our previous reports. Our experiments demonstrated that C. ‘Ponkan’ limonin compounds are closest to C. ‘Dahongpao’. In this experiment, we first determined hesperidin, naringin, and neohesperidin levels in Juhe. Naringin only exists in some C. ‘Dahongpao’ samples and Fortunella margarita (Lour.) Swingle sample. Most C. sinensis samples do not contain neohesperidin, This finding may be due to inherent factors such as genetic material and seed maturity[34–35]. Some C. ‘Dahongpao’ samples lack naringin and a portion of C. ‘Ponkan’ samples lack of neohesperidin. These finding may be attributed to their growth environment and studies have shown that some environmental factors, such as light, temperature, irrigation, and fertilization, can affect flavonoids content in plant seeds. We also found that flavonoids content in C. ‘Ponkan’ and C. ‘Dahongpao’ samples from some places was similar, which was consistent with the research results base on shape and size. This funding may indicate that the seeds’ chemical composition was closely related to their physicochemical properties, which is consistent with previous research and further illustrates that C. ‘Ponkan’ is the most equivalent to C. ‘Dahongpao’, which proves that C. ‘Ponkan’ is a qualified emerging medical variety.
Cluster analysis revealed close relationships between C. ‘Dahongpao’ samples from Neijiang, Ziyang, Zigong, and Meishan, and the relationships among the C. ‘Ponkan’ samples obtained from these four places are also close. These four cities are geographically adjacent, indicating that mutual introduction may exist. Given similar natural conditions and cultivation techniques, the relationship remains close after many years of cultivation.
In general, most C. ‘Dahongpao’ samples are closely related to the C. ‘Ponkan’ samples, indicating that C. ‘Ponkan’ has the potential to become a new Chinese medicinal material.