Chaenomeles speciosa (Sweet) Nakai, also known as Tiegeng Haitang and Tiegeng Mugua in Chinese, is a genus of Chaenomeles which belongs to the family of Rosaceae. It is used as medicine in dried ripe fruit, which is rich in a large number of organic acids such as malic acid, tartaric acid and citric acid. It also contains catalase, tannins, pectin, flavonoids and other substances (Ros et al., 2004; Du et al., 2013; Miao et al., 2016; Zhang et al., 2016; Zhang and Shen, 2018). Chaenomeles speciosa (Sweet) Nakai was originally produced in the southwest of China, with continuous changes, now Hubei, Sichuan, Anhui, Shandong, Zhejiang, Shanxi, Yunnan and other provinces are planted, has formed Ziqiu papaya, Xuan papaya and Sichuan papaya and many other major production areas (Wang et al., 2009; Yang, 2011; Shao et al., 2017). The Chaenomeles speciosa (Sweet) Nakai has a sour taste and a warm nature and has high medicinal and nutritional value (Xie et al., 2015; Li et al., 2018; Zhu et al., 2019).

Chaenomeles speciosa (Sweet) Nakai has a long history and wide planting area in China and has great potential for development. However, due to the similar pharmacognostic characters of Chaenomeles, there are many counterfeit products in the market, and the quality of medicinal materials is extremely unstable, which brings great inconvenience to the species identification and drug safety. Therefore, the rapid and accurate identification of medicinal plants of Chaenomeles is particularly important and urgent. In this study, mature Chaenomeles speciosa (Sweet) Nakai from 12 major producing areas in China were selected as experimental materials to extract their genomic DNA. On the basis of obtaining appropriate DNA extraction methods and ISSR-PCR optimized amplification system, appropriate primers were screened for PCR amplification of genomic DNA of these 12 species of Chaenomeles speciosa (Sweet) Nakai. Through these studies, we hope to clarify the genetic diversity and genetic relationships of 12 Chaenomeles speciosa (Sweet) Nakai resources and provide research basis for the identification and classification of medicinal resources of Chaenomeles in the later stage.

1 Results and Analysis

1.1 Genomic DNA extraction and detection

The extracted genomic DNA bands of 12 Chaenomeles speciosa (Sweet) Nakai germplasm were clear and had no tail. The genomic DNA was amplified with ITS₂ primers, and the bands were bright and clear, and the size of the bands was basically the same (Figure 1; Figure 2). The above results indicated that the extracted genomic DNA of Chaenomeles speciosa (Sweet) Nakai was of high quality and could be used for subsequent ISSR genetic diversity analysis.

1.2 ISSR primer amplification polymorphism

The optimized ISSR-PCR system was used to screen out 10 polymorphism primers from 100 ISSR primers for genetic diversity analysis of Chaenomeles speciosa (Sweet) Nakai (Figure 3; Table 1), the 10 primers amplified 511 bands in 12 samples, among which the number of polymorphic bands was 355, and the percentage of polymorphic bands was 75.3%. A total of 87 loci were amplified, among which 74 loci were polymorphic, and the percentage of polymorphic loci was 85%. The percentage of polymorphism of each primer ranged from 63% to 100%, among which primer U873 had the highest number of amplification and polymorphism sites (both 16), and primer U847, U848, U850, U858, U873 had the highest percentage of polymorphism bands and polymorphism sites (all 100%). The results of amplification showed that the genotypes of Chaenomeles speciosa (Sweet) Nakai from 12 producing areas were different, and there was rich genetic diversity.

Figure 3 Amplification results of twelve Chaenomeles speciosa (Sweet) Nakai resources by primer U873 Note: M: DL2000DNA Marker; 1~12: Amplification results of twelve Chaenomeles speciosa (Sweet) Nakai resources

Table 1 Polymorphism ISSR primer information and amplification

1.3 Genetic similarity and genetic distance analysis

The genetic similarity coefficient (GS) and genetic distance (GD) of 12 Chaenomeles speciosa (Sweet) Nakai germplasm from different producing areas were analyzed by using the software of NTSYS-pc2.10e. The GS value of 12 Chaenomeles speciosa (Sweet) Nakai from different producing areas ranged from 0.5057 to 0.8621, with an average of 0.713 7 and a range of 0.356 4. The GS values of most germplasms ranged from 0.750 0 to 0.820 0, and the maximum GS and minimum GD values were between M4 and M7, which were 0.862 1 and 1.463 5, respectively, indicating that M4 and M7 had the highest degree of genetic similarity and the closest genetic relationship. The GS value between M1 and M10 is the smallest, while the GD value is the largest, indicating the lowest degree of genetic similarity and the farthest relationship between them (Table 2).

Table 2  Genetic similarity coefficient and genetic distance of twelve Chaenomeles speciosa (Sweet) Nakai materials Note: M1-M12 materials coding name are shown in Table 3,Genetic distance (above diagonal)and genetic similarity coefficient (below diagonal)

1.4 Clustering analysis

Clustering analysis was performed on 12 samples of Chaenomeles speciosa (Sweet) Nakai with NTSYS-pc2.10e software (Figure 4). The systematic clustering genetic consistency ranged from 0.56 to 0.86, with an average value of 0.71. Therefore, the 12 materials can be divided into three groups from top to bottom, and the first group can be divided into two subgroups. The first subgroup includes M1, M2, M3 and M6. The second subgroup consisted of the M4, M7, M12, M5, M9 and M8. The second group is M11, and the third group is M10.

2 Discussion

Research on genetic diversity of Chaenomeles germplasm resources is very important for solving the problem of provenance hybridification, establishing variety classification system, and further breeding research (Wang et al., 2010). Compared with the traditional morphological, cytological and biochemical markers, the new molecular marker technology has the advantages of more markers, higher polymorphism and easier detection. For example, ISSR molecular labeling technology has been applied in the analysis of genetic diversity of many species, such as plants and microorganisms. However, there are few reports on the genetic diversity of Chaenomeles. Wang et al. (2010) used SRAP molecular marker technology to divide 32 cultivated materials of papaya into four groups, namely C. cathayensis system, C. thibetica, C. speciosa system and C. japonica system. By revealing the genetic relationships among the four strains, they found that the degree of differentiation of C. speciosa system was relatively high. Xia et al. (2010) screened 28 pairs of primers with clear bands and rich polymorphism based on 21 Chaenomeles germplasm materials in Tibet, laying a foundation for genetic diversity analysis. At present, the research on Chaenomeles speciosa (Sweet) Nakai is mainly focused on indirectly revealing the differences of Chaenomeles speciosa (Sweet) Nakai germlines by means of quality character evaluation methods. It was found that there were significant differences in organic acids, flavonoids, polyphenols and polysaccharides in different producing areas (Chen et al., 2019). The research provides a basis for the use of molecular marker techniques to reveal the genetic diversity of Chaenomeles speciosa (Sweet) Nakai.

In this study, we used ISSR molecular markers to analyze the genetic diversity and genetic relationships of 12 Chaenomeles speciosa (Sweet) Nakai resources in China. The number of polymorphic ISSR primers (U847, U848, U850, U858, U873) and the percentage of polymorphic ISSR primers (U847, U848, U850, U858, U873) reached 100%, and the percentage of each primer was≥63%, indicating that 12 samples of Chaenomeles speciosa (Sweet) Nakai had rich genetic information. The genetic similarity analysis showed that the GS value of 12 Chaenomeles speciosa (Sweet) Nakai germplasm resources ranged from 0.5057 to 0.8621, and the average GS value was 0.7137, indicating that there were certain genetic differences among the 12 Chaenomeles speciosa (Sweet) Nakai germplasm resources, which was mutually verified with the differences of Chaenomeles speciosa (Sweet) Nakai from different producing areas revealed by Chen et al. (2019) through quality trait evaluation method. Studies have shown that geographical distribution is correlated with genetic relationship, and germplasm in close geographical locations tends to cluster into one group (Wilson et al., 2001). In this study, 12 Chaenomeles speciosa (Sweet) Nakai germplasm resources were divided into 3 groups from top to bottom. Group 1 included 2 subgroups, among which M4 (Sangzhi County-1, Hunan Province) and M7 (Sangzhi County-2, Hunan Province) had the closest genetic relationship, while M1 (Eryuan County, Yunnan Province) and M10 (Yunxi County, Hubei Province) had the farthest genetic relationship. M1 (Eryuan County, Yunnan Province) and M2 (Yun County, Yunnan Province), M4 (Sangzhi County-1, Hunan Province) and M7 (Sangzhi County-2, Hunan Province), M5 (Hedong District-1, Shandong Province) and M8 (Zheng'an County, Guizhou Province) were clustered together, indicating that the closer the origin of papaya varieties is, the closer the genetic relationship is, which further confirmed the reliability of the results of this study. These studies will provide a basis for the identification and screening of Chaenomeles speciosa (Sweet) Nakai germplasm resources.

3 Materials and Methods

3.1 Experimental materials

The test materials included Chaenomeles speciosa (Sweet) Nakai samples from 12 major producing areas, including Hubei, Hunan and Anhui, and the sample number, producing area, geographical location and other information (Table 3).

3.2 Genomic DNA extraction and detection

The new plant genomic DNA kit (Beijing Tiangen Biotechnology Co., Ltd.) was used to extract the genomic DNA from fresh Chaenomeles speciosa (Sweet) Nakai. The concentration and quality of the DNA were detected by spectrophotometry and agarose electrophoresis, and stored at -20℃ for later use. The ITS2 primers (F: 5'-ATGCGATACTTGGTGTGAAT-3'; R: 5'-GACGCTTCTCCAGACTACAAT-3') to detect DNA for the presence of PCR reaction inhibitors.

3.3 Selection and amplification of polymorphic ISSR primers

In this study, primers were synthesized by Shanghai Yingjun Company, and 10 primers with good polymorphism, good repeatability and clear bands were screened by the optimized ISSR PCR system. The optimized 20 μL PCR system was: Template DNA 50 ng, 2×Taq mix 10 μL, 2.5 μmol /L primer 2 μL, ddH₂O 7 μL.

3.4 Data processing

At the same level position on the glue map, the stripe was marked as 1, and the stripe was marked as 0. Excel software was used to sort out the data, and NTSYS-pc2.1 software was used to calculate the genetic similarity coefficient (GS) and the sum genetic distance (GD). Based on the genetic similarity coefficient matrix, UPGMA method was used for cluster analysis.

Authors’ contributions

JXG is the executor of this research and has completed the writing of the first draft of the paper. LXM and ZMD helped complete the experiment; WH assisted in data analysis and processing; GKY is the architect and the person in charge of the project, guiding the experimental design, data analysis, paper writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This study was jointly funded by the Special Project for the Construction of Modern Agricultural Industrial Technology System (CARS-21), the Third Batch of "Hubei Youth Talents Development Program", the Special Project for Technological Innovation of Hubei Province (Special Project for Western Hubei Nationalities 2019AKB092) and the Research and Development Project of Enshi Prefecture Science and Technology Plan (D20180016).

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