There are many species of Dendrobium, and the genetic diversity among them is rich. It is difficult to identify Dendrobium by a single morphological index. Only with more accurate and stable technical methods, Dendrobium can be identified effectively. It has been found that molecular marker technology can effectively analyze the genetic diversity of Dendrobium and identify species quickly and accurately (Ren et al., 2015; Fu et al., 2017). At present, molecular markers of RFLP, AFLP, RAPD, SRAP, ISSR, SSR are widely used. ISSR (inter simple sequence repeat) is a molecular marker technique established by Zietkiewicz et al. (1994). This technique has the advantages of simple primer design, low DNA consumption, high polymorphism, simple operation, rapid sensitivity and low experimental cost (Lu et al., 2013; Yang et al., 2019). It has been widely used in the study of genetic diversity, genetic relationship and genetic map construction.

At present, ISSR Molecular markers have been widely used to analyze the genetic diversity of orchids. Tikendra et al. (2019) evaluated the fidelity of Dendrobium chrysotoxum tissue culture seedlings by using RAPD and ISSR markers respectively. Pastel in Solano et al. (2019) used ISSR Molecular marker technology to evaluate the variation of somatic cells in different subcultures of vanilla planifolia, and divided the clustering results into two categories. The first specie was donor plants and subculture 1~5 generations of tissue culture seedlings, the genetic distance was small; The second specie was 6~10 generations of tissue culture seedlings, the genetic distance was larger. Lu et al. (2019) analyzed the genetic diversity and genetic relationship of 24 Dendrobium officinale germplasm resources from different regions by ISSR molecular marker technology. The results showed that 24 cultivated populations of Dendrobium officinale were divided into three groups, and the germplasm of Dendrobium officinale had rich genetic diversity. Gomes et al. (2018) evaluated the genetic variation and structure of Cattleya lobata population by using ISSR molecular marker technology, indicating that the population has abundant genetic variation. Li et al. (2018) used RAPD and ISSR molecular markers to analyze the genetic diversity and genetic relationship of 21 varieties of 7 Cymbidium SW. The genetic distance of 21 cultivars ranged from 1.91 to 6.60, and the clustering results were basically consistent with the traditional morphological classification. Li et al. (2016) studied the genetic diversity of 7 populations of Paphiopedilum micranthum by using ISSR molecular marker technology, and found that 7 populations clustered into two groups. Cluster analysis showed that there was no significant correlation between genetic differentiation and geographical distance. Jiang et al. (2016) used ISSR molecular marker technology to study the genetic diversity of 12 populations of Dendrobium in Qinba area. The results showed that the 12 populations could be divided into 3 groups. Li et al. (2015a) analyzed the genetic relationship of 36 Dendrobium officinale germplasm resources by ISSR molecular marker technology, and found that the genetic similarity coefficient was 0.70, which could be divided into 6 groups. He et al. (2014) used ISSR molecular marker to analyze the genetic diversity and genetic relationship of 36 Phalaenopsis aphrodite cultivars. It was found that the Phalaenopsis aphrodite could be divided into 7 groups at the genetic similarity coefficient of 0.644, and the clustering was consistent with the flower color characteristics. At present, there are few studies on genetic diversity analysis, DNA fingerprint construction and germplasm resources identification based on ISSR molecular marker technology. In this study, 22 species of wild Dendrobium were used as test materials. ISSR molecular marker technology was used to analyze their genetic diversity and construct DNA fingerprints, which provided theoretical basis for species identification, genetic map construction and cross breeding of Dendrobium.

1 Results and Analysis

1.1 Polymorphism analysis of primer amplification

Six ISSR primers with clear amplification bands, high polymorphism and good repeatability were selected from 100 ISSR primers for PCR amplification (Figure 1). It can be seen from the amplification results of 6 primers (Table 1): the total number of amplified bands was 241, of which 241 were polymorphic, and the polymorphism ratio was 100%. The number of bands amplified by each primer ranged from 30 to 48. Among them, UBC842 had the largest number of amplification sites, 48 amplification sites, the minimum number of UBC811, and 30 amplification sites. The average number of polymorphic bands per primer was 40.2. The results showed that the genetic diversity of 22 species of Dendrobium was rich, and it was feasible to detect the genetic diversity of Dendrobium by ISSR molecular markers.

1.2 Analysis of interspecific relationship and genetic diversity among species of dendrobium

The genetic similarity coefficients (Table 2) of 22 Dendrobium species were calculated by NTSYS 2.10 software with the marker information generated by 6 ISSR primers, and the variation range was 0.698 4 ~ 0.878 7. Among them, the genetic similarity coefficient of Dendrobium affine and Dendrobium bigbibum was the largest, which was 0.878 7, indicating that the genetic relationship between them was the closest and the genetic difference was relatively small. The genetic similarity coefficient of Dendrobium biggibum and Dendrobium aurantiacum was the smallest, which was 0.698 4, indicating that the genetic relationship between them was the farthest and the genetic difference was relatively large. The genetic diversity index (Table 3) of 22 Dendrobium species was calculated by gGenAlEx 6.5 software. The average number of observed alleles (NA), effective alleles (NE), Nei's genetic diversity index (he) and Shannon information diversity index (I) were 1.983, 1.167, 0.133 and 0.247, respectively.

1.3 Cluster analysis of dendrobium

In order to determine the genetic relationship among the tested materials, NTSYS 2.10 analysis software was used to cluster the genetic matrix according to UPGMA, and to construct the tree-like clustering map (Figure 2). The results showed that 22 species of Dendrobium germplasm resources could be divided into ten categories at the similarity coefficient of 0.795. Class I consists of Dendrobium thyrsiflorum and Dendrobium chrysotoxum in section Callista; Class II consists of Dendrobium bullenianum and Dendrobium secundum in section Pedilonum. Class Ⅲ consists of Dendrobium lowii and Dendrobium ochraceum in section Formosae. Class Ⅳ consists of Dendrobium hercoglossum and Dendrobium aduncum in section Breviflores. Class Ⅴ consists of Dendrobium auriculatum, Dendrobium amethystoglossum, Dendrobium acinaciforme, Dendrobium leonis. In traditional classification, Dendrobium auriculatum and Dendrobium amethystoglossum belong to section Calcarifera, Dendrobium acinaciforme and Dendrobium leonis belong to section Aporum. Class Ⅵ consists of Dendrobium strongylanthum and Dendrobium delacourii in section Stachyobium. Class Ⅶ consists of Dendrobium loddigesii, Dendrobium officinale, Dendrobium nobile in the group of section Dendrobium. Class Ⅷ consists of Dendrobium nemorale in section Conostalix. Class Ⅸ consists of Dendrobium fimbriatum and Dendrobium aurantiacum in section Holochrysa. Class Ⅹ consists of Dendrobium affine and Dendrobium biggibum in section Phalaenanthe. From the cluster diagram, we can see that the group Ⅹ is separated from nine groups and clustered into a branch, which indicates that it has a relatively distant relationship with the first nine categories. This is consistent with the actual results, because the Ⅹ category is Dendrobium hybrida, while the first nine categories are all Dendrboium. There are significant differences between Dendrobium hybrida and Dendrboium in plant morphological characteristics and flowering period. The above clustering results by using molecular markers were consistent with the traditional classification, which fully indicated that ISSR Molecular markers could be applied to the classification and identification of dendrobium germplasm resources and the study of genetic relationship.

1.4 Construction of DNA fingerprint of Dendrobium

Six ISSR primers were selected to construct DNA fingerprints of 22 primary species of dendrobium. The results showed that 22 species of dendrobium primers could be identified separately by the primer UBC834, UBC836 and UBC868 by comparison. In this study, DNA fingerprints of 22 species of dendrobium were constructed by primer UBC834 (Figure 3), which can be used for the classification and identification of 22 species of dendrobium.

2 Discussion

At present, R. schlechter's classification system is mainly used in the international classification of Dendrobium, which divides Dendrobium into 4 subgenera and 41 groups (Ren et al., 2015). Based on the classification system of R. schlechter, Wang et al. (2014) slightly changed the classification of Dendrobium, and classified Dendrobium into 5 subgenera and 40 groups. In volume 19 of 《Flora of China》, 74 species and 2 varieties of Dendrobium from China were divided into 12 groups (Ji et al., 1999). The classification of Dendrobium from China by Wang et al. (2014) is consistent with that in 《Flora of China》, but the group names of different groups are slightly different. For example, aection Aporum group is the sword leaf group of 《Flora of China》, section Breviflores is thin axis group, section Callista is parietal leaf group, and section Dendrobium is Dendrobium group. There were 22 native species of Dendrobium candidum,11 species were native species in China and 11 species were native species abroad. The morphological identification of native species in China refers to the 19th volume of 《Flora of China》 compiled by Ji et al. (1999). The morphological identification of foreign native species is based on 《Dendrobium》 published by Wang et al. (2014). Due to the existence of foreign native species, the classification of Dendrobium in this study mainly refers to Wang et al. (2014). Cluster analysis showed that Dendrobium was divided into 10 groups at the genetic similarity coefficient of 0.795, and the clustering result was the same as that of Wang et al. (2014). Wang et al. (2014) reported that some primary species, such as Dendrobium fimbriatum and Dendrobium aurantiacum, were previously classified into the group of section Dendrobium. However, due to the cross incompatibility between these species and the species of section Dendrobium, they were divided into one group by taxonomists. According to the cluster analysis results of this study, D. fimbriatum and D. aurantiacum were indeed far from the species of the section Dendrobium, which also provides a molecular theoretical basis for their separate grouping.

Germplasm resource is the basis of breeding. Only when we have a deep understanding of the genetic relationship between germplasm resources, can we select hybrid parents and breed excellent varieties (Ge et al., 2012). In this study, ISSR molecular markers were used to analyze the genetic diversity and genetic relationship of 22 collected species of Dendrobium germplasm resources. A total of 241 loci were amplified by 6 selected primers, with an average of 40 loci amplified by each primer, indicating that ISSR molecular markers are widely distributed in Dendrobium genome. There are 241 polymorphic loci and the proportion of polymorphism was 100%. The primer polymorphism was consistent with the results of Li et al. (2015b), Lu et al. (2013) and Song et al. (2016). Results showed that the genetic diversity among different species of Dendrobium was rich, and ISSR Molecular marker technology was feasible for genetic diversity analysis of Dendrobium. In the aspect of genetic relationship, the results of this study showed that Dendrobium affinis and Dendrobium biggibum were the closest. Both of them belonged to section Phalaenopsis group, both originated from Australia, and their plant morphology and flowering period were almost the same. The genetic relationship between Dendrobium biggibum and Dendrobium aurantiacum was quite far away, which was consistent with the research results of Lin et al. (2018). Dendrobium biggibum belongs to Dendrobium hybrida in autumn, while Dendrobium aurantiacum belongs to Dendrobium chungii in spring, and there are obvious differences in phenotypic characteristics between them. Dendrobium has a large number of species, and its differentiation is complex and diverse. It is not comprehensive to analyze its genetic relationship only by ISSR molecular markers. Therefore, although the molecular classification results of this study are consistent with the classical morphological classification results, the genetic relationship between groups needs to be further verified. In the future, we should study the morphology and anatomy, palynology, molecular biology and other aspects to objectively reveal the genetic relationship of species in Dendrobium.

DNA fingerprinting is a general term for DNA samples treated with specific molecular marker technology and showing specific DNA fragments (Vos et al., 1995). As DNA is less affected by natural environment, relatively stable and highly polymorphic, DNA as a template for PCR amplification of molecular markers has become a common technical means for flower species identification. At present, other marker methods such as SSR and SRAP have been used to construct dendrobium fingerprints (Liu et al., 2017; Li et al., 2017). However, there are few reports on the construction of DNA fingerprints of dendrobium by ISSR molecular marker technology. Three of the six primers selected in this study can be used to construct fingerprints by amplified bands, and identified the tested materials by chromatogram. Results indicate that ISSR is an effective molecular marker technology to construct dendrobium germplasm fingerprints. However, due to the diversity of dendrobium species, there are still some limitations in the identification of all dendrobium based on a single molecular marker or a combination of different markers. At present, Ding Xiaoyu's research team has conducted in-depth research on molecular identification of dendrobium resources. They have constructed genetic relationship maps of different species of dendrobium by using ITS, rbcL, matK, psbA-trnH, trnL, SRAP, SSR and their combinations (Ding et al., 2002; Ding et al., 2003; Ding et al., 2008). It was found that most of them showed high specificity, but some species with very similar genetic backgrounds cannot be distinguished by these molecular markers. Therefore, the team has recently developed an effective identification technology for dendrobium species that are difficult to identify, that is, complete plastome comparative analysis technology. By sequencing the plastid genome sequences of different species and analyzing the differences, similar species can be identified (Zhu et al., 2018). This provides a more reliable and effective reference method for accurate identification of dendrobium resources.

3 Materials and Methods

3.1 Materials

The materials were collected from 22 native species of Dendrobium, which were planted in orchid germplasm resource nursery of Institute of flowers, Guangxi Academy of Agricultural Sciences (Table 4).

The reagents were as follows: 2×EasyTaq® PCR SuperMix (-dye)、Trans2K® DNA Marker (BM101-01) purchased from Beijing quanshijin Biotechnology Co., Ltd, Agarose, Tris-HCl and TAE buffer were purchased from Shenggong Bioengineering (Shanghai) Co., Ltd.

The instrument were as follows: Smart Spec^TM Plus nucleic acid protein analyzer (BIO-RAD, USA); PCR amplification instrument T1Thermocycle (Bio-DL); Normal temperature centrifuge (Xiangyi H1650-W); Constant temperature water bath (HWS-26); Electrophoresis apparatus (Beijing Liuyi DYY-6C); GEL DOCXR Automatic gel imaging system (BIO-RAD, USA).

3.2 Extraction and detection of genomic DNA

Genomic DNA of dendrobium was extracted by EasyPure Genomic DNA Kit (Beijing Quanshijin Biotechnology Co., Ltd.). The integrity of DNA was detected by 1% agarose gel electrophoresis, and the concentration and purity were detected by UV spectrophotometer, the extracted DNA was diluted to 20 ng/μL, with TE buffer, and stored in the refrigerator at - 20℃.

3.3 Primer screening and ISSR-PCR amplification

According to the ninth ISSR primer sequence published by Columbia University of Canada, primers with clear amplification band, high polymorphism and strong stability were selected from the synthesized primers by PCR amplification. 20 μL PCR reaction system includes the following steps: 2×EasyTaq® PCR SuperMix 10 μL, template DNA 1 μL, primer 1 μL, sterile water 8 μL. The PCR amplification procedure was as follows: pre denaturation at 94℃ for 4 min, denaturation at 94℃ for 1 min, annealing at 48℃ for 45 s (Table 1), 72℃ 1 min, 35 cycles were performed, extend at 72℃ for 7 min, the amplified products were stored in refrigerator at 4℃. The amplified products of 10 L PCR were detected by electrophoresis with 1% agarose gel.

3.4 Data statistics and analysis

According to the electrophoretic patterns of the amplified products, the bands in the same migration position were marked as "1", and those without bands were marked as "0". We constructed "0,1" matrix from the bands recorded by the amplified primers. We used NTSYS 2.10 statistical software for data analysis and UPGMA clustering. The effective allele number (NE), allele number (NA), Nei's genetic diversity index (he) and Shannon information diversity index (I) were calculated by genalex 6.51 software. According to the clustering results, the primers which can identify the tested materials were selected. According to the "0,1" matrix of the primer combination, the DNA fingerprint was constructed manually.

Authors’ contributions

Cui Xueqiang and Tang Xuan were the executors of the experimental design and research; Huang Changyan, Deng Jieling and Li Xiuling participated in the experimental design and part of the experiment; Lu Jiashi and Li Xianmin participated in the experimental data collation and analysis; Zhang Zibin guided the experimental design, data analysis, and essay writing and revision. All authors read and agree to the final text.

Acknowledgement

This research is jointly funded by the basic scientific research business project of Guangxi Academy of Agricultural Sciences (No.: Guinongke 2020YM32), Guangxi innovation driven development project (No.: Guike AA17204045-6), Guangxi key R & D plan (No.: Guike AB16380061), Guangxi innovation driven development project (No.: Guike AA17204026) and basic scientific research business project of Guangxi Academy of Agricultural Sciences (No.: Guinongke 2015YT90).

Reference

Ding G., Zhang D.Z., Ding X.Y., Zhou Q., Zhang W.C., and Li, X.X., 2008, Genetic variation and conservation of the endangered Chinese endemic herb Dendrobium offificinale based on SRAP analysis, Plant Syst. Evol., 276(3): 149-156
https://doi.org/10.1007/s00606-008-0068-1

Ding X.Y., Wang Z.T., Zhou K.Y., Xu L.S., Xu H., and Wang Y.Q., 2003, Allele-specifific primers for diagnostic PCR authentication of Dendrobium offificinale, Planta Med., 69 (6): 587-588
https://doi.org/10.1055/s-2003-40651
PMid:12865990

Ding X.Y., Xu L.S., Wang Z.T., Zhou K.Y., Xu H., and Wang Y.Q., 2002, Authentication of stems of Dendrobium offificinale by rDNA ITS region sequence, Planta Med., 68(2): 191-192
https://doi.org/10.1055/s-2002-20239
PMid:11859481

Fu T., Hu Z.Y., He Y.Q., Li W., and Lin L., 2017, Development of EST-SSR molecular markers in Dendrobium officinale, Henongxue Bao (Journal of Nuclear Agricultural Sciences), 31(4): 663-670

Ge Y.Y., Zhang F., Shen X.L., Liu J.X., Wang W.Y., Tian D.Q., and Zhang Z., 2012, Analysis of genetic diversity and construction of fingerprint of Vriesea by ISSR, Zhongguo Nongye Kexue (Scientia Agricultura Sinica), 45(4): 726-733

Gomes P.C.L., Smidt E.D.C., de Fraga C.N., and Silva-Pereira V., 2018, High genetic variability is preserved in relict populations of Cattleya lobata (Orchidaceae) in the Atlantic Rainforests inselbergs, Brazilian Journal of Botany, 41(1): 185-195
https://doi.org/10.1007/s40415-017-0422-z

Jiang A.M., Cao J., and Cai G.L., 2016, Genetic relationship of Dendrobium and genetic diversity of Dendrobium nobile in Qinling-Data mountains revealed by ISSR, Xibei Zhiwu Xuebao (Acta Botanica Boreali-Occidentalia Sinica), 36(10): 1977-1983

Li L.H., Hu Y., Li H.G., Zhang Y.L., Zhang Y., and Wu H.Y., 2018, Study on genetic diversity of China orchid resources using RAPD and ISSR markers, Zhongguo Nongxue Tongbao (Chinese Agricultural Science Bulletin), 34(29): 42-47

Li Y.Q., Jiang J.L., Ye W., Lei F.G., and Hua S.Z., 2015a, Relationships among 37 germplasm resources of medicinal Dendrobium based on ISSR, Fujian Nongye Xuebao (Fujian Journal of Agricultural Sciences), 30(2): 131-135

Li Y.Q., Ye W., Jiang J.L., and Lei F.G., 2015b, Analysis of genetic diversity of germplasm resources of Dendrobium officinale by ISSR, Xinan Nongye Xuebao (Southwest China Journal of Agricultural Sciences), 28(4): 1530-1534

Li Z.J., Wang Y.C., Han B., Yang Y.B., Wang Z., and Sun Z.Y., 2019, Research progress on constituents of alkaloids in plants from Dendrobium Sw., Zhongcaoyao (Chinese Traditional and Herbal Drugs), 50(13): 3246-3254

Li Z.Y., Guan M.Y., Li J., and Li M.Y., 2016, Genetic diversity of Paphiopedilum micranthum detected by ISSR data, Xibao Zhiwu Xuebao (Acta Botanica Boreali-Occidentalia Sinica), 36(7): 1351-1356

Lin R.Y., Ye X.X., Zhong Z.Q., Lin B., and Huang M.L., 2018, Genetic diversity of Dendrobium germplasm accessed by SRAP molecular markers, Fujian Nongye Xuebao (Fujian Journal of Agricultural Sciences), 33(5): 469-473

Lu J.S., Bu Z.Y., Lv W.L., Su J.M., Huang C.Y., and Li C.N., 2013, ISSR analysis on genetic diversity of germplasms resources in Dendrobium SW. from different habitats, Zhongcaoyao (Chinese Traditional and Herbal Drugs), 44(1): 96-100

Pastelín Solano M.C., Salinas Ruíz J., González Arnao M.T., Castañeda Castro O., Galindo Tovar M.E., and Bello Bello J.J., 2019, Evaluation of in vitro shoot multiplication and ISSR marker based assessment of somaclonal variants at different subcultures of vanilla (Vanilla planifolia Jacks), Physiol. Mol. Biol. Plants, 25(2): 561-567
https://doi.org/10.1007/s12298-019-00645-9
PMid:30956436 PMCid:PMC6419708

Ren Y., Wang C.D., Lu X.S., Zhang Z.Q., and Yun J.M., 2015, Study on cross fruitfulness of ornamental Dendrobium based on molecular classification, Henongxue Bao (Journal of Nuclear Agricultural Sciences), 29(10): 1909-1916

Song S., Zhou Y.F., Liu Z.J., Zhao M.F., Yang J., Xu S.Z., and Wen G.S., 2016, Genetic diversity among Dendrobium with known origins based on the ISSR and AFLP marker, Yunnan Nongye Daxue Xuebao (Journal of Yunnan Agricultural University (Natural Science)), 31(4): 688-695

Tikendra L., Koijam A.S., and Nongdam P., 2019, Molecular markers based genetic fidelity assessment of micropropagated Dendrobium chrysotoxum Lindl, Meta Gene, 20: 100562
https://doi.org/10.1016/j.mgene.2019.100562

Vos P., Hogers R., Bleeker M., Reijans M., van de Lee T., Hornes M., Frijters A., Pot J., Peleman J., Kuiper M., 1995, AFLP: a new technique for DNA fingerprinting, Nucleic Acids Res., 23(21): 4407-4414
https://doi.org/10.1093/nar/23.21.4407
PMid:7501463 PMCid:PMC307397

Yang H.C., Su W.Y., Tan Y.L., Qin Y.Y., Ma T., Pu H.C., and Zhou Z.L., 2019, Germplasm evaluation of Pleurotus eryngii based on ISSR markers, Xinan Nongye Xuebao (Southwest China Journal of Agricultural Sciences), 32(7): 1480-1486

Zhu S.Y., Niu Z.T., Xue Q.Y., Wang H., Xie X.Z., and Ding X.Y., 2018, Accurate authentication of Dendrobium officinale and its closely related species by comparative analysis of complete plastomes, Acta Pharmaceutica Sinica B, 8(6): 969-980
https://doi.org/10.1016/j.apsb.2018.05.009
PMid:30505665 PMCid:PMC6251949

Zietkiewicz E., Rafalski A., and Labuda D., 1994, Genome fingerprinting by simple sequence repeat (SSR) -anchored polymerase chain reaction amplification, Genomics, 20(2): 176-183
https://doi.org/10.1006/geno.1994.1151
PMid:8020964