TrnL-F and ISSR Primer-based Molecular Identification and Genetic Diversity Determination on Camellia impressinervis and Three Closely Related Species
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摘要:目的 凹脉金花茶是一种具有较高价值的观赏与药用两用植物，与其同属中的金花茶、显脉金花茶、东兴金花茶外形相似。通过分子指纹技术分析这4个近源种之间的遗传多样性，可为凹脉金花茶的鉴定及其种质资源保护和利用提供理论指导。方法 利用金花茶、凹脉金花茶、显脉金花茶、东兴金花茶的19个样品评估TrnL-F条形码在这4种山茶中鉴定凹脉金花茶的潜力，并利用简单序列间重复（ISSR）引物检测4个山茶组种的遗传多样性。结果 基于TrnL-F序列构建的进化树与检测到的单核苷酸多态性位点的差异相一致，所有凹脉金花茶样品单独聚为一类。ISSR分析显示，4个凹脉金花茶植物样本单独聚为一类，与其他3个山茶组物种的相似指数为0.57。结论 TrnL-F可有效鉴别凹脉金花茶与3种近缘种。本研究揭示了这4个近缘种的分子亲缘关系，从分子水平证实显凹脉金花茶具有异于其他3种山茶的遗传特性。Abstract:Objective Molecular identification and genetic diversity of Camellia impressinervis were studied to improve the preservation and utilization of the endangered species.Method A highly valuable ornamental and medicinal, but extremely endangered as classified by the International Union for the Conservation of Nature, C. impressinervis was compared with the closely related C. nitidissima, C. euphlebia, and C. tunghinensis. Using 19 specimens of the 4 species, validity of applying trnL-F barcode to confidently identify them was evaluated, and their genetic diversity examined based on the inter-simple sequence repeat (ISSR) primers.Result TrnL-F could accurately differentiate C. impressinervis from those closely related camellia cultivars and clustered all in a same group. The ISSR analysis grouped the 4 C. impressinervis specimens together and showed a similarity index of 0.57 with the other 3 camellia species.Conclusion The genetic fingerprint in terms of trnL-F barcode and ISSR primers successfully identified the close relationship of C. impressinervis with, but distinguishable from, C. nitidissima, C. euphlebia, and C. tunghinensis.
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Keywords:
- Camellia /
- trnL-F /
- molecular identification /
- genetic diversity /
- ISSR
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图 3 引物UBC846聚合酶链反应扩增
M：Marker；1、8；9、10、12、14为金花茶；2、6；11、18为显脉金花茶，3、4、7、15为凹脉金花茶，5、13、16、17、19为东兴金花茶。
Figure 3. PCR amplification with primer UBC846
M: Marker; 1,8,9,10,12,14 are C . nitidissima; 2, 6, 11,18 are C.euphlebia; 3,4,7,15 are C. impressinervis; 5,13,16,17,19 are C.tunghinensis.
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图 4 4种金花茶种质资源的分类鉴定及亲缘关系
Figure 4. Classification, identification, and genetic relationship of 4 camellia germplasms
表 1 ISSR扩增引物
Table 1 Amplified ISSR primers
引物
Primer引物序列（3′-5′）
Primer sequence（3′-5′）退火温度
Annealing
temperature/℃UBC811 GAGAGAGAGAGAGAGAC 51.2 UBC815 CTCTCTCTCTCTCTCTG 50.5 UBC825 ACACACACACACACACT 50.5 UBC834 AGAGAGAGAGAGAGAGYT 49 UBC835 AGAGAGAGAGAGAGAGYC 56 UBC836 AGAGAGAGAGAGAGAGYA 52 UBC840 GAGAGAGAGAGAGAGAYT 49 UBC841 GAGAGAGAGAGAGAGAYC 52 UBC844 CTCTCTCTCTCTCTCTRC 52 UBC845 CTCTCTCTCTCTCTCTRG 50.5 UBC846 CACACACACACACACART 51.2 UBC848 CACACACACACACACARG 55 UBC851 GTGTGTGTGTGTGTGTYG 52 UBC856 ACACACACACACACACYA 52 
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表 2 ISSR引物扩增产物的多态性
Table 2 Polymorphisms of ISSR primers amplified products
引物
Primer扩增条带总数
Total number of amplified bands/条多态性条带数
Polymorphic strips/条多态性条带占比
Percentage of polymorphic bands/%UBC811 20 20 100.0 UBC815 12 12 100.0 UBC825 18 16 88.9 UBC834 13 13 100.0 UBC835 15 15 100.0 UBC836 16 13 81.3 UBC840 12 12 100.0 UBC841 12 10 83.3 UBC844 15 15 100.0 UBC845 12 12 100.0 UBC846 16 16 100.0 UBC848 3 3 100.0 UBC851 9 8 88.9 UBC856 9 8 88.9 总数
Total182 173 95.1
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