利用PCR-RFLP技术鉴定传粉榕小蜂隐种混合样品的物种组成

doi:10.3724/SP.J.1003.2010.414

摘要/Abstract

摘要：

隐种(cryptic species)是指形态上几乎完全相同但遗传组成存在显著分化的物种。在榕树–榕小蜂一对一共生系统中, 传粉榕小蜂隐种的发现对协同进化、物种共存等重要的生态和进化理论提出了严峻的挑战。因为很难从形态上直接区分隐种, 所以, 相关研究中的一个迫切需要解决的问题就是如何快速而准确地鉴定隐种。本文采用PCR-RFLP方法分析了mtDNACOI基因片段, 对木瓜榕(Ficus auriculata)和鸡嗉果榕(F. semicordata)的传粉榕小蜂隐种进行了区分。结果表明为木瓜榕传粉的大果榕小蜂(Ceratosolen emarginatus)存在两个隐种(A和B), 分别包含1个XhoI和1个BssSI酶切位点。将两个隐种的样品按不同比例混合, 提取基因组DNA, PCR扩增mtDNA COI片段, 经XhoI和BssSI分别酶切, 均能通过酶切图谱准确检测出混合样品的隐种组成。鸡嗉果榕小蜂(C. gravelyi)两个隐种的mtDNA COI基因序列也存在较大差别, 分别包含1个BmrI和1个AvaI酶切位点, 隐种混合样品经BmrI和AvaI分别酶切的结果也能准确鉴定混合样品的物种组成。我们的结果表明基于PCR和DNA酶切技术能快速而准确地区分传粉榕小蜂的隐种。

关键词: 隐种, PCR-RFLP, COI, 传粉榕小蜂, 物种鉴定

Abstract

Cryptic species are morphologically undetected yet genetically divergent species. The presence of cryptic pollinating-fig wasp species has potentially important implications for understanding fig/fig-wasp mutualisms and the coexistence of cryptic species. It is essential to identify cryptic species quickly and accurately using molecular markers since the species are morphologically undistinguishable. We used PCR-RFLP to analyze the mtDNA COI gene in cryptic species of Ceratosolen emarginatusandC. gravelyi. The PCR-RFLP was useful for determining the composition of cryptic pollinating-fig wasp species. Ceratosolen emarginatus included two cryptic species (A and B) with an XhoI and a BssSI digesting site in the COIgene, respectively. Ceratosolen gravelyi included two cryptic species with a BmrI and an AvaI digesting site, respectively. Genomic DNA samples were extracted from a mixture of cryptic species A and B withinC. emarginatus, and XhoI andBssSI endonucleases were used to digest the amplified COIfragments. XhoI-specific fragments were found when cryptic species A was present, and BssSI-specific fragments were found when cryptic species B was present. The digestion of COI gene with BmrI andAvaI in two cryptic species ofC. gravelyi showed similar results. Taken together, PCR-RFLP, based on PCR and DNA restriction enzyme digestion, can quickly and accurately identify cryptic pollinating-fig wasp species.

Key words: cryptic species, PCR-RFLP, COI, pollinating-fig wasp, species identification

隆沂峄, 杨丽媛, 廖万金 (2010) 利用PCR-RFLP技术鉴定传粉榕小蜂隐种混合样品的物种组成. 生物多样性, 18, 414-419. DOI: 10.3724/SP.J.1003.2010.414.

Yiyi Long, Liyuan Yang, Wanjin Liao (2010) Identifying cryptic species in pollinating-fig wasps by PCR-RFLP on mtDNA COI gene. Biodiversity Science, 18, 414-419. DOI: 10.3724/SP.J.1003.2010.414.

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链接本文: https://www.biodiversity-science.net/CN/10.3724/SP.J.1003.2010.414

https://www.biodiversity-science.net/CN/Y2010/V18/I4/414

图/表 4

表1 混合样品中大果榕小蜂隐种A和隐种B的头数与鸡嗉果榕小蜂隐种A和隐种B的头数

Table 1 Composition of cryptic species (A and B) in Ceratosolen emarginatus and C. gravelyi used in this study

样品编号 Sample No.	大果榕小蜂 C. emarginatus		鸡嗉果榕小蜂 C. gravelyi
样品编号 Sample No.	隐种A头数 Number of A	隐种B头数Number of B	隐种A头数 Number of A	隐种B头数 Number of B
1	1	10	6	2
2	6	2	10	2
3	10	2	20	2
4	20	2	1	10
5	2	20	2	20
6	2	10	2	10
7	2	6	2	6
8	4	4	4	4
9	0	2	0	2
10	2	0	2	0

图1 大果榕小蜂(a)与鸡嗉果榕小蜂(b)COI片段PCR扩增结果。M表示DNA marker, 数字表示样品编号, 对应的隐种组成比例如表1所示。

Fig. 1 The PCR products of COI gene in Ceratosolen emarginatus (A) and C. gravelyi (B). M indicates the DNA marker, and the numbers indicate the sample number. See Table 1 for the composition of cryptic species for each sample.

图2 大果榕小蜂混合样品经XhoI(a和b)和BssSI(c和d)酶切电泳结果。数字表示样品编号, 对应的隐种组成比例如表1所示。M表示DNA marker (DL 2000)。a和c是PCR产物经胶回收纯化后酶切图谱, b和d是PCR产物直接酶切图谱。

Fig. 2 The digested products by XhoI (a and b) and BssSI (c and d) in Ceratosolen emarginatus. a and c show the digested results of purified PCR products; b and d, unpurified. M is the DNA marker (DL 2000). 1-10 correspond to the sample numbers in Table 1.

图3 鸡嗉果榕小蜂隐种混合样品经BmrI(a和b)和AvaI(c和d)酶切电泳结果。数字表示样品编号, 对应的隐种组成比例如表1所示。M表示DNA marker (DL 2000)。a和c是PCR产物经胶回收纯化后酶切图谱, b和d是PCR产物直接酶切图谱。

Fig. 3 The digested products by BmrI (a and b) and AvaI (c and d) in Ceratosolen gravelyi. a and c show the digested results of purified PCR products; b and d, unpurified. 1-10 correspond to the sample numbers in Table 1.

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