Applying pollen DNA metabarcoding to the study of plant-pollinator interactions.
Applying pollen DNA metabarcoding to the study of plant-pollinator interactions.
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DOI：
10.3732/apps.1600124
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发表时间：
2017-06
影响因子：
3.6
通讯作者：
Brosi BJ
中科院分区：
文献类型：
--
作者：
Bell KL;Fowler J;Burgess KS;Dobbs EK;Gruenewald D;Lawley B;Morozumi C;Brosi BJ
为了研究环境变化下的传粉网络，我们需要精确、高通量的方法。先前的研究表明，相对于实地观察，通过研究蜜蜂的花粉负荷可以构建更高分辨率的网络。与传统方法（如光学显微镜）相比，DNA元条形码可能允许更快和更精细的花粉分类分辨率，但尚未应用于授粉网络。我们从佛罗里达州不同森林管理地点收集了38种蜜蜂的花粉样本。我们从花粉混合物中分离DNA，并在Illumina MiSeq平台上对所有混合物中的rbcL和ITS2基因区域进行测序。我们使用综合rbcL和ITS2数据库从序列数据中确定物种。我们成功地利用花粉元条形码建立了一个概念验证的定量授粉网络。我们的工作强调花粉元条形码不是定量的，但可以基于相互作用个体的数量构建定量网络。由于污染和假阳性读数的频率，每个反应都应采用分离和PCR阴性对照。与花粉显微鉴定相比，DNA元条形码在效率和分辨率方面具有优势，可望在未来植物与传粉者相互作用的研究中具有广泛的应用价值。
To study pollination networks in a changing environment, we need accurate, high-throughput methods. Previous studies have shown that more highly resolved networks can be constructed by studying pollen loads taken from bees, relative to field observations. DNA metabarcoding potentially allows for faster and finer-scale taxonomic resolution of pollen compared to traditional approaches (e.g., light microscopy), but has not been applied to pollination networks. We sampled pollen from 38 bee species collected in Florida from sites differing in forest management. We isolated DNA from pollen mixtures and sequenced rbcL and ITS2 gene regions from all mixtures in a single run on the Illumina MiSeq platform. We identified species from sequence data using comprehensive rbcL and ITS2 databases. We successfully built a proof-of-concept quantitative pollination network using pollen metabarcoding. Our work underscores that pollen metabarcoding is not quantitative but that quantitative networks can be constructed based on the number of interacting individuals. Due to the frequency of contamination and false positive reads, isolation and PCR negative controls should be used in every reaction. DNA metabarcoding has advantages in efficiency and resolution over microscopic identification of pollen, and we expect that it will have broad utility for future studies of plant–pollinator interactions.