Comparison of different kits for the extraction of mixed pollen DNA carried by pollinators

Author of the article:LI Yue, WEI Wei, LIU Wen-Ping, ZHAO Kai-Xuan, ZHOU Ze-Yang, ZHU Chao-Dong, LUO A-Rong, HUANG Dun-Yu

Author's Workplace：Key Laboratory of Conservation and Utilization of Pollinator Resources in the Upper Reaches of the Yangtze River of Ministry of Agriculture and Rural Affairs, College of Life Sciences, Chongqing Normal University, Chongqing 401331, China

Key Words：insect pollinator; mixed pollen; pollen source plants; DNA kits; DNA metabarcoding

Abstract：[Objectives]  To compare the performance of different kits in extracting DNA from micro mixed pollen and determine which kits meet the minimum demands of DNA extraction from mixed pollen samples. [Methods]  Pollen carried on the body surface of Apis cerana cerana, Osmia excavata and Amegachile strupigera from different regions was mixed. Genomic DNA was extracted using the Shanghai BioTeke Magnetic Bead Extraction Kit (CZ), the Beijing BioTeke Centrifugal Column Genomic DNA Extraction Kit (BT), the Omega E.Z.N.A.® MicroElute Genomic DNA Extraction Kit (EZ), the Beijing Tiangen Genomic DNA Extraction Kit (TG) and the Jiangsu Jirui Genloci® TNA Extraction Kit (GL). Sequencing data were obtained based on ITS macro-barcoding and the results obtained from the five kits were compared in terms of variance and analyzed using linear discrimination and hierarchical clustering analysis. The extraction method of the BT kit was adjusted to reduce the amount of mixed pollen required. [Results]  The concentration of DNA obtained using the TG and GL kits was 5-15 times higher than that obtained using the CZ, BT and EZ kits. The purity of the DNA obtained using the EZ and TG kits was significantly different from that obtained using the other kits. The number of plant species detected by each kit was as follows: (167 ± 8) (BT), (161 ± 16) (GL), (160 ± 10) (EZ), (160 ± 6) (TG), (143 ± 12) (CZ). Some kits were significantly better at extracting DNA from specific plant orders, families and genera. Indeed, some plant DNA could only be extracted to templates by specific kits. The smallest quantity of mixed pollen that could be extracted to a qualified DNA template was 0.02 mg. [Conclusion]  Different kits varied in their ability to extract the DNA of different plant orders, families and genera. DNA could be extracted from mixed pollen samples as low as 0.02 mg. These findings provide information to guide the selection of kits for extracting DNA from mixed pollen samples.