Comparisons On The Efficiency Of Various DNA Extraction Techniques From Benthic Foraminifera And Molecular Diversity Of Benthic Foraminifera In The Tidal Flat Of Qingdao Bay

Benthic foraminifera consist of many types, including hyaline, porcelain, agglutinated or organic tests, and the first three types play significant role in study of paleoceanography, geology, biomonitoring, ecological system and so on. For better application of foraminifera in these fields, we must indentify the species accurately. However, the vast diversity and wide morphological variability of foraminifera make it very difficult to clarity their taxonomic status. The application of molecular biology techniques will help solve some misidentification problems that derived from morphological taxonomy. However, there was limited study on molecular biological research of domestic foraminifera. In this study, we compared the efficiency of different methods for DNA-extracting and DNA-preserving from benthic foraminifera. Furthermore, we utilized the improved molecular methods to investigate the molecular diversity of benthic foraminifera in intertidal zone of Qingdao Bay using high throughput sequencing.In order to establish an efficient method for preserve, extraction and amplification of DNA from hyaline and porcelain benthic foraminifera, and to extract and amplify agglutinated benthic foraminiferal DNA efficiently, some experimental series were set up: 1) We used the partial SSU rDNA as DNA barcode to test the different efficiency of five DNA extraction methods for the representative hyaline foraminifera Ammonia spp. The DNA extracting methods include DOC lysis buffer method, Guanidine lysis buffer method, Qiagen Dneasy Plant Mini Kit, Dneasy Blood & Tissue Kit and REDExtract-N-AmpTM Tissue PCR Kit. In order to establish efficient methods for preserving DNA from hyaline benthic foraminifera Ammonia spp., the partial LSU rDNA was used as DNA barcode and three experimental series of DNA preservation were set up including air-dried(from 20℃ to 120℃); frozen(-20℃、-80℃, with or without seawater); DOC lysis buffer with vs. without EDTA. 2) We used the partial SSU rDNA as DNA barcode to test the different efficiency of five DNA extraction methods for Quinqueloculina spp. which represent porcelain foraminifera, and three methods for DNA preservation. 3) We also used the partial SSU rDNA as DNA barcode to test the different efficiency of five DNA extraction methods for Trochammina spp. which represent agglutinated foraminifera.And the result showed that 1) DOC method, Guanidine method and REDExtract-N-AmpTM Tissue PCR Kit were all effective for hyaline foraminifera, but the DOC method was more economic and convenient; DNA was better preserved under the condition of air-dried under 20℃and 30℃ than 40℃(P<0.05); Frozen without seawater group was better than with seawater group(P<0.05); There was no significant difference between the DOC lysis buffer with or without EDTA treatment(P>0.05); And the best results were obtained in the treatment with frozen samples. 2) For porcelain foraminifera, Guanidine lysis buffer was the best method for DNA extracting; And DNA was better preserved under the condition of air-dried under 110℃and 30℃ but the difference was not significant(P>0.05); There was no significant difference among frozen groups(P>0.05); Samples in Guanidine lysis buffer with EDTA and stored in-20℃ were better preserved(P<0.05); And the best results were obtained in the treatment with samples stored in Guanidine lysis buffer. 3) For agglutinated foraminifera, Guanidine lysis buffer was the best method for DNA extracting.Benthic foraminifera is an important component of Marine micro-food-cycle, occuping an important position in the Marine ecosystem. The traditional method to investigate the diversity of foraminifera is time-consuming and occasionally omits some species. High-throughput sequencing technology is helpful to solve this problem. Targeting a short hypervariable region of foraminiferal SSU rRNA gene, we used the next-generation sequencing method to examine foraminiferal molecular diversity in Qingdao Bay intertidal sediments sampled from May to October, and the relationship between foraminiferal diversity and environmental factors(temperature, salinity, pH) was also assessed.We got 506 251 tags which were assembled from reads and 1527 OTUs(Operational Taxonomic Units) for 6 samples. Preliminary analysis demonstrated that 1) the OTUs mainly belonged to Rotaliida and Miliolida; 2) The diversity of foraminiferal communities in intertidal zone of Qingdao Bay is highest in July(summer), moderate in September and October(autumn), and lowest in May(spring); 3) pH was the main environmental factor affecting benthic foraminiferal richness; Among the top five genus, the relative abundance of Ammonia, Epistominella, Operculina, Bulimina(hyaline foraminifera) were positively associated with pH; On the contrary, the relative abundance of Textularia(agglutinated foraminifera) was negatively associated with pH.In summary, an optimal DNA-preservation and-extraction protocols were tested respectively for hyaline, porcelain and agglutinated foraminifera. Moreover, we optimized the method to apply HTS to investigate the molecular diversity of benthic foraminifera in intertidal zone of Qingdao Bay and analyzed the relationship between the molecular diversity and the environment.