Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae
Symbiodiniaceae community structure in corals is crucial for understanding the plasticity of different holobionts under environmental stress. While this relies on molecular analyses, accuracy of molecular quantification, as influenced by DNA extraction efficiency and rDNA copy number variations in p...
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doaj-b435e1c25dee4166a34ac2ebb3a3a8862020-11-25T03:18:55ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2020-05-011110.3389/fmicb.2020.00847528552Genome Size, rDNA Copy, and qPCR Assays for SymbiodiniaceaeOsama S. Saad0Osama S. Saad1Xin Lin2Tsz Yan Ng3Ling Li4Put Ang5Put Ang6Senjie Lin7Senjie Lin8State Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, ChinaDepartment of Biological Oceanography, Red Sea University, Port-Sudan, SudanState Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, ChinaMarine Science Laboratory, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, ChinaState Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, ChinaMarine Science Laboratory, School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, ChinaInstitute of Space and Earth Information Science, The Chinese University of Hong Kong, Hong Kong, ChinaState Key Laboratory of Marine Environmental Science, College of Ocean and Earth Sciences, Xiamen University, Xiamen, ChinaDepartment of Marine Sciences, University of Connecticut, Groton, CT, United StatesSymbiodiniaceae community structure in corals is crucial for understanding the plasticity of different holobionts under environmental stress. While this relies on molecular analyses, accuracy of molecular quantification, as influenced by DNA extraction efficiency and rDNA copy number variations in particular, has rarely been systematically investigated. Here, we report the development of a set of genus-specific qPCR assays. First, a protocol for efficient DNA isolation and accurate measurements of genome size and rDNA copy number was established. Second, seven newly designed genus-specific ITS2 primer sets were validated using computational and empirical analyses and qPCR assays were developed. We find that while the genome size ranges between 1.75 ± 0.21 and 4.5 ± 0.96 Gbp, rDNA copy number shows over 10-fold variation among Symbiodiniaceae species. Our protocol produced standard curves with high efficiencies (89.8–99.3%; R2 ≥ 0.999) and tight Cq values over different PCR conditions, illustrating high specificity and sensitivity of the qPCR assays. Tested on mock communities of mixed culture species, our qPCR results agreed well with microscopic counts and facilitated calibration of metabarcoding data. To test the applicability of our protocol for field samples, we analyzed three different Hong Kong coral samples. Six Symbiodiniaceae genera were detected in Acropora valida, Oulastrea crispata, and Platygyra acuta, with Breviolum, Effrenium, Fugacium, and Gerakladium sp. being reported for the first time. Our results suggest that aggressively disrupting cells to ensure thorough cell lysis, estimating cell loss and DNA loss, and validating qPCR assays are critical for success. The number of species examined here is limited, but the primers are potentially applicable to most species in respective genera, and the protocol and the approach to develop it provide a base and template toward a standardized procedure for quantitatively characterizing Symbiodiniaceae communities in corals.https://www.frontiersin.org/article/10.3389/fmicb.2020.00847/fullSymbiodiniaceaegenome sizerDNASpecific-qPCRHTScoral |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Osama S. Saad Osama S. Saad Xin Lin Tsz Yan Ng Ling Li Put Ang Put Ang Senjie Lin Senjie Lin |
spellingShingle |
Osama S. Saad Osama S. Saad Xin Lin Tsz Yan Ng Ling Li Put Ang Put Ang Senjie Lin Senjie Lin Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae Frontiers in Microbiology Symbiodiniaceae genome size rDNA Specific-qPCR HTS coral |
author_facet |
Osama S. Saad Osama S. Saad Xin Lin Tsz Yan Ng Ling Li Put Ang Put Ang Senjie Lin Senjie Lin |
author_sort |
Osama S. Saad |
title |
Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae |
title_short |
Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae |
title_full |
Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae |
title_fullStr |
Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae |
title_full_unstemmed |
Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae |
title_sort |
genome size, rdna copy, and qpcr assays for symbiodiniaceae |
publisher |
Frontiers Media S.A. |
series |
Frontiers in Microbiology |
issn |
1664-302X |
publishDate |
2020-05-01 |
description |
Symbiodiniaceae community structure in corals is crucial for understanding the plasticity of different holobionts under environmental stress. While this relies on molecular analyses, accuracy of molecular quantification, as influenced by DNA extraction efficiency and rDNA copy number variations in particular, has rarely been systematically investigated. Here, we report the development of a set of genus-specific qPCR assays. First, a protocol for efficient DNA isolation and accurate measurements of genome size and rDNA copy number was established. Second, seven newly designed genus-specific ITS2 primer sets were validated using computational and empirical analyses and qPCR assays were developed. We find that while the genome size ranges between 1.75 ± 0.21 and 4.5 ± 0.96 Gbp, rDNA copy number shows over 10-fold variation among Symbiodiniaceae species. Our protocol produced standard curves with high efficiencies (89.8–99.3%; R2 ≥ 0.999) and tight Cq values over different PCR conditions, illustrating high specificity and sensitivity of the qPCR assays. Tested on mock communities of mixed culture species, our qPCR results agreed well with microscopic counts and facilitated calibration of metabarcoding data. To test the applicability of our protocol for field samples, we analyzed three different Hong Kong coral samples. Six Symbiodiniaceae genera were detected in Acropora valida, Oulastrea crispata, and Platygyra acuta, with Breviolum, Effrenium, Fugacium, and Gerakladium sp. being reported for the first time. Our results suggest that aggressively disrupting cells to ensure thorough cell lysis, estimating cell loss and DNA loss, and validating qPCR assays are critical for success. The number of species examined here is limited, but the primers are potentially applicable to most species in respective genera, and the protocol and the approach to develop it provide a base and template toward a standardized procedure for quantitatively characterizing Symbiodiniaceae communities in corals. |
topic |
Symbiodiniaceae genome size rDNA Specific-qPCR HTS coral |
url |
https://www.frontiersin.org/article/10.3389/fmicb.2020.00847/full |
work_keys_str_mv |
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