Data_Sheet_1_Genome Size, rDNA Copy, and qPCR Assays for Symbiodiniaceae.zip

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.

珊瑚中Symbiodiniaceae群落结构对于理解不同全生物体在环境压力下的可塑性至关重要。尽管这依赖于分子分析，但受DNA提取效率和rDNA拷贝数变异等因素影响的分子定量准确性，却鲜有系统性的研究。本研究中，我们报告了一组属特异性qPCR检测方法的开发。首先，建立了一种高效DNA分离和准确测量基因组大小及rDNA拷贝数的方案。其次，通过计算和实证分析验证了七套新设计的属特异性ITS2引物集，并开发了qPCR检测方法。我们发现，基因组大小介于1.75 ± 0.21至4.5 ± 0.96 Gbp之间，而rDNA拷贝数在Symbiodiniaceae物种间显示出超过10倍的变异。我们的方案产生了高效率的标准曲线（89.8–99.3%；R2 ≥ 0.999）和在不同PCR条件下的紧密Cq值，展现了qPCR检测方法的高特异性和敏感性。在混合培养物种的模拟群落中测试，我们的qPCR结果与显微镜计数结果吻合良好，并促进了元条形码数据的校准。为了测试本方案在野外样本中的应用性，我们分析了三个不同的香港珊瑚样本。在Acropora valida、Oulastrea crispata和Platygyra acuta中检测到六种Symbiodiniaceae属，其中Breviolum、Effrenium、Fugacium和Gerakladium sp.首次被报道。我们的结果提示，破坏细胞以确保彻底裂解、估计细胞损失和DNA损失、以及验证qPCR检测方法是成功的关键。虽然在此处检验的物种数量有限，但这些引物可能适用于各自属中的大多数物种，且本方案及其开发方法为建立标准化程序、定量描述珊瑚中Symbiodiniaceae群落提供了一个基础和模板。