Precise measurement of the fitness effects of spontaneous mutations by droplet digital PCR in Burkholderia cenocepacia
Precise measurement of the fitness effects of spontaneous mutations by droplet digital PCR in Burkholderia cenocepacia
复制标题
液滴数字PCR精确测量新洋葱伯克霍尔德菌自发突变的适应度效应
DOI：
10.1093/genetics/iyab117
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发表时间：
2021
期刊：
影响因子：
3.3
通讯作者：
Sung, Way
中科院分区：
文献类型：
--
作者：
Rana, Anita;Patton, David;Turner, Nathan T;Dillon, Marcus M;Cooper, Vaughn S;Sung, Way
了解突变如何影响生存能力是了解生物体和复杂特征如何进化的关键组成部分。然而，大多数突变对适应度的影响很小，这些影响很难用传统的分子技术来解决。因此，迫切需要更准确和精确的体能测量方法。在这里，我们用滴状数字聚合酶链式反应(DdPCR)检测了伯克霍尔德氏菌HI2424突变积累(MA)系的适合度效应。总体而言，DdPCRMA的适合度与传统表型标记法得出的适合度呈正相关(r= 0.297，P= 0.0 5)，但显示出一些差异。首先，在对照实验中，ddPCR的适合度的测量方差显著较低(F= 3.78，P<； 2.6×10−13)。其次，DDPCRMA测量的平均适合度显著低于表型标记物分析(−0.0041vs−0.0071，P= 0.006)。与表型标记分析一致，ddPCR-MA测量观察到多个(27/43)谱系显著偏离平均适合度，表明大多数突变是中性的或略有有害的，并与少数具有极大影响的突变混合在一起。在这些突变中，我们发现DNA核酸外切酶和Lys R转录调节因子中有大量的突变，这些突变具有极端有害和有益的影响，表明对转录和复制的修改可能对生物体的适应性有很大的影响。这项研究证明了ddPCR作为一种普遍存在的方法在基于DNA和RNA的生物体中进行高通量适应性测量的能力，而不考虑细胞类型或生理。
Understanding how mutations affect survivability is a key component to knowing how organisms and complex traits evolve. However, most mutations have a minor effect on fitness and these effects are difficult to resolve using traditional molecular techniques. Therefore, there is a dire need for more accurate and precise fitness measurements methods. Here, we measured the fitness effects inBurkholderia cenocepaciaHI2424 mutation accumulation (MA) lines using droplet-digital polymerase chain reaction (ddPCR). Overall, the fitness measurements from ddPCR-MA are correlated positively with fitness measurements derived from traditional phenotypic marker assays (r= 0.297,P= 0.05), but showed some differences. First, ddPCR had significantly lower measurement variance in fitness (F= 3.78,P< 2.6 × 10−13) in control experiments. Second, the mean fitness from ddPCR-MA measurements were significantly lower than phenotypic marker assays (−0.0041vs−0.0071,P= 0.006). Consistent with phenotypic marker assays, ddPCR-MA measurements observed multiple (27/43) lineages that significantly deviated from mean fitness, suggesting that a majority of the mutations are neutral or slightly deleterious and intermixed with a few mutations that have extremely large effects. Of these mutations, we found a significant excess of mutations within DNA excinuclease and Lys R transcriptional regulators that have extreme deleterious and beneficial effects, indicating that modifications to transcription and replication may have a strong effect on organismal fitness. This study demonstrates the power of ddPCR as a ubiquitous method for high-throughput fitness measurements in both DNA- and RNA-based organisms regardless of cell type or physiology.