Evolutionary resequencing ( Evolve and Resequence, E&R ) is to study the adaptation process of the population to the new environment under laboratory conditions , and analyze the changes of genes during the adaptation process through the second-generation sequencing technology. It can monitor the molecular evolution process in real time at the genome level. E&R has been applied to research in RNA , bacteria, yeast and fruit flies. Although their studies are not completely the same, these differences can be explained to a large extent by using existing population genetics theories, including factors such as the number of initial populations, the level of initial gene variation, recombination rates, and the scope of adaptation.
The adaptation of organisms to the new environment has resulted in unparalleled biodiversity on earth. But how does this adaptation happen at the molecular level? Faced with such a problem, it is still difficult for us to give a reasonable and accurate answer. It is already a big challenge for us to identify favorable mutation sites that lead to species differentiation, let alone to speculate on the role and dynamic process of natural selection. Many issues related to adaptation remain controversial, such as the rate of evolution of favorable mutations, the size of their effects, and how they interact with each other, and how the environment affects the frequency of alleles.
In the past century, biologists in different fields have tried to study the process of adaptation through evolution in the laboratory. They found that the adaptation of different populations to the new laboratory environment can well reflect the adaptation process of organisms. However, due to the limitations of experimental technical conditions, these studies are only for phenotypes, and it is difficult to do it at the molecular level. Of course, with the advancement of sequencing technology, especially the second-generation sequencing technology, it is possible for people to study the process of adaptation at the molecular level. People have successively carried out evolutionary resequencing ( E&R ) studies on RNA molecules, viruses, bacteria, yeast and fruit flies . Through E&R research, people can monitor the evolution process in real time. This kind of real-time monitoring can help us understand some questions about the adaptation process, for example, does the existing mutation ( standing genetic variation ) play a large role in the adaptation , or the new mutation play a large role? Is evolution at the molecular level repeatable? In the process of adaptation, will the natural selection coefficient of favorable variation change with the process of adaptation? In the process of adaptation, what is the role of protein coding and regulatory regions? Wait.
There are four research systems for E&R research:
· In vitro single nucleotide research ( RNA or DNA )
· Monoclonal populations of bacteria and yeast populations,
· Originated from hybrid yeast populations,
• Originated from hybrid fruit fly populations.
The results of these four research systems are not completely consistent. For example, in vitro experiments often get many different results. The asexual reproductive system of microorganisms can usually observe a small number of favorable mutations fixed in the population, while the sexual reproductive system usually shows traits. Polygenic.
E&R : Research System Overview
In vitro studies Single Nucleotide system: usually derived from the short DNA sequences by DNA transcribed into RNA , then the RNA is exposed to different chemical systems selected, then expand and reverse transcriptase. Cycle 10-20 times in sequence . This can also be used for molecular screening. Since the "genome" of the in vitro system is relatively small, Sanger sequencing can identify the last RNA domain selected .
Asexually reproduced monoclonal population: usually a population of 10^6-10^8 bacteria or yeasts that have evolved through asexual reproduction. Because of the rapid growth of microorganisms, they can multiply for hundreds of generations within a few weeks to several months. Some people have E. coli for as long as 25 evolution experiments, a total of over more than 6 generations. In contrast, if human evolution 6 generations, you will need 120 million years, which is 120 million years ago, Homo sapiens has not yet appeared.
Yeast population system originating from hybridization: This population has both the characteristics of microbial populations, such as a large population, but also many new characteristics, such as sexual reproduction and genetic variation in the initial population. For the entire evolutionary population, sequencing can be performed by Pool-seq to obtain population gene frequencies.
A sexual eukaryotic system originating from hybridization: Drosophila is a representative of this system. The initial population comes from the wild, which means that there are many rare variations in the population. The population size can range from 100-1000 . As compared to microbes, flies a limited number of generations, usually 1-3 years of experiments can only breed 25-75 generations.
Dynamic changes of sites during selection
Although population genetics theory has many kinds of predictions and explanations about allele frequency changes, there are few real experimental data to support it. The E&R experiment can clearly understand the changes in the allele frequency during the adaptation process, including the rate of change, the amplitude of change, etc., by studying the gene frequency of the population at different time points.
In in vitro experiments, a site with an initial allele frequency of 10^-16 can be fixed in the population after 10 rounds of selection, which indicates that the favorable mutation is subject to strong positive selection, and the selection coefficient exceeds 50% , The genetic diversity in the population is rapidly lost, and the adaptability of the population is quickly increased.
In the in vitro experimental system, the haploid type in the population decreases rapidly with the process of evolutionary selection, and the gene heterozygosity also decreases rapidly.
For bacteria and fruit flies in monoclonal populations, the changes in allele frequencies can be complicated. Due to the lack of effective gene recombination, the interference effect between mutations is very obvious ( clonal interference ), and favorable mutations must compete fiercely with favorable mutations on other haploids. Only lucky ones can their genes be fixed in the population. Compared with in vitro experiments, the selection pressure of asexual reproduction sites is usually very good, between 1%-20% , which may be limited by the multiple effects of genes.
In the monoclonal population, the competition between different mutation sites is very fierce. The evolution process is always accompanied by the emergence of new and more favorable mutations. Each time point has a different dominant haplotype, and the population as a whole is heterozygous. The level of sex is very low.
For the hybrid yeast population that reproduces asexually, there are a large number of mutation types in the starting population, and the genetic diversity is high, so the population responds very quickly to selection. It is estimated that the selection pressure is only ~1% based on changes in allele frequency . The changes of alleles in the population can reflect how genes are recombined during adaptation. If a sequence of thousands of kb contains harmful or beneficial genes, this sequence can be clearly identified through this process. Moreover, almost all adaptation processes involve the participation of existing gene mutations rather than new mutation sites .
The initial population of the hybrid yeast population contains a large number of existing mutation types. Due to the effect of near-moderate selection, the frequency of one of the mutation types increases rapidly in the population; asexual reproduction lacks genetic recombination, so this haplotype is in the population It can exist for a long time; the heterozygosity of the group also decreases rapidly.
For sexual reproduction of fruit flies, the starting population is a group containing hundreds of natural haploids, but due to the existence of sexual reproduction and genetic recombination, interference effects between sites are unlikely to hinder evolution. Two different gene regions can independently respond to natural selection. Certain regions of the genome may have reduced heterozygosity due to natural selection, but it is difficult to reduce heterozygosity to zero , which also means that the selection occurs on the basis of existing mutations rather than new mutations ( soft selective sweep ).
Top image: Changes in the frequency of SNPs in the population before and after adaptation ; Middle image: Changes in haploids during evolution. Due to sexual reproduction and genetic recombination, genes in different regions have been recombined to form multiple New haploid; bottom picture: During the evolution process, heterozygosity changes, the heterozygosity near the selected site decreases (marked by the black arrow), and the heterozygosity changes from red to green
Through the comparison of the above several experimental systems, it can be seen that different experimental subjects have very different responses to selection, and there are also certain differences in the evolution process.
E&R实验中的突变
对于体外实验系统，分子的多样性通常受限于DNA合成平台，比如一个DNA合成平台通常能够产生10^16种分子，而一个60个碱基长度的序列的分子多样性可以达到4^60，这个理论数量要比实际分子数量多20个数量级。所以选择可以发挥作用的空间很大。
对于无性单克隆系统，尽管基因之间的相互干扰会延长基因固定的时间，但是和其他系统相比，它的基因多样性仍然是偏低的。由于其起源群体遗传背景单一，所以群体中的变异类型多是新突变产生的。而且对于每一种新环境，似乎总有很多有利突变。在细菌和酵母的E&R中，发现了很多不同类型的突变。尽管点突变依旧是多数，但是小的插入缺失突变和较大的重复序列及缺失，以及转座子都对新环境的适应发挥了重要作用。很多受选择的突变都涉及到某些功能缺失，比如提前出现的终止密码子、转座子在基因中插入等。似乎选择压力倾向于某些细胞功能的丢失。
在无性繁殖的单克隆酵母群体中，通过E&R实验发现大量重复序列可以用来应对环境压力。通过增加基因拷贝数，能够补偿有害突变的作用或者提升个体对资源有限的外界环境的适应。但是对于杂交的微生物群体，其适应环境主要是在现有变异基础之上发生的，在E&R实验中，并没有观测到大规模的基因结构突变和拷贝数突变。一个可能的解释是，相对于新的突变，现有基因变异在群体中存在了一定的时间，其对群体带来的负面作用比突变要小很多，所以现有基因变异更容易被选择下来。
关于新突变和现有变异哪一个对适应更重要的争论还有没有结束。在对人类基因组的研究中，显示现有变异对适应的意义更大，不过在对果蝇的研究中，却得到了相反的结论，认为新突变对果蝇适应新的环境更有意义。也就是说，对于一个种群数量较大的群体而言，新突变的进化的意义可能比现有变异的意义更大。
目前为止，针对果蝇的E&R的实验还不能精确的识别出是哪一个基因和环境适应有关系，通常只能够识别出数百Kbp的一段序列，该序列中杂合度水平降低，SNP频率改变。此外，果蝇的E&R实验能否识别出适应的发生很大程度上依赖于实验设计，比如实验的重复数、群体数量、经历的世代数等。重复数多、群体大、世代数多能够大大提升对受选择基因的识别能力和在基因组上的定位能力。
平行进化（Parallel evolution）
平行进化是指来自同一个群体的两个分支独立进化，但进化过程和结果很相似。与自然条件下的进化不同，E&R能够精确的控制环境条件，能够同时进行多个重复，因而E&R是研究平行进化的最有效的工具。
对于无性繁殖的单克隆的酵母和细菌群体，进化依赖于新出现的突变，但是突变的随机性导致了在突变水平上，很难观测到平行进化现象；不过在更高一级的基因水平和细胞功能水平，是可以看到平行进化现象的，即某些基因或细胞功能是不同分支群体相同的进化靶标，虽然这些基因或细胞功能内有具体不同的突变情况。比如影响细胞代谢的某些基因，或者影响细胞形状的基因，亦或者某些应对外界环境压力的基因。
对于杂交E&R研究系统，起始群体中已经存在了大量的多态性位点，但是可能只有其中的一小部分是进化选择的靶标。要识别出这些靶标，E&R的设计很关键，即增加实验的重复数量能够很有效的提升识别这些靶标的能力。在果蝇的E&R研究中，平行进化是很常见的。首先因为其实群体有很多相同的变异位点，其中的很多变异位点，可能是不同实验重复共同的作用靶标。其次，由于存在有性生殖和基因重组，因而位点之间的相互干扰很微弱，也就意味着不同的有利位点可以同时且相互独立地提升在群体中的频率。
不过也要注意到，在观察平行进化的同时，重复之间可能存在的基因流可能对观察结果造成影响，虽然这些基因流可能是无意中发生的，但哪怕每一代只有一个个体的基因交换，那么这两个重复之间的中性等位基因频率也会被同质化。
上位效应（Epistasis）
上位效应是指基因之间的相互影响。在体外E&R系统（如体外RNA分子刷选实验）中，上位效应是普遍存在的，而且这种上位效应能够在很短的序列内发生，所以二代测序的一个读长通常就足够来了解不同变异之间的相互影响。不过对于杂交E&R系统（果蝇），观察到基因之间的上位效应很困难。这可能是由于杂交系统的有利变异可以存在于多种单倍体上，而自然选择可以直接作用在这些单倍体上。在杂交E&R系统中，最简单识别上位效应的方法就是识别出非关联位点的连锁不平衡（LD），不过如果是用Pool-seq池测序的方法来测序，只能得到等位基因频率，无法得到不同位点的关联性，所以在方法上，也很难识别出杂交E&R系统的上位效应。
总结
E&R research can be used for 30bp nucleic acid sequences, and can be used for multicellular eukaryotes like fruit flies. The evolutionary principles are the same. The biggest difference comes from their response speed to the new environment and the molecular basis of evolution. The characteristics of the initial population and the pressure of natural selection are the main reasons for the above differences. Asexual reproduction in the population, the mutual interference between the gene ( clonalinterference ) determines the different types of competing haploid, sexual reproduction in populations, may be relatively independent of pressure response between gene selection. In the E&R system (hybridization system) that originated from existing mutations , the process of adaptation is relatively certain, and all experimental repeats have similar mutation sites for natural selection; while for monoclonal populations, evolution is based on new The resulting mutations, so the evolution process is full of uncertainty and randomness.
Finally, although E&R research is based on the laboratory simulation of natural selection process, it has brought great convenience to our research; but we must see that there are still certain differences between laboratory simulation and the real natural selection process.
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文献来源：Long, A., Liti, G., Luptak, A., & Tenaillon, O. (2015). Elucidating the molecular architecture of adaptation via evolve and resequence experiments. Nature Reviews Genetics, 16(10), 567.
