Combined genotype and phenotype analyses reveal patterns of genomic adaptation to local environments in the subtropical oak Quercus acutissima

Understanding the effects of the demographic dynamics and environmental heterogeneity on the genomic variation of forest species is important not only for uncovering the evolutionary history of the species but also for predicting their ability to adapt to climate change. In this study, we combined a common garden experiment with range-wide population genomics analyses to infer the demographic history and characterize patterns of local adaptation in a subtropical oak species, Quercus acutissima. We scanned about 8% of the oak genome using a balanced representation of both genic and non-genic regions and identified a total of 55,361 SNPs in 167 trees. Genomic diversity analyses revealed an east-west split in the species distribution range. Coalescent-based model simulations inferred a late Pleistocene divergence in Q. acutissima between the east and west groups as well as subsequent pre-glaciation population expansion events. Consistent with observed genetic differentiation, morphological traits also showed east-west differentiation and the biomass allocation in seedlings was significantly associated with precipitation. Environment was found to have a significant and stronger impact on the non-neutral than the neutral SNPs, and also significantly associated with the phenotypic differentiation, suggesting that apart from the geography, environment had played a role in determining non-neutral and phenotypic variation. Our approach, which combined a common garden experiment with landscape genomics data, validated the hypothesis of local adaptation of this long-lived oak tree of subtropical China. Our study joins the small number of studies that have combined genotypic and phenotypic data to detect patterns of local adaptation.

Methods
The RAD-seq library was prepared using 200ng DNA from each individual tree; after extraction, DNA samples were digested with the restriction enzyme EcoRI. Illumina sequencing adaptor and a unique barcode were ligated to EcoRI-digested fragments. All the individual libraries were pooled and randomly sheared, and amplified by PCR using Illumina sequencing primers. DNA fragments with insert fragment sizes from 200–400 bp were isolated from a 2.0% agarose gel using QiaQuick gel extraction kits (Qiagen, Inc., Hilden, Germany). These fragments were then double–end sequenced on an Illumina HiSeq2500 (Illumina Inc., San Diego, USA).

We scored the phenotypic traits of three 1-year-old seedlings that were randomly selected from each population grown in the common garden.The traits that we measured included total mass, leaf area, leaf mass fraction (LMF, g leaf mass g-1 total mass), stem mass fraction (SMF, g stem mass g-1 total mass), root mass fraction (RMF, g root mass g-1 total mass), and specific taproot length (STRL, mm root length g-1 root mass), which measures the root extension achieved per unit root mass. LMF, SMF, and RMF represent biomass partitioning among the major organs, and STRL represents the efficiency of soil depth penetration to access deeper water.

解析种群动态与环境异质性对林木基因组变异的影响，不仅有助于揭示物种的演化历史，还能为预测其适应气候变化的能力提供重要依据。本研究将同质园实验（common garden experiment）与全分布区种群基因组学分析相结合，旨在推断亚热带栎属物种麻栎（Quercus acutissima）的种群历史，并解析其本地适应模式。

本研究通过兼顾编码区与非编码区的均衡取样，对该栎类基因组约8%的区域进行了扫描，在167个单株中共鉴定出55361个单核苷酸多态性位点（SNPs）。基因组多样性分析显示，该物种的分布区存在东西分化格局。基于溯祖模型的模拟分析表明，麻栎东西两个类群的分化发生在更新世晚期，且随后经历了冰期前的种群扩张事件。与观测到的遗传分化格局一致，该物种的形态性状同样呈现东西分化，且幼苗的生物量分配与降水量显著相关。

研究发现，环境对非中性SNPs的影响显著强于中性SNPs，且与表型分化显著相关，这表明除地理因素外，环境在非中性遗传变异与表型变异的塑造中发挥了重要作用。本研究将同质园实验与景观基因组学数据相结合，验证了中国亚热带地区这一长寿栎类物种的本地适应假说。本研究是少数结合基因型与表型数据以解析本地适应格局的研究之一。

## 材料与方法
### RAD测序文库构建与测序
本研究采用每份单株样本200ng基因组DNA进行RAD-seq（Restriction-site Associated DNA sequencing）文库构建：DNA提取完成后，使用限制性内切酶EcoRI对DNA样品进行酶切；将Illumina测序接头与唯一分子条形码连接至经EcoRI酶切的DNA片段。将所有个体文库混合后进行随机打断，再使用Illumina测序引物通过PCR扩增文库。通过2.0%琼脂糖凝胶电泳分离插入片段长度为200~400 bp的DNA片段，并使用QiaQuick凝胶回收试剂盒（Qiagen公司，德国希尔德）回收目标片段。随后在Illumina HiSeq2500测序平台（Illumina公司，美国圣地亚哥）上对这些DNA片段进行双端测序。

### 表型性状测定
我们对同质园栽培的每个种群随机选取的3株1年生幼苗进行表型性状测定。所测定的性状包括：总生物量、叶面积、叶质量分数（Leaf Mass Fraction, LMF，单位：g 叶质量/g 总生物量）、茎质量分数（Stem Mass Fraction, SMF，单位：g 茎质量/g 总生物量）、根质量分数（Root Mass Fraction, RMF，单位：g 根质量/g 总生物量）以及比主根长（Specific Taproot Length, STRL，即单位根质量对应的根延伸长度，单位：mm 根长/g 根质量）。其中LMF、SMF与RMF反映了植物主要器官间的生物量分配格局，STRL则表征了根系穿透土层以获取深层水分的效率。