Polymorphisms in two key anthocyanic genes of clivia (Clivia miniata L.) reveal evidence of selection and possible association with flower pigmentation

Members of the genus Clivia show considerable variation in flower pigmentation and morphology. Such variation is effected by mutations that emerge in candidate flower development genes over time. Besides population history, mutations can further illuminate the effects of demographic events in populations in addition to population genetic parameters including selection, recombination, and linkage disequilibrium (LD). The current study aimed to find sequence variants in two anthocyanin biosynthetic genes (DFR and bHLH) of Clivia miniata and use the data to assess population genetic factors from a random collection of orange/red- and yellow-flowered specimens. Overall, average nucleotide diversity in the two anthocyanic genes was moderate (π = 0.00646), whereas haplotypes differed significantly (Hd ≥ 0.9). Gene evolution was seemingly driven by mutations (CmiDFR) or recombination (CmibHLH001). LD decayed swiftly within the analyzed gene regions and supported the feasibility of assessing trait-variant associations via the association/linkage mapping approach. In the end, most associations were found to be spurious, but one haplotype in CmibHLH001 showed a promising correlation to the orange/red flower phenotype in Clivia specimens. In all, the present study is the first to measure gene-level diversity in C. miniata – data that had never been reported so far. Further, the study also identified allelic and haplotypic variants that may be beneficial in future association genetic studies of Clivia. Such studies, however, consider large diverse populations to control for statistical bias intrinsic to the analysis of small datasets.

Methods
Plants were randomly sourced from a private collector's pool as well as landscaped gardens of a university campus. After RNA isolation and cDNA synthesis, regions of two pigmentation-related genes, DFR and an ortholog of TRANSPARENT TESTA8, were PCR amplified and sequenced. The generated sequences were manually checked, edited and used to phase haplotypes using PHASE algorithms in the software DnaSP v5. Haplotype sequences were then used to calculate various population genetic parameters as well as to test for the possible correlation between the identified sequence variants and flower colour through case/control association tests.

君子兰属（Clivia）植物在花色素沉着与形态上存在显著变异。此类变异由随时间推移在候选花发育基因中产生的突变所介导。除种群历史外，突变还可进一步阐明种群中人口统计学事件的影响，同时也能用于解析包括选择、重组以及连锁不平衡（linkage disequilibrium, LD）在内的群体遗传参数。本研究旨在检测大花君子兰（Clivia miniata）的两个花青素生物合成基因（DFR与bHLH）中的序列变异，并利用这些数据对随机采集的橙红色花与黄色花样本的群体遗传因素进行评估。整体而言，这两个花青素相关基因的平均核苷酸多样性处于中等水平（π = 0.00646），而单倍型多样性差异显著（Hd ≥ 0.9）。基因演化似乎分别由突变（CmiDFR）或重组（CmibHLH001）驱动。在所分析的基因区域内，连锁不平衡快速衰减，这支持了通过关联/连锁作图方法评估性状-变异关联的可行性。最终，多数关联被证实为虚假关联，但CmibHLH001中的一个单倍型与君子兰样本的橙红色花表型呈现出具有前景的相关性。综上，本研究首次对大花君子兰的基因水平多样性进行了测定——此前尚无相关报道。此外，本研究还鉴定出了等位基因与单倍型变异，这些变异有望在未来君子兰的关联遗传研究中发挥作用。不过此类研究需要使用大型多样化种群，以控制小数据集分析固有的统计偏差。

材料与方法
研究材料随机采集自私人收藏种群以及大学校园的景观花园。在完成RNA提取与cDNA合成后，对两个色素相关基因（DFR以及透明种皮8（TRANSPARENT TESTA8）的同源基因）的区域进行PCR扩增并测序。对获得的序列进行人工校验与编辑后，利用DnaSP v5软件中的PHASE算法进行单倍型分型。随后利用单倍型序列计算多项群体遗传参数，并通过病例/对照关联检验，探究所鉴定的序列变异与花色之间的潜在关联。