桂花的全基因组重测序为花色进化提供了见解

Whole-genome resequencing of Osmanthus fragrans provides insights into flower color evolution

Abstract  抽象的

Osmanthus fragrans is a well-known ornamental plant that has been domesticated in China for 2500 years. More than 160 cultivars have been found during this long period of domestication, and they have subsequently been divided into four cultivar groups, including the Yingui, Jingui, Dangui, and Sijigui groups. These groups provide a set of materials to study genetic evolution and variability. Here, we constructed a reference genome of O. fragrans ‘Liuyejingui’ in the Jingui group and investigated its floral color traits and domestication history by resequencing a total of 122 samples, including 119 O. fragrans accessions and three other Osmanthus species, at an average sequencing depth of 15×. The population structure analysis showed that these 119 accessions formed an apparent regional cluster. The results of linkage disequilibrium (LD) decay analysis suggested that varieties with orange/red flower color in the Dangui group had undergone more artificial directional selection; these varieties had the highest LD values among the four groups, followed by the Sijigui, Jingui, and Yingui groups. Through a genome-wide association study, we further identified significant quantitative trait loci and genomic regions containing several genes, such as ethylene-responsive transcription factor 2 and Arabidopsis pseudoresponse regulator 2, that are positively associated with petal color. Moreover, we found a frameshift mutation with a 34-bp deletion in the first coding region of the carotenoid cleavage dioxygenase 4 gene. This frameshift mutation existed in at least one site on both alleles in all varieties of the Dangui group. The results from this study shed light on the genetic basis of domestication in woody plants, such as O. fragrans.

桂花（Osmanthus fragrans）是一种著名的观赏植物，在中国已有2500年的驯化历史。在这漫长的驯化过程中，人们发现了160多个栽培品种，它们被进一步划分为四个栽培品种群，包括银桂（Yingui）、金桂（Jingui）、丹桂（Dangui）和四季桂（Sijigui）群。这些品种群为研究遗传进化和变异提供了丰富的材料。

在此，我们构建了金桂（Jingui）群中的‘柳叶金桂’（Liuyejingui）的参考基因组，并通过对122个样本进行重测序（包括119个桂花品种和3个其他桂花物种，平均测序深度为15×），研究了其花色性状和驯化历史。群体结构分析表明，这119个品种形成了明显的区域聚类。连锁不平衡（LD）衰减分析结果显示，丹桂群中橙色/红色花色的品种经历了更多的人工定向选择；这些品种在四个品种群中具有最高的LD值，其次是四季桂、金桂和银桂群。

通过全基因组关联分析，我们进一步鉴定了与花瓣颜色显著相关的数量性状位点和基因组区域，其中包括乙烯响应转录因子2和拟南芥假反应调节器2等基因。此外，我们还发现了一个34个碱基对的缺失导致的移码突变，该突变发生在类胡萝卜素裂解双加氧酶4基因的第一个编码区。这种移码突变在丹桂群的所有品种中至少存在于两个等位基因中的一个位点上。

本研究的结果为木本植物（如桂花）的驯化遗传基础提供了新的见解。

Introduction  介绍

Sweet osmanthus (Osmanthus fragrans Lour.), belonging to the family Oleaceae, is a well-known ornamental germplasm native to the Sino-Himalayan region1. It has been cultivated in China for more than 2500 years. More than 160 cultivars of O. fragrans have been classified based on phenotypes, such as flower color and blooming season. They have been divided into four cultivar groups, including the Yingui group (Albus group), which has white to pale yellow flowers; the Jingui group (Luteus group), which has yellow flowers; the Dangui group (Aurantiacus group), which has orange/red flowers that bloom mainly in autumn for commercial harvest; and the Sijigui group (Asiaticus group), which has pale yellow to yellow flowers that bloom throughout most of the year2–4. It is thought that varieties in the Sijigui group and Yingui group are less differentiated from wild O. fragrans than the other two groups, which probably originated earlier2. The results of microsatellite marker analysis indicate that the varieties in the Jingui and Dangui groups, which displayed more significant genetic differentiation, might have diverged earlier4. Thus, the evolutionary relationships of varieties with different colors are still not clear.

桂花（Osmanthus fragrans Lour.）属于木犀科（Oleaceae），是一种原产于中喜马拉雅地区的著名观赏植物。它在中国已有超过2500年的栽培历史。根据表型特征（如花色和开花季节）对桂花品种进行了分类，目前已发现160多个品种，并被划分为四个品种群：银桂群（Albus group），花色为白色至淡黄色；金桂群（Luteus group），花色为黄色；丹桂群（Aurantiacus group），花色为橙色/红色，主要在秋季开花并用于商业采摘；四季桂群（Asiaticus group），花色为淡黄至黄色，全年大部分时间开花。据推测，四季桂群和银桂群的品种与野生桂花的分化程度较低，可能起源较早。微卫星标记分析的结果表明，金桂群和丹桂群的品种显示出更显著的遗传分化，可能较早分化。因此，不同花色品种的进化关系仍不明确。

Due to their ornamental and commercial value, flowers have long been a focus of interest in the study of O. fragrans. It has been reported that α-ionone and β-ionone are the main floral components of O. fragrans5–7. The accumulation of α-ionone and β-ionone in the cultivars of the Yingui, Jingui, and Sijigui groups is higher than that in the Dangui group, mainly due to the higher efficiency of carotenoid cleavage8,9. Furthermore, the presence of white, yellow, and orange color varieties is primarily attributable to the level of carotenoids, whereas flavonoids are speculated to provide only the background color10. Thus, the main differences in flower color and floral fragrance among varieties in different groups of O. fragrans are determined mainly by the degree of carotenoid accumulation and cleavage. Carotenoid cleavage dioxygenase 1 (CCD1) and CCD4 are crucial contributors to the cleavage of α-carotene and β-carotene into α-ionone and β-ionone9,11. The most critical factor determining the diversity of carotenoid concentrations was the differential expression level of CCD410,12. This leads to the question, what role does the CCD4 gene play in the evolution of O. fragrans flower color?

由于桂花的观赏和商业价值，其花朵一直是桂花研究的焦点。据报道，α-紫罗兰酮（α-ionone）和β-紫罗兰酮（β-ionone）是桂花的主要花香成分。银桂、金桂和四季桂品种中α-紫罗兰酮和β-紫罗兰酮的积累量高于丹桂品种，这主要是因为它们的类胡萝卜素裂解效率更高。此外，白色、黄色和橙色品种的存在主要归因于类胡萝卜素的水平，而黄酮类化合物被认为仅提供背景颜色。因此，桂花不同品种群之间的花色和花香差异主要由类胡萝卜素的积累和裂解程度决定。

类胡萝卜素裂解双加氧酶1（CCD1）和CCD4是将α-胡萝卜素和β-胡萝卜素裂解为α-紫罗兰酮和β-紫罗兰酮的关键酶。决定类胡萝卜素浓度多样性的最关键因素是CCD4的差异表达水平。这引发了以下问题：CCD4基因在桂花花色进化中扮演了什么角色？

More recently, genome sequencing of O. fragrans ‘Rixianggui’ (OFR) in the Sijigui group, which blooms for most of the year, has been performed at the chromosome level13. However, a systematic study to chart the genetic architecture of ornamental traits in a large population using a genome-wide association (GWA) method has not yet been performed. As most cultivars of O. fragrans bloom in autumn, we generated a reference genome for O. fragrans ‘Liuyejingui’ (OFL) from the Jingui group. In addition to the flowering time, OFR has fewer flowers at each blooming event, with a typical complete pedicel and pale yellow flower color. OFL produces many flowers that typically bloom for a week twice per year on average; the flowers are lemon yellow in color, with a strong fragrance and high essential oil contents, and are harvested for ornamental use as well as food and industrial uses14,15. We also reported on genomic variations and population evolution by resequencing 119 O. fragrans accessions with different colors from the four groups. We further sequenced the transcriptomes of different tissues of OFL, such as the rhizomes, leaves, flowers, and flowers, in different flowering stages to validate the quantitative trait loci (QTLs) and functional CCD4s through the expression of candidate genes between transcriptomes. For the first time, the present study explains the origin and evolutionary relationship of varieties in different groups of O. fragrans and color formation in the different varieties in terms of the deletion of the CCD4 gene structure.

最近，对四季桂群中全年大部分时间开花的“日香桂”（OFR）进行了染色体水平的基因组测序。然而，尚未进行利用全基因组关联（GWA）方法对大量种群的观赏性状遗传结构进行系统研究。由于大多数桂花品种在秋季开花，我们构建了金桂群中“柳叶金桂”（OFL）的参考基因组。除了开花时间外，OFR每次开花数量较少，具有典型的完整花梗和淡黄色花色。而OFL每年平均开花两次，每次持续一周，花朵呈柠檬黄色，具有浓郁的香气和高精油含量，既用于观赏，也用于食品和工业用途。

我们还通过对119个来自四个品种群且花色不同的桂花品种进行重测序，研究了它们的基因组变异和群体进化。此外，我们进一步测序了OFL不同组织（如根茎、叶片、花朵以及不同开花阶段的花朵）的转录组，通过候选基因在转录组之间的表达验证了数量性状位点（QTLs）和功能性的CCD4基因。

本研究首次解释了桂花不同品种群的起源和进化关系，以及不同品种花色形成的机制，这主要与CCD4基因结构的缺失有关。

Materials and methods  材料和方法

Plant materials  植物材料

For genome sequencing, leaf samples were collected from OFL on the campus of Huazhong Agricultural University (Wuhan, China) (114°21′ W, 30°29′ N). For resequencing, leaves were collected from 119 representative O. fragrans landraces and three close relatives of osmanthus, including O. cooperi, O. × fortunei, and O. heterophyllus (G. Don) P. S. Green var. Heterophyllus (Supplementary Table 1).
 

为了进行基因组测序，我们从位于中国武汉的华中农业大学校园内（114°21′E，30°29′N）的“柳叶金桂”（OFL）植株上采集了叶片样本。为了进行重测序，我们从119个具有代表性的桂花地方品种以及三种桂花的近缘种（包括O. cooperi、O. × fortunei和O. heterophyllus (G. Don) P. S. Green var. Heterophyllus）的植株上采集了叶片样本（补充表1）。

基因组测序和重测序 Genome sequencing and resequencing

Fresh, healthy leaves were harvested from the best-growing individuals and immediately frozen in liquid nitrogen, followed by preservation at −80 °C in the laboratory prior to DNA extraction. High-quality genomic DNA was extracted using a modified Cetyltrimethyl Ammonium Bromide method16. For genome sequencing, single-molecule real-time (SMRT) libraries were constructed and sequenced using a PacBio Sequel II instrument (Pacific Biosciences, Menlo Park, CA, USA) at Frasergen Bioinformatics Co., Ltd. (Wuhan, China). For resequencing, 1 μg DNA per sample was used as the input material, and sequencing libraries were generated using the VAHTS Universal DNA Library Prep Kit for MGI (Vazyme, Nanjing, China) following the manufacturer’s recommendations. Library quantification and size measurement were performed using a Qubit 3.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and a Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Subsequently, libraries of 122 accessions were constructed and sequenced on an MGI-SEQ 2000 platform at Frasergen Bioinformatics Co., Ltd.

从生长良好的植株上采集新鲜健康的叶片，并立即将其放入液氮中速冻，随后在实验室中于-80°C保存，