BSA-seq、BSR-seq 和 RNA-seq 的关联分析揭示了茶群体（Camellia sinensis）中紫叶形成所涉及的关键基因

Association analysis of BSA-seq, BSR-seq, and RNA-seq reveals key genes involved in purple leaf form

Abstract  抽象的

Purple tea, rich in anthocyanins, has a variety of health benefits and is attracting global interest. However, the regulation mechanism of anthocyanin in purple tea populations has not been extensively studied. In this experiment, RNA-seq, BSA-seq, and BSR-seq were performed using 30 individuals with extreme colors (dark-purple and green) in an F1 population of ‘Zijuan’ and ‘Jinxuan’. The results show that 459 genes were differentially expressed in purple and green leaves, among which genes involved in the anthocyanin synthesis and transport pathway, such as CHS, F3H, ANS, MYB75, GST, MATE, and ABCC, were highly expressed in purple leaves. Moreover, there were multiple SNP/InDel variation sites on chromosomes 2 and 14 of the tea plant, as identified by BSA-seq. The integrated analysis identified two highly expressed genes (CsANS and CsMYB75) with SNP/InDel site variations in the purple tea plants. By silencing leaves, we proved that CsMYB75 could positively regulate anthocyanin accumulation and expression of related structural genes in tea plants. A 181-bp InDel in the CsMYB75 promoter was also found to be co-segregating with leaf color. The results of this study provide a theoretical reference for the molecular mechanism of anthocyanin accumulation in purple tea plants and contribute to the creation of new tea cultivars with high anthocyanin content.
紫茶富含花青素，具有多种健康益处，正在吸引全球的关注。然而，紫茶群体中花青素的调控机制尚未得到广泛研究。在本实验中，使用“紫鹃”和“锦轩”F 1 群体中的 30 个具有极端颜色（深紫色和绿色）的个体进行 RNA-seq、BSA-seq 和 BSR-seq 。结果表明，459个基因在紫色和绿色叶片中差异表达，其中涉及花青素合成和转运途径的基因，如CHS、F3H、ANS、MYB75、GST、MATE和ABCC在紫色叶片中高表达。此外，经BSA-seq鉴定，茶树2号和14号染色体上存在多个SNP/InDel变异位点。综合分析确定了紫茶树中两个具有 SNP/InDel 位点变异的高表达基因（CsANS 和 CsMYB75）。通过沉默叶片，我们证明CsMYB75可以正向调节茶树花青素的积累和相关结构基因的表达。 CsMYB75 启动子中的 181 bp InDel 也被发现与叶色共分离。本研究结果为紫茶树花青素积累的分子机制提供了理论参考，有助于创建高花青素含量的茶树新品种。

Introduction  介绍

The tea plant originated in China and is extensively cultivated worldwide as a valuable cash crop. The leaves of regular tea plants are green, while purple tea emerged through long-term natural evolution. Purple tea, known for its high level of anthocyanins, is made from the buds of purple tea plants and is a natural health beverage rich in secondary metabolites of flavonoids [1]. Studies have shown that purple tea extract can regulate the intestinal microbiota and improve obesity and metabolic disorders caused by high fat intake [2]. Its anti-inflammatory and anti-proliferative properties may also be used for cancer prevention [3]. In addition, purple tea has a strong antioxidant capacity, which shows benefits in anti-aging [4], neuroprotection [5], and other aspects. Taking these properties together, the purple tea plant has high research value as a potential resource of natural raw materials for functional tea beverages.
茶树起源于中国，作为一种有价值的经济作物在世界范围内广泛种植。普通茶树的叶子是绿色的，而紫茶是经过长期的自然进化而出现的。紫茶以花青素含量高而著称，由紫茶树的芽制成，是一种富含黄酮类次级代谢产物的天然保健饮料[1]。研究表明，紫茶提取物可以调节肠道菌群，改善高脂肪摄入引起的肥胖和代谢紊乱[2]。其抗炎和抗增殖特性也可用于预防癌症[3]。此外，紫茶具有很强的抗氧化能力，在抗衰老[4]、神经保护[5]等方面表现出功效。综合这些特性，紫茶树作为功能性茶饮料天然原料的潜在资源具有很高的研究价值。

Anthocyanins belong to the flavonoids, a class of natural pigments widely distributed in nature, and their accumulation is the primary cause of purple coloration in plants [6]. Studies have shown that the significant upregulation of structural genes, such as anthocyanidin synthase (ANS), dihydroflavonol reductase (DFR), and flavonoid-3-O-glycosyltransferase (UFGT), and key regulatory genes like MYB and bHLH can promote the transfer of metabolites towards anthocyanin biosynthesis [7–9]. Furthermore, He et al. isolated the transcription factor gene CsMYB6A in purple leaves, and its overexpression could activate the expressions of flavonoid-related structural genes [10]. Sun et al. activated the transcription factor gene CsAN1, which specifically upregulated the late anthocyanin biosynthesis genes, leading to the ectopic accumulation of purple pigment [11]. Wei et al. found that the transcription factor gene CsMYB75 and the glutathione transferase gene CsGSTF1 are related to the purple leaf color phenotype [12, 13]. These studies provide valuable resources for the evolution and breeding of purple tea plants. However, the complex genetic background of various tea cultivars increases the complexity of pigment biosynthesis, and the results obtained for a single-purple cultivar as a research object cannot be generally applied to tea plant populations. The precise exploration of the molecular mechanism of purple formation therefore remains an ongoing objective.
花青素属于黄酮类化合物，是自然界中广泛分布的一类天然色素，其积累是植物呈现紫色的主要原因[6]。研究表明，花青素合酶（ANS）、二氢黄酮醇还原酶（DFR）、类黄酮-3-O-糖基转移酶（UFGT）等结构基因以及MYB、bHLH等关键调控基因的显着上调可以促进花青素生物合成的代谢物[7-9]。此外，He 等人。等人在紫色叶片中分离出转录因子基因 CsMYB6A，其过表达可激活黄酮类相关结构基因的表达[10]。孙等人。激活转录因子基因CsAN1，特异性上调晚期花青素生物合成基因，导致紫色素异位积累[11]。魏等人。发现转录因子基因CsMYB75和谷胱甘肽转移酶基因CsGSTF1与紫色叶色表型相关[12, 13]。这些研究为紫茶树的进化和育种提供了宝贵的资源。然而，各茶品种复杂的遗传背景增加了色素生物合成的复杂性，以单一紫色品种为研究对象所获得的结果不能普遍应用于茶树群体。因此，对紫色形成的分子机制的精确探索仍然是一个持续的目标。

In recent years, various combinations of different high-throughput technologies, such as quantitative trait locus (QTL) analysis, bulked segregant RNA (BSR) analysis, bulked segregant analysis (BSA), and RNA-seq, have been used to explore the potential mechanism of plant color and more accurately locate related genes. Relative studies on oilseed rape red flower [14] and barley black skin [15] show that the combinations of these methods are highly efficient and powerful in the identification of key genes. Therefore, the above multi-technique combined analysis also presents a new approach for identifying potential genes associated with anthocyanin accumulation in tea plants.
近年来，不同高通量技术的各种组合，如数量性状位点（QTL）分析、批量分离RNA（BSR）分析、批量分离分析（BSA）和RNA-seq等，已被用来探索潜在的潜力。植物颜色机制，更准确地定位相关基因。对油菜红花[14]和大麦黑皮[15]的相关研究表明，这些方法的组合在关键基因的鉴定方面是高效且强大的。因此，上述多技术联合分析也为鉴定茶树花青素积累相关的潜在基因提供了一种新的方法。

In this study, we obtained a hybrid population of purple tea cultivar ‘Zijuan’ (ZJ) and green leaf tea cultivar ‘Jinxuan’ (JX), from which a total of 30 tea individuals with extreme leaf colors (15 dark-purple leaf individuals and 15 green leaf individuals) were selected (Fig. 1A). BSA-seq and BSR-seq were used to reveal the candidate regions and gene mutation sites associated with purple leaf formation in tea plants, and RNA-seq was used to identify the differentially expressed genes (DEGs) among different leaf colors. The integrated analysis aimed to find the key genes and variation patterns for purple leaf formation in the tea population.
在本研究中，我们获得了紫娟茶品种（ZJ）和绿叶茶品种金轩（JX）的杂交群体，其中共有30个具有极端叶色的茶树个体（15个深紫叶个体）和15个绿叶个体）被选择（图1A）。 BSA-seq和BSR-seq用于揭示茶树紫色叶片形成相关的候选区域和基因突变位点，RNA-seq用于识别不同叶色之间的差异表达基因（DEG）。综合分析旨在寻找茶群体紫叶形成的关键基因和变异模式。

图1A 

Results  结果

Differentially expressed genes between purple and green individuals in the tea population
茶群体中紫色和绿色个体之间的差异表达基因

A hybrid population of purple tea cultivar ZJ and green leaf cultivar JX, which includes 127 individuals, was used in this study. This population varied in their leaf coloration from being totally green (including 18 individuals) to dark purple (32 individuals) (Supplementary Data Fig. S1). A total of 30 tea individuals with extreme leaf colors (15 dark purple and 15 green leaf individuals) were used for subsequent analysis. To identify genes potentially associated with the leaf color phenotype, autumn samples were subjected to RNA-seq analysis. More than 42 011 640 raw reads were obtained and 39 532 308 clean reads were obtained after removing the adapter and low-quality reads. The sequencing shows that the RNA-seq result was of high quality (Q30⩾ 93.57%, Q20 ⩾ 97.82%) (Supplementary Data Table S1), and >90.50% of clean read segments were successfully mapped to the ‘Tieguanyin’ reference genome (Supplementary Data Table S2). Principal component a