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The genome of Eustoma grandiflorum reveals the whole-genome triplication event contributing to ornamental traits in cultivated lisianthus
Plant Biotechnology Journal ( IF 13.8 ) Pub Date : 2022-07-25 , DOI: 10.1111/pbi.13899
Yuwei Liang 1 , Fan Li 2 , Qiang Gao 1 , Chunlian Jin 2 , Liqing Dong 3 , Qi Wang 1 , Min Xu 1 , Fuhui Sun 1 , Bo Bi 1, 4 , Peng Zhao 3 , Shenchong Li 2 , Jiwei Ruan 2 , Xiaofan Zhou 5 , Liangsheng Zhang 1, 6 , Jihua Wang 2
Affiliation  

Introduction

Eustoma grandiflorum (lisianthus) is a Gentianaceae-family ornamental plant. Because of its enormous rose-like blossoms, long stems and extended vase life, its sales have increased dramatically in recent years, earning it the title of ‘next rose’. Selective breeding has produced commercial lisianthus with a wide range of flower colours and shapes (Figure S1a; Li et al., 2022). In polyploid crops including wheat, cotton, peanuts and others, polyploidy is critical for the development of high-quality traits (Cheng et al., 2018). Polyploidy may also contribute to the development of desirable traits in cultivated lisianthus. Here, we report a high-quality chromosome-scale genome assembly for E. grandiflorum (2n = 6x = 72) using a combination of PacBio HiFi reads and Hi-C scaffolding technology and reveal that polyploidy domestication of lisianthus contributes to ornamental traits in cultivated lisianthus.

A total of 32.05 Gb (~23.56X) of PacBio HiFi data and 140.14 Gb (~103.04X) Hi-C data were generated for de novo whole-genome sequencing. The total length of the assembly was 1.71 Gb, comprising 1056 contigs with a corresponding N50 of 7.33 Mb (Table S1), and 36 pseudo-chromosomes were assembled (Figures S1 and S2, Table S2). BUSCO revealed a completeness rate of 94.7% and a duplication rate of 31.6% (Table S5). A total of 54 305 high-quality protein-coding genes were predicted (Tables S3–S8). In addition, 77.85% of the genome was annotated to be repeat sequences (Tables S9–S12).

Genome collinearity identified a large number of collinear blocks with a ratio of 6 : 1 between E. grandiflorum and Gelsemium sempervirens (Figure 1a), and a ratio of 2 : 1 between E. grandiflorum and E. grandiflorum (Figure S3), indicative of polyploidy events' existence. To this end, the 36 pseudo-chromosomes could be divided into three subgenomes, then 12 homologous groups with three sets of monoploid chromosomes: A, B and C were obtained according to the transposable element profiles (Figure S4, Table S10–S12). The reasonable collinearity within subgenomes suggested that E. grandiflorum had experienced a whole-genome duplication (WGD) event in the recent history of E. grandiflorum (Figure S5). The Ks distribution further confirmed that E. grandiflorum experienced a WGD event (Ks peak value = 0.93) and a whole-genome triplication (WGT) event (Ks peak value = 0.21) after divergence from Calotropis gigantea, which is consistent with the ratio of 6 : 1 (E. grandiflorum: G. sempervirens) and the evolutionary relationships depicted by the phylogenetic tree (Figures 1a, S6–S7).

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Figure 1
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(a) Alignment of Eustoma grandiflorum chromosomes with Gelsemium sempervirens scaffolds. (b) Histograms showing expression profiles of essential regulators of flower coloration in lisianthus. S1, bud stage; S2, turning stage; S3, blooming stage. FLS, flavonol synthase; CHS, chalcone synthase; ANS, anthocyanidin synthase; F3′5′H, flavonoid 3′,5′-hydroxylase; CRD, Mg-protoporphyrin IX monomethyl ester cyclase; CHLH/D/I, Mg-chelatase subunits H, D and I; PORA/B/C, protochlorophyllide reductase. (c) A proposed model of flower coloration in lisianthus. The potential simplified anthocyanin biosynthesis pathway in lisianthus is depicted. CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; DFR, dihydroflavonol-4-reductase; GT, glucosyltransferase; AT, acyltransferase. Essential regulators of flower coloration in lisianthus are in red fonts. The main pigments refer to the reference (Gao and Li, 2020). (d) A proposed floral development model of lisianthus based on the expression patterns of floral identity genes. Heatmap shows the number of MADS genes across various species. (e) Histograms showing the expression levels of C-class MADS genes in lisianthus. In all samples, AGL1d is zero in FPKM, hence it is not displayed. [Colour figure can be viewed at wileyonlinelibrary.com]

A total of 15 436 genes belonging to syntenic gene groups resulting from the WGT event were found based on collinearity across E. grandiflorum subgenomes, which were enriched in processes of external stimulus response, anatomical structure morphogenesis and biosynthetic process (Figure S8, Table S13). Substantial copy number variations of transcriptional factors (TFs) and structural genes participating in flavonoid/anthocyanin biosynthesis were discovered as a result of WGT including MYBs, bHLHs, CHIs, CHSs, DFRs, GTs and FLSs (Figures S9–S15). Thus, it was hypothesized that the polyploidy event would enable the breeding of colourful lisianthus varieties by providing enhanced genetic materials for anthocyanin production. We also found that numerous TFs regulating organ/flower development were generated by WGT, including floral identity genes in the MADS family, and members in the TCP family and HD-Zip III family, which might influence the formation of E. grandiflorum's flower morphology (Figures 1d, S16–S17).

To examine the genetic pathways regulating flower pigmentation in lisianthus, we performed RNA-seq of petals in the purple lisianthus (Purple01), the green lisianthus (Green01), the yellow lisianthus (Yellow01), the red lisianthus (Red01) and ‘Rosita White’ at different stages (bud stage, S1; turning stage, S2; blooming stage, S3) and conducted weighted correlation network analysis (WGCNA) (Figure S18). Genes involved in flavonoid biosynthesis were identified, and the putative anthocyanin biosynthesis pathway was depicted (Figures 1c, S11–S15, S19). We found that FLSa was highly expressed in S1 and S2, and was barely expressed in S3 (Figure 1b). In S3, we found that CHSa expression level was significantly higher in all coloured cultivars than in white cultivars, and ANSa/b displayed much higher expression levels in both pink/red and blue/violet cultivars (Figures 1b, S20). Moreover, compared to pink/red lisianthus, blue/violet lisianthus showed higher F3′5′Ha/b levels (Figures 1b, S20). Co-expression network reconstruction of the module specific to S3 of lisianthus Purple01 (‘darkorange2’ module in Figure S18) identified several MYBs co-expressed with F3′5′Ha and ANSa/b; among them, MYB32a, MYB32b and MYB8b showed high expression levels in S3 of lisianthus Purple01 similar to those of F3′5′Ha and ANSa/b during flower development (Figures 1b, S21). As a result, we suggested that in lisianthus CHSa, ANSa/b, F3′5′Ha/b, MYB32a/b and MYB8b codetermine blue/violet flower coloration, CHSa and ANSa/b codetermine pink/red flower coloration and CHSa determines yellow flower coloration (Figure 1c).

Instead of accumulating anthocyanins in the blooming stage, green lisianthus varieties synthesize and accumulate large amounts of chlorophylls, leading to green phenotypes. CHLMa, CRDa and PORA/B/Ca were assumed to be important regulators of chlorophyll biosynthesis in petals since they had the highest expression levels in S3 in lisianthus Green01 (Figure 1b). Multiple genes involved in photosynthesis, including those encoding Chlorophyll a/b-binding proteins (CABs), were found to be co-expressed with CHLMa, CRDa and PORA/B/Ca, suggesting that these CABs might also play important roles in green petal formation by inhibiting chlorophyll degradation via forming antenna complexes with free chlorophylls (Figures S22–S24). Based on the same expression patterns of genes involved in chlorophyll synthesis and photosynthesis (e.g. CABs) in the tan lisianthus with those in lisianthus Green01, it is possible that chlorophylls were also pigments in the tan lisianthus (Figures 1c, S24–S25).

In lisianthus, a total of 120 MADS-box genes were identified, including 27 floral organ identity genes (three A-class genes, nine B-class genes, four C-class genes, one D-class genes and 10 E-class genes; Figures 1d, S26–S28). The floral development model in E. grandiflorum was constructed according to floral identity genes' expression profiles (Figures 1d, S30). We found that multiple floral organ identity genes were generated by WGT, including B-class AP3s, C-class AGL1s and E-class AGL9s, which might influence the evolution of E. grandiflorum's flower morphology (Li et al., 2022; Figure S29). ‘Rosita White’ and ‘Wavy White’ are two popular lisianthus cultivars, which have flat-shaped petals and wave-shaped petals respectively. We found that AGL1a, a C-class MADS gene, was highly expressed in the stamen and carpel of ‘Rosita White’, while it was barely expressed in all the flower tissues of ‘Wavy White’, potentially due to differences in cis-acting regulatory elements between their promoters (Figures 1e, S30–S31). It was reported that the absence of C-class MADSs could result in the double-flower phenotype. Thus, we speculated that AGL1a could be linked to the more apparent double-flower phenotype with more petal numbers of ‘Wavy White’ (Figure S32).

In summary, we present the chromosome-level genome of E. grandiflorum and identify the key candidate genes involved in flower coloration and morphology, which will speed up the molecular breeding of E. grandiflorum in the future.



中文翻译:

洋桔梗基因组揭示了栽培桔梗观赏性状的全基因组三重复制事件

介绍

Eustoma grandiflorum (lisianthus) 是龙胆科观赏植物。由于其巨大的玫瑰般的花朵,长长的茎和延长的花瓶寿命,近年来其销量急剧增加,赢得了“下一个玫瑰”的称号。选择性育种已经生产出具有多种花色和形状的商业桔梗(图 S1a;Li 等人,  2022 年)。在包括小麦、棉花、花生等在内的多倍体作物中,多倍体对于开发高质量性状至关重要(Cheng 等人,  2018 年)。多倍体也可能有助于培养桔梗的理想性状。在这里,我们报告了一种用于E. grandiflorum的高质量染色体规模基因组组装(2 n  = 6x  = 72) 使用 PacBio HiFi 读取和 Hi-C 支架技术的组合,揭示了桔梗的多倍体驯化有助于栽培桔梗的观赏性状。

为从头全基因组测序生成了总共 32.05 Gb (~23.56X) 的 PacBio HiFi 数据和 140.14 Gb (~103.04X) Hi-C 数据。组装的总长度为 1.71 Gb,包括 1056 个重叠群,相应的 N50 为 7.33 Mb(表 S1),并组装了 36 条假染色体(图 S1 和 S2,表 S2)。BUSCO 显示完整率为 94.7%,重复率为 31.6%(表 S5)。共预测了 54 305 个高质量的蛋白质编码基因(表 S3-S8)。此外,77.85% 的基因组被注释为重复序列(表 S9-S12)。

基因组共线性识别了大量的共线性块,E之间的比例为 6:1 。GrandiflorumGelsemium sempervirens (图 1a), E之间的比例为 2:1 。大花E . 大花(图S3),表明多倍体事件的存在。为此,可以将 36 条假染色体分为三个亚基因组,然后根据转座因子谱获得具有三组单倍体染色体 A、B 和 C 的 12 个同源组(图 S4,表 S10-S12)。亚基因组内的合理共线性表明E大花在E. grandiflorum的近期历史中经历了全基因组重复(WGD)事件(图 S5)。K s 分布进一步证实,大花桉在与大花豆分化后经历了WGD事件(K s 峰值= 0.93)和全基因组三倍体( WGT)事件(Ks峰值= 0.21),这与6 : 1 的比例(E. grandiflorum : G. sempervirens)和系统发育树描述的进化关系(图 1a,S6-S7)。

详细信息在图片后面的标题中
图1
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(a)洋桔梗染色体与Gelsemium sempervirens的比对脚手架。( b )直方图显示桔梗中花色的基本调节因子的表达谱。S1,芽期;S2,转弯阶段;S3,开花期。FLS,黄酮醇合酶;CHS,查尔酮合酶;ANS,花青素合酶;F3'5'H,类黄酮3',5'-羟化酶;CRD,Mg-原卟啉 IX 单甲酯环化酶;CHLH/D/I,Mg-螯合酶亚基 H、D 和 I;PORA/B/C,原叶绿素还原酶。(c) 桔梗花着色模型。描述了桔梗中潜在的简化花青素生物合成途径。CHI,查尔酮异构酶;F3H,黄烷酮 3-羟化酶;F3'H,类黄酮3'-羟化酶;DFR,二氢黄酮醇-4-还原酶;GT,葡糖基转移酶;AT,酰基转移酶。桔梗花色的基本调节剂是红色字体。主要颜料参考文献(高和李, 2020 年)。(d) 基于花身份基因表达模式的桔梗花发育模型。热图显示了不同物种中 MADS 基因的数量。( e )显示桔梗中C类MADS基因表达水平的直方图。在所有样本中,FPKM中的 AGL1d 为零,因此不显示。[可以在wileyonlinelibrary.com查看彩色图]

基于跨E的共线性,共发现了 15 436 个属于由 WGT 事件产生的同线基因组的基因。大花亚基因组,在外部刺激反应、解剖结构形态发生和生物合成过程中富集(图S8,表S13)。由于 WGT 包括MYBsbHLHsCHIsCHSsDFRsGTsFLSs,发现转录因子 (TFs) 和参与黄酮类/花青素生物合成的结构基因的大量拷贝数变异(图 S9-S15)。因此,假设多倍体事件将通过为花青素生产提供增强的遗传物质来培育五颜六色的桔梗品种。我们还发现WGT产生了许多调节器官/花发育的TFs,包括MADS家族的花身份基因,以及TCP家族和HD-Zip III家族的成员,这可能会影响大花的花形态的形成。图 1d,S16-S17)。

为了检查调控桔梗花朵色素沉着的遗传途径,我们对紫色桔梗(Purple01)、绿色桔梗(Green01)、黄色桔梗(Yellow01)、红色桔梗(Red01)和'Rosita White'的花瓣进行了RNA-seq '在不同阶段(芽期,S1;转折期,S2;开花期,S3)并进行加权相关网络分析(WGCNA)(图S18)。鉴定了参与类黄酮生物合成的基因,并描绘了推定的花青素生物合成途径(图 1c,S11-S15,S19)。我们发现FLSa在 S1 和 S2 中高度表达,而在 S3 中几乎没有表达(图 1b)。在 S3 中,我们发现所有有色品种的CHSa表达水平显着高于白色品种,而ANSa/ b在粉红色/红色和蓝色/紫色栽培品种中表现出更高的表达水平(图 1b,S20)。此外,与粉色/红色桔梗相比,蓝色/紫色桔梗显示出更高的F3'5'Ha / b水平(图 1b,S20)。桔梗 Purple01 的 S3 特异性模块的共表达网络重建(图 S18 中的“darkorange2”模块)确定了几个与F3'5'HaANSa / b共表达的MYB;其中,MYB32aMYB32bMYB8b在桔梗Purple01的S3中呈高表达水平,与F3′5′HaANSa相似。/ b在花发育期间(图 1b,S21)。因此,我们建议在桔梗中CHSaANSa / bF3′5′Ha / bMYB32a / bMYB8b共同决定蓝色/紫色花色,CHSaANSa / b共同决定粉红色/红色花色,CHSa决定黄色花着色(图1c)。

不是在开花期积累花青素,而是绿色桔梗品种合成并积累大量叶绿素,导致绿色表型。CHLMaCRDaPORA / B / Ca被认为是花瓣中叶绿素生物合成的重要调节因子,因为它们在桔梗 Green01 的 S3 中具有最高的表达水平(图 1b)。发现参与光合作用的多个基因,包括编码叶绿素 a/b 结合蛋白 (CAB) 的基因与CHLMaCRDaPORA / B / Ca共表达,表明这些 CAB 也可能通过与游离叶绿素形成天线复合物来抑制叶绿素降解,从而在绿色花瓣形成中发挥重要作用(图 S22-S24)。基于与桔梗Green01中涉及叶绿素合成和光合作用的基因(例如CABs)的相同表达模式,叶绿素可能也是桔梗中的色素(图1c,S24-S25)。

在桔梗中,共鉴定出120个MADS-box基因,其中花器官同一性基因27个(A类基因3个,B类基因9个,C类基因4个,D类基因1个,E类基因10个) ; 图 1d,S26-S28)。E中的花发育模型。根据花身份基因的表达谱构建大花(图1d,S30)我们发现WGT产生了多个花器官身份基因,包括B类AP3s、C类AGL1s和E类AGL9s,这可能会影响大花的花形态进化( Li et al.,  2022 ); 图 S29)。'Rosita White'和'Wavy White'是两种流行的桔梗品种,花瓣分别为扁平形和波浪形。我们发现C类MADS基因AGL1a在'Rosita White'的雄蕊和心皮中高度表达,而在'Wavy White'的所有花组织中几乎不表达,可能是由于顺式作用的差异它们的启动子之间的调节元件(图1e,S30-S31)。据报道,缺乏 C 类MADS可能导致双花表型。因此,我们推测AGL1a可能与具有更多“波浪白色”花瓣数的更明显的重瓣表型有关(图 S32)。

综上所述,我们展示了大花的染色体水平基因组,确定了涉及花色和形态的关键候选基因,这将在未来加速大花的分子育种。

更新日期:2022-07-25
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