Polyploidy underlies co-option and diversification of biosynthetic triterpene pathways in the apple tribe Wenbing Sua,1,2, Yi Jingb,1, Shoukai Linc,1, Zhen Yueb,1, Xianghui Yanga,1, Jiabao Xub, Jincheng Wuc, Zhike Zhanga, Rui Xiaa, Jiaojiao Zhud, Ning And, Haixin Chenb, Yanping Honga, Yuan Yuana, Ting Longa, Ling Zhanga, Yuanyuan Jianga, Zongli Liua, Hailan Zhanga, Yongshun Gaoa, Yuexue Liua, Hailan Linc, Huicong Wanga, Levi Yante, Shunquan Lina,3, and Zhenhua Liud,3 aState Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture and Rural Affairs, College of Horticulture, South China Agricultural University, Guangzhou 510642, China; bResearch Cooperation Department, Beijing Genomics Institute Genomics, Shenzhen 518083, China; cKey laboratory of Loquat Germplasm Innovation and Utilization (Fujian Province), Putian University, Putian 351100, China; dJoint Center for Single Cell Biology, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240, China; and eFuture Food Beacon and School of Life Sciences, University of Nottingham, Nottingham NG7 2RD, United Kingdom Edited by Richard A. Dixon, University of North Texas, Denton, TX, and approved April 5, 2021 (received for review January 28, 2021) Whole-genome duplication (WGD) plays important roles in plant loquat, that is, ursolic acid (UA) and corosolic acid (CA), are evolution and function, yet little is known about how WGD un- a major class of bioactive compounds with anti-inflammatory, derlies metabolic diversification of natural products that bear sig- antidiabetic, and anticancer activities (15–17). However, gene nificant medicinal properties, especially in nonmodel trees. Here, pathways encoding the biosynthesis of these bioactive triterpenes we reveal how WGD laid the foundation for co-option and differ- have not been identified and characterized in loquat. It has been entiation of medicinally important ursane triterpene pathway du- proposed that members in the apple tribe such as loquat, apple, plicates, generating distinct chemotypes between species and and pear have been derived from an ancient Rosaceae species between developmental stages in the apple tribe. After generat- via WGD (autopolyploidization), although others have suggested ing chromosome-level assemblies of a widely cultivated loquat allopolyploidizations between sister species (18, 19). High-quality variety and Gillenia trifoliata, we define differentially evolved, du- genome assemblies for species within (apple and pear) (20–22) plicated gene pathways and date the WGD in the apple tribe at and outside (strawberry and peach) (23, 24) the apple tribe have PLANT BIOLOGY 13.5 to 27.1 Mya, much more recent than previously thought. We been generated, yet genome sequences from the tribe’sclosest then functionally characterize contrasting metabolic pathways re- outgroup Gillenieae are still not available. sponsible for major triterpene biosynthesis in G. trifoliata and lo- Here, we merge comparative genomics, transcriptomics, metab- quat, which pre- and postdate the Maleae WGD, respectively. Our olomics, and functional assays to understand how WGD under- work mechanistically details the metabolic diversity that arose pinned the diversification of biosynthetic pathways encoding major post-WGD and provides insights into the genomic basis of medic- inal properties of loquat, which has been used in both traditional Significance and modern medicines. Plants are a primary source of both traditional and modern drugs polyploidy | comparative genomics | evolution | triterpene biosynthesis | due to their astounding capability to synthesize diverse molecules. apple tribe The fruit tree loquat in the apple tribe has been long used in medicine to treat cough, chronic bronchitis, and asthma, yet why lants generate a vast array of specialized metabolites, which loquat—but not its relatives—evolved these medicinal properties Pdiffer by species and tissue. This astounding diversity has is unknown. Here, we generate high-quality genomes of loquat long been thought to arise largely by gene duplications, followed and a relative, which are separated by a recent whole-genome by differentiation of gene expression and function between du- duplication (WGD) around 13.5 to 27.1 Mya. We revealed the post- plicates (1–3). Whole-genome duplication (WGD) contributes to WGD diversification of triterpene biosynthesis and the excep- the generation of single gene duplicates and has played impor- tionally high levels of bioactive ursane-type triterpenes specifically tant roles in plant genome function and evolution (4–6). Ex- in loquat. Our work underscores the importance of WGD- amples for WGD-associated metabolic diversity are known for associated metabolic diversification underlying the bioactivity of the innovation of glucosinolates in the Brassicales and for oil some medicinal plants. biosynthesis in wild olive trees (7–9). In addition to underlying change in enzyme functions, WGD also plays a role in the evolution Author contributions: W.S., Y. Jing, Shoukai Lin, Z.Y., X.Y., Shunquan Lin, and Zhenhua of metabolic gene expression, which further impacts the biosynthesis Liu designed research; W.S., Y. Jing, Shoukai Lin, Z.Y., and X.Y. performed research; Y.Y., of specialized metabolites (3, 10, 11). However, tracing the evo- T.L., L.Z., Y. Jiang, Zongli Liu, H.Z., Y.G., Y.L., H.L., H.W., and Zhenhua Liu contributed new reagents/analytic tools; W.S., Y. Jing, Shoukai Lin, Z.Y., X.Y., J.X., J.W., Z.Z., R.X., J.Z., N.A., lution of a particular metabolic pathway and characterizing func- H.C., Y.H., Shunquan Lin, and Zhenhua Liu analyzed data; and W.S., Y. Jing, L.Y., Shun- tional impacts of WGDs are still challenging, mainly due to the quan Lin, and Zhenhua Liu wrote the paper. ancient status of relevant WGDs and the formidable complexity of The authors declare no competing interest. metabolic pathways. This article is a PNAS Direct Submission. Within the apple tribe there exist medicinally important but This open access article is distributed under Creative Commons Attribution-NonCommercial- understudied species harboring metabolic innovations that we NoDerivatives License 4.0 (CC BY-NC-ND). hypothesized were based on the foundation laid by the tribe- 1W.S., Y. Jing, Shoukai Lin, Z.Y., and X.Y. contributed equally to this work. Eriobotrya specific WGD. A native species from China, loquat ( 2Present address: Fruit Research Institute, Fujian Academy of Agricultural Science, Fuzhou japonica Lindl), has been cultivated as a fruit tree worldwide and 350013, China. used in both traditional and modern medicines (12, 13). Uses in- 3To whom correspondence may be addressed. Email: [email protected] or loquat@ clude treatment of coughing, documented as early as 1590 CE in the scau.edu.cn. Chinese Encyclopedia of Botany and Medicines [Li Shi-Zhen, Ben This article contains supporting information online at https://www.pnas.org/lookup/suppl/ Cao Gang Mu (14)] and recent commercial herbal syrups treating doi:10.1073/pnas.2101767118/-/DCSupplemental. throat pain. Recent work showed that ursane-type triterpenes in Published May 13, 2021. PNAS 2021 Vol. 118 No. 20 e2101767118 https://doi.org/10.1073/pnas.2101767118 | 1of11 Downloaded by guest on September 25, 2021 triterpenes within and outside of theappletribe.Wefirstgenerate To determine species groupings in relation to the Maleae WGD, chromosome-level assemblies of a widely cultivated loquat cul- we used 661 single-copy orthologs from 10 related species for tivar E. japonica cultivar (cv.) Jiefangzhong (which produces high phylogenetic analysis. This revealed loquat grouping with pear and levels of ursane-type triterpene) and Gillenia trifoliata.This apple and separated from G. trifoliata, peach (Prunus persica), and allowed us to first identify a single WGD marking the origin of the other Rosaceae species (Fig. 1A). Molecular clock analysis indi- apple tribe at 13.5 to 27.1 Mya, much more recent than previously cated that the apple tribe (Maleae) diverged from Gillenieae be- thought (18, 22). We then identified a well-conserved group of tween 13.5 and 27.1 Mya (Fig. 1A), suggesting that the emergence enzymatic genes, which we functionally confirm to be responsible of the apple tribe was much more recent than previously thought for the biosynthesis of bioactive ursane-type triterpenes differen- (18, 22). Using intra- and intergenomic syntenic analysis, we ob- tially in species within (apple, pear, and loquat) and outside (G. served a 2:1 syntenic depth ratio when comparing loquat with the trifoliata and peach) the apple tribe. Our analysis shows how WGD species (G. trifoliata and P. Persica) that diverged before the split underlaid the diversification of metabolic pathways responsible for with Maleae but showed a 1:1 syntenic depth ratio within Maleae major triterpene biosynthesis. In addition, WGD provided a basis (apple versus loquat) (Fig. 1B). Furthermore, the spectrum of for the evolution of functionally critical gene expression differenti- synonymous substitutions per synonymous site (Ks) of these syn- ation and co-option for biosynthesis of triterpenes. Taken together, tenic blocks confirmed a single WGD peak (Ks around 0.16) for our results provide a clear example of the post-WGD diversification the two Maleae species (loquat and apple) but not for G. trifoliata of an important metabolic pathway and identify the genomic basis and the outgroup