【作者】 张丹； 李伟； 夏恩华； 张群洁； 张昀； 刘源； 佟岩； 赵原； 牛永超； 许珈欢； 高立志；

【Author】 Dan Zhang;Wei Li;En-hua Xia;Qun-jie Zhang;Yun Zhang;Yuan Liu;Yan Tong;Yuan Zhao;Yong-chao Niu;Jia-huan Xu;Li-zhi Gao;Institution of Genomics and Bioinformatics, South China Agricultural University;Plant Germplasm and Genomics Center, Germplasm Bank of Wild Species in Southwestern China, Kunming Institute of Botany, Chinese Academy of Sciences;Agrobiological Gene Research Center, Guangdong Academy of Agricultural Sciences;Yunnan Agricultural University;Genosys Inc.;

【机构】 华南农业大学基因组学与生物信息学中心； 中国科学院昆明植物研究所中国西南野生种质库植物种质和基因组学中心； 广东农业科学院农学基因研究中心； 云南农业大学； 基诺生物科技公司；

【摘要】 三七(Panaxnotoginseng)是我国特有的传统名贵中药材,与人参(P. ginseng)、西洋参(P. quinquefolius)同属于五加科(Araliaceae)之人参属(Panax),具有非常重要的医药产业价值。然而,目前三七的遗传信息十分有限,对人参皂苷生物合成途径研究还不够深入,抗病性差、连作障碍、药材品质退化严重以及优良新品种匮乏长期以来制约着三七产业的发展。本研究选用来自云南省南涧县无量山的三七(2n=2x=24)植株,通过Illumina HiSeq2000测序平台,使用全基因组鸟枪法测序获得了大约385.28Gb的原始数据集,序列覆盖度高达192.63倍,经过从头组装,首次获得了大约1.85 Gb三七基因组序列。在获得的高质量三七参考基因组中,共预测得到34,369个蛋白编码基因,其中有2,513个转录因子(TFs),还鉴定得到了347个参与三萜皂苷代谢生物合成途径的基因。我们对三七、葡萄、猕猴桃、胡萝卜、咖啡、辣椒、土豆、番茄、可可和水稻共10个物种进行了比较基因组学研究。通过基因家族进化的分析鉴定得到了3,712个三七特有的基因家族,它们的功能显著地富集在三萜皂苷合成通路上两个关键的乙酰辅酶-A和GT(糖基转移酶)中。在10,973个存在于上述10个物种的共同祖先的基因家族中,1,200个基因家族(包括4,548个基因)在三七中大量显著地扩增,扩增的基因家族大多为CYP450s和GTs两个家族的编码基因,这两个基因家族编码最终催化形成多种人参皂苷的关键酶,进而支持了三萜皂苷在三七这种药用植物中大量产生的生物学特性。系统发育基因组学分析发现三七与胡萝卜进化关系最近,大约在7,140万年前发生分化。计算同义突变率(Ks)的年龄分布结果表明,在三七和猕猴桃分化之后大约2,600万年前发生了一次全基因组重复事件(WGD)。基于高质量的三七基因组参考序列,我们在上述10个物种中鉴定得到了参与人参皂苷生物合成通路的23个基因家族。分析发现,14个基因家族在三七中发生了特异性扩增,这些重复基因的功能分化可能促进了一些三七特有人参皂苷的产生,决定了与三七药效息息相关的生物化学成分多样性。进一步对三七中参与人参皂苷代谢的基因拷贝数变异和基因组位置聚集分析发现,两个最大的基因家族GTs和CYP450s的基因数目分别是127和145,其中分别鉴定得到17个GT和11个CYP450基因簇,每个基因簇包含2-4个基因,它们中的大多数基因在系统发育树上也聚在一起,说明它们可能起源于串联基因重复。为了深入地认识三七中人参皂苷生物合成的机制,我们采用RNA-Seq和高效液相色谱(HPLC)对参与人参皂苷生物合成通路的23个基因家族在三七不同组织的表达模式和人参皂苷含量进行了比较分析。结果表明,与三七皂苷生物合成相关的大多基因可能主要在花和叶里表达与合成,然后才转运并储存在根部。总之,三七这一人参属中重要二倍体植物参考基因组的首次获得和相关代谢通路的解析,对指导三七、人参和西洋参的育种、种植与深加工产业的发展具有十分重要的意义。更多还原

【Abstract】 Panax notoginseng(Burk) F. H. Chen is one of the rare traditional medicinal herbs in China and has very significant value in the pharmaceutical industry. Together with Asian ginseng(P. ginseng C.A. Mey) and American ginseng(P. quinquefolius L.), it belongs to the genus Panax(Araliaceae).However, we still lack an overall knowledge of ginsenoside biosynthesis in P.notoginseng; poor disease resistance, continuous cropping obstacle, medicinal quality degradation and the lack of improved new varieties have long limited sustainable development of P.notoginseng industry. Here we present the first high-quality draft nucleotide sequence of the P.notoginseng genome. The genomic DNA was extracted from a P. notoginseng(2 n = 2 x = 24 chromosomes) individual plant, which was collected from Wuliang Mountains, Nanjian County,Yunnan Province, China. Raw sequence data of ～385.28 Gb were generated by a whole-genome shotgun sequencing analysis with the Illumina HiSeq 2000 platform, thus yielding approximately192.63-fold high quality sequence coverage. We de-novo assembled the genome and finally yielded a ～1.85-Gb high-quality draft nucleotide sequence of P. notoginseng. We predicted 34,369 protein-coding genes in the P. notoginseng genome, among which 2,513 genes encode transcription factors(TFs). Besides, we identified a total of 347 genes involved in the ginsenoside biosynthesis.We performed comparative genomic analyses of P. notoginseng, grape, kiwifruit, carrot,coffee, pepper, potato, tomato, cacao, and rice. Analysis of gene family evolution among ten species showed that the 3,712 P. notoginseng-specific gene families were significantly enriched in functions associated with the first step of terpenoid backbone biosynthesis, hydroxyl methylglutaryl-coenzyme A reductase and GTs(Gycosyl transferases). Our results further revealed that, of the 10,973 gene families inferred to be present in the most recent common ancestor(MRCA) of the ten studied plant species, a total of 1,200 gene families comprising 4,548 genes exhibited significant expansions in the P. notoginseng lineage. Intriguingly, functional annotation indicated that they were significantly enriched in a number of genes encode the two large families of key enzymes, CYP450 s, and GTs, which are ultimately involved in the catalysis of terpenoids to generate diverse ginsenosides, enlightening a high production of abundant triterpene saponins in this medicinal plant. Phylogenomic analysis showed that P. notoginseng was most closely related to carrot, with an estimated divergence time of ～71.4 million years ago(MYA). Furthermore, our results suggestone round of WGD that we estimate to have occurred～26.15 mya in P. notoginseng and this recent WGD event distinctly occurred in P. notoginseng after the P. notoginseng-kiwifruit divergence by calculated an age distribution of synonymous substitution rates(Ks).Based on previous studies on the biosynthesis of ginsenosides, we identified 23 gene families involved in the ginsenoside biosynthesis of P. notoginseng and their orthologs in grape, kiwifruit,carrot, coffee, pepper, potato, tomato, cacao, and rice. Phylogenetic analyses suggested that P.notoginseng experienced independent lineage-specific duplications in 14 gene families compared with the other nine species. The functional divergence of these duplicated genes, known as neofunctionalization, may enhance the evolution of a specified ginsenoside biosynthesis. We further investigated the copy number variation and physical gathering of genes involved in the ginsenoside biosynthesis of P. notoginseng, of which GT and CYP450 are the two largest gene families with 127 and 145 genes, respectively. We examined physical gathering of GTs and CYP450 s and totally identified 17 and 11 gene clusters containing 2-4 genes, respectively.Phylogenetic analysis indicated that the majority of these gene clusters are closely grouped together, implying that they probably originated from tandem duplication events.To gain insights into the molecular mechanisms underlying ginsenosides metabolites in P.notoginseng, we examined tissue-specific expression patterns of genes potentially involved in the ginsenoside biosynthesis using RNA-Seq together with high-performance liquid chromatography analysis(HPLC). Our results showed that ginsenosides may be actively synthesized in flowers/leaves besides roots and then accumulated in roots.In conclusion, we present the first draft genome assembly of the diploid P. notoginseng species widely grown in Southwestern China. We obtained novel insights into ginsenoside biosynthesis that will efficiently guide the future genetic breeding programs, cultivation and deep processing industries of P. notoginseng, P. ginseng and P. quinquefolius.更多还原