《蒙古沙冬青的参考级基因组揭示了其在豆科植物中的进化历史和耐旱机制》

A reference-grade genome of the xerophyte Ammopiptanthus mongolicus sheds light on its evolution history in legumes and drought tolerance mechanisms

Abstract

Plants that grow in extreme environments represent unique sources of stress-resistance genes and mechanisms. Ammopiptanthus mongolicus (Leguminosae) is a xerophytic evergreen broadleaf shrub native to semi-arid and desert regions; however, its drought-tolerance mechanisms remain poorly understood. Here, we report the assembly of a reference-grade genome for A. mongolicus, describe its evolutionary history within the legume family, and examine its drought-tolerance mechanisms. The assembled genome is 843.07 Mb in length, with 98.7% of the sequences successfully anchored to the nine chromosomes of A. mongolicus. The genome is predicted to contain 47 611 protein-coding genes, and 70.71% of the genome is composed of repetitive sequences; these are dominated by transposable elements, particularly long-terminal-repeat retrotransposons. Evolutionary analyses revealed two whole-genome duplication (WGD) events at 130 and 58 million years ago (mya) that are shared by the genus Ammopiptanthus and other legumes, but no species-specific WGDs were found within this genus. Ancestral genome reconstruction revealed that the A. mongolicus genome has undergone fewer rearrangements than other genomes in the legume family, confirming its status as a “relict plant”. Transcriptomic analyses demonstrated that genes involved in cuticular wax biosynthesis and transport are highly expressed, both under normal conditions and in response to polyethylene glycol-induced dehydration. Significant induction of genes related to ethylene biosynthesis and signaling was also observed in leaves under dehydration stress, suggesting that enhanced ethylene response and formation of thick waxy cuticles are two major mechanisms of drought tolerance in A. mongolicus. Ectopic expression of AmERF2, an ethylene response factor unique to A. mongolicus, can markedly increase the drought tolerance of transgenic Arabidopsis thaliana plants, demonstrating the potential for application of A. mongolicus genes in crop improvement.

生长在极端环境中的植物是独特抗逆基因和机制的来源。蒙古沙冬青（*Ammopiptanthus mongolicus*，豆科）是一种原产于半干旱和沙漠地区的旱生常绿阔叶灌木；然而，其耐旱机制尚未被充分理解。在此，我们报告了蒙古沙冬青的参考级基因组组装，描述了其在豆科中的进化历史，并研究了其耐旱机制。组装的基因组长度为843.07 Mb，其中98.7%的序列成功锚定到蒙古沙冬青的九条染色体上。该基因组预测包含47611个蛋白编码基因，且70.71%的基因组由重复序列组成，其中以转座元件为主，尤其是长末端重复序列逆转录转座子。

进化分析揭示了蒙古沙冬青与其他豆科植物共享的两次全基因组复制（WGD）事件，分别发生在约1.3亿年前和5800万年前，但在该属内未发现物种特有的全基因组复制事件。祖先基因组重建表明，蒙古沙冬青的基因组比豆科其他基因组发生了更少的重排，证实了其作为“孑遗植物”的地位。

转录组分析表明，参与角质蜡生物合成和运输的基因在正常条件下以及在聚乙二醇诱导的脱水条件下均表现出高表达。在脱水胁迫下，叶片中与乙烯生物合成和信号传导相关的基因也表现出显著诱导，表明增强的乙烯响应和厚蜡质角质层的形成是蒙古沙冬青耐旱的两大主要机制。蒙古沙冬青特有的乙烯响应因子 *AmERF2* 的异位表达可以显著提高转基因拟南芥（*Arabidopsis thaliana*）的耐旱性，证明了蒙古沙冬青基因在作物改良中的应用潜力。

Introduction

The genus Ammopiptanthus (family Leguminosae or Fabaceae, subfamily Papilionoideae) comprises only two species, Ammopiptanthus mongolicus and Ammopiptanthus nanus (Cheng, 1959). The model species, A. mongolicus (Figure 1A), is distributed throughout the southern Gobi Desert and adjacent areas in central Asia, whereas A. nanus is restricted to Kashgar, Xinjiang Province, China (Zhao and Zhu, 2003). Geographical research suggests that deserts have existed in eastern central Asia for approximately 22 million years (Xie and Yang, 2012). The genus Ammopiptanthus, an endemic plant, is considered a relict descendant of the ancient Tethyan flora that evolved to survive in desert environments (Xie and Yang, 2012). Despite growing in extremely arid and cold environments, Ammopiptanthus spp. are evergreen broadleaf shrubs (Zhao et al., 2016a). These evergreen shrubs play an essential role in maintaining the semi-desert ecosystem, as they can obstruct drifting sand and prevent further desertification (Liu and Qiu, 1982). However, the harsh environment of the Gobi Desert leads to low seed germination rates and increased growth pressure. Therefore, both Ammopiptanthus species have been listed as endangered and second-class state-protected wild plants in China (Ge et al., 2005).

引言  
蒙古沙冬青属（Ammopiptanthus，隶属于豆科或蝶形花科，亚科为蝶形花亚科）仅包含两个物种：蒙古沙冬青（Ammopiptanthus mongolicus）和新疆沙冬青（Ammopiptanthus nanus）（程，1959）。模式物种蒙古沙冬青（图1A）分布于中亚的南戈壁沙漠及其周边地区，而新疆沙冬青仅限于中国新疆省喀什地区（赵和朱，2003）。地理研究表明，东亚的沙漠大约在2200万年前就已经存在（谢和杨，2012）。蒙古沙冬青属是特有植物，被认为是古代特提斯植物区系的孑遗后代，适应了沙漠环境的生存（谢和杨，2012）。尽管生长在极度干旱和寒冷的环境中，蒙古沙冬青属植物仍是常绿阔叶灌木（赵等，2016a）。这些常绿灌木在维持半沙漠生态系统中发挥着重要作用，因为它们可以阻挡流沙，防止进一步的沙漠化（刘和丘，1982）。然而，戈壁沙漠的恶劣环境导致种子萌发率低和生长压力增加。因此，蒙古沙冬青属的两个物种均被列为中国的濒危物种和国家二级保护野生植物（葛等，2005）。

图1A

Phylogenetic analyses based on the nuclear ribosomal internal transcribed spacer (nrITS) marker and the plastid matK gene have shown that Ammopiptanthus is monophyletic and has a sibling genetic relationship with a clade including Sophora and Ammodendron (Xie and Yang, 2012). A study of chloroplast intergenic spacers and ITS markers estimated the divergence time between A. mongolicus and A. nanus at approximately 0.77 million years ago (mya) (Su et al., 2016). Our recent chloroplast and mitochondrial sequence analyses revealed that these two species have almost the same organelle gene content, with only a few insertions and deletions in non-coding regions, suggesting that they diverged very recently (Feng et al., 2017, 2019).
 

基于核糖体内部转录间隔区（nrITS）标记和质体matK基因的系统发育分析表明，蒙古沙冬青属是单系的，并且与包含刺槐属（Sophora）和沙拐枣属（Ammodendron）的类群具有姐妹群遗传关系（谢和杨，2012）。对叶绿体基因间隔区和ITS标记的研究估计，蒙古沙冬青（Ammopiptanthus mongolicus）和新疆沙冬青（Ammopiptanthus nanus）的分化时间约为77万年前（Su等，2016）。我们最近对叶绿体和线粒体序列的分析显示，这两种植物的细胞器基因含量几乎相同，仅在非编码区存在少量插入和缺失，这表明它们的分化时间非常近（Feng等，2017, 2019）。

Mechanisms of abiotic stress tolerance in this genus are receiving increasing attention. Morphological studies have shown that its leaf surfaces are covered with an exceptionally thick waxy cuticle (up to 18 μm) that helps to limit transpiration and maintain plant water status (Han and Li, 1992; Li et al., 2023). There are dense silvery trichomes on both leaf surfaces that protect against heat and cold (Liu et al., 2004). A. mongolicus leaves also have a lower stomatal density than leaves of Arabidopsis (Li et al., 2015), further reducing the rate of transpiration. Notably, the palisade mesophyll of A. mongolicus has nine or 10 cell layers, resulting in succulent leaves and thus an increased water storage capacity. Gene expression profiling of this genus under drought, cold, and salinity stress has identified hundreds of abiotic-stress-inducible genes (Zhou et al., 2012; Liu et al., 2013; Pang et al., 2013, 2015; Wu et al., 2014; Gao et al., 2015; Yang et al., 2022b). However, the exact stress-tolerance mechanisms of this unique desert flora remain unclear.

该属植物的非生物胁迫耐受机制正受到越来越多的关注。形态学研究表明，其叶片表面覆盖着异常厚的蜡质角质层（可达18微米），有助于限制蒸腾作用并维持植物的水分状态（韩和李，1992；李等，2023）。叶片两面都有密集的银白色绒毛，可抵御高温和寒冷（刘等，2004）。蒙古沙冬青的叶片气孔密度比拟南芥叶片低（李等，2015），这进一步降低了蒸腾速率。值得注意的是，蒙古沙冬青的栅栏组织有9层或10层细胞，导致叶片肉质化，从而增加了水分储存能力。在干旱、寒冷和盐胁迫下，该属植物的基因表达分析已鉴定出数百个非生物胁迫诱导基因（周等，2012；刘等，2013；庞等，2013, 2015；吴等，2014；高等，2015；杨等，2022b）。然而，这种独特沙漠植物的确切耐逆机制仍然不清楚。

Legumes (family Fabaceae) form the third largest angiosperm family in the plant kingdom, with 751 genera and approximately 19 500 species (Bruneau et al., 2013). With recent advances in genome sequencing technologies, genomes of more than 20 legume species have been sequenced (Wang et al., 2017; Bertioli et al., 2019; Xie et al., 2019; Zhuang et al., 2019; Xu et al., 2020; Yang et al., 2022a; Chang et al., 2022). These sequenced genomes are invaluable resources for functional studies of individual plant species and comparative and evolutionary studies of eudicots. However, the sequenced genomes represent only a small portion of the large legume family. An 823-Mb draft genome of A. nanus was recently assembled using only Pacific Biosciences (PacBio) sequencing data (Gao et al., 2018); it provides basic information on the genome but is insufficient for a detailed understanding of its genomic features, downstream functional studies, and comparative genomic analyses.

豆科植物（Fabaceae）是植物界中第三大被子植物科，包含751个属和大约19500个物种（Bruneau等人，2013）。随着基因组测序技术的最新进展，已有超过20种豆科植物的基因组被测序（Wang等人，2017；Bertioli等人，2019；Xie等人，2019；Zhuang等人，2019；Xu等人，2020；Yang等人，2022a；Chang等人，2022）。这些测序的基因组是研究个别植物物种功能以及进行真双子叶植物比较和进化研究的宝贵资源。然而，这些测序的基因组仅代表庞大豆科家族的一小部分。最近，利用太平洋生物科学公司（PacBio）测序数据组装了一个823 Mb的新疆沙冬青（Ammopiptanthus nanus）草图基因组（Gao等人，2018）；它为基因组提供了基本信息，但不足以深入了解其基因组特征、下游功能研究和比较基因组分析。

Here, we assembled a high-quality reference-grade genome sequence for A. mongolicus by combining Illumina shotgun sequencing, PacBio long-read sequencing, Bionano optical mapping, and high-throughput chromosome conformation capture (Hi-C) sequencing. The final assembly is 843.07 Mb in length, with ∼98.7% anchored to nine chromosomes. Transcriptome sequencing and gene expression analyses revealed that ethylene signaling and cuticular wax biosynthesis play important roles in the drought tolerance of this desert plant. Our results provide new insights into the genomic characteristics, evolutionary history, and drought-tolerance mechanisms of A. mongolicus.

在此研究中，我们通过结合Illumina鸟枪法测序、PacBio长读长测序、Bionano光学图谱分析以及高通量染色体构象捕获（Hi-C）测序技术，组装了蒙古沙冬青（*Ammopiptanthus mongolicus*）的高质量参考级基因组序列。最终的组装基因组长度为843.07 Mb，其中约98.7%的序列锚定到九条染色体上。转录组测序和基因表达分析表明，乙烯信号传导和角质蜡生物合成在该沙漠植物的耐旱性中发挥着重要作用。我们的研究结果为蒙古沙冬青的基因组特征、进化历史以及耐旱机制提供了新的见解。