De novo genome assembly of the stress tolerant forest species Casuarina equisetifolia provides insight into secondary growth

Casuarina equisetifolia (C. equisetifolia), a conifer-like angiosperm with resistance to typhoon and stress tolerance, is mainly cultivated in the coastal areas of Australasia. C. equisetifolia, making it a valuable model to study secondary growth associated genes and stress-tolerance traits. However, the genome sequence isunavailable and therefore wood-associated growth rate and stress resistance at the molecular level is largely unexplored. We therefore constructed a high-quality draft genome sequence of C. equisetifolia by a combination of Illumina second-generation sequencing reads and Pacific Biosciences single-molecule real-time (SMRT) long reads to advance the investigation of this species. Here, we report the genome assembly, which contains approximately 300 megabases (Mb) and scaffold size of N50 is 1.06 Mb. Additionally, gene annotation, assisted by a combination of prediction and RNA-seq data, generated 29 827 annotated protein-coding genes and 1983 non-coding genes, respectively. Furthermore, we found that the total number of repetitive sequences account for one-third of the genome assembly. Here we also construct the genome-wide map of DNA modification, such as two novel forms N6-adenine (6mA) and N4-methylcytosine (4mC) at the level of single-nucleotide resolution using single-molecule real-time (SMRT) sequencing. Interestingly, we found that 17% of 6mA modification genes and 15% of 4mC modification genes also included alternative splicing events. Finally, we investigated cellulose, hemicellulose, and lignin-related genes, which were associated with secondary growth and contained different DNA modifications. The high-quality genome sequence and annotation of C. equisetifolia in this study provide a valuable resource to strengthen our understanding of the diverse traits of trees.

木麻黄（Casuarina equisetifolia，简称C. equisetifolia）是一类类针叶被子植物，兼具抗台风与胁迫耐受特性，主要栽培于澳大拉西亚沿海地区。该物种是研究次生生长相关基因与抗胁迫性状的珍贵模式材料。然而，由于其基因组序列尚未获取，学界对其木材相关生长速率与抗胁迫特性的分子层面研究仍存在大量空白。为此，本研究结合Illumina第二代测序读段与太平洋生物科学（Pacific Biosciences）的单分子实时（single-molecule real-time, SMRT）长读段测序技术，构建了高质量的木麻黄草图基因组，以推动该物种的相关研究。

本研究报道该基因组组装结果：其总长度约300兆碱基（Mb），支架序列（scaffold）N50长度为1.06 Mb。此外，结合基因预测与RNA测序（RNA-seq）数据辅助注释，共得到29827个蛋白编码基因与1983个非编码基因。进一步分析发现，重复序列总占比约为基因组组装序列的三分之一。

本研究同时利用单分子实时测序技术，在单核苷酸分辨率水平上构建了全基因组DNA修饰图谱，涵盖两类新型修饰形式：N6-腺嘌呤（N6-adenine, 6mA）与N4-甲基胞嘧啶（N4-methylcytosine, 4mC）。有趣的是，研究发现17%的6mA修饰基因与15%的4mC修饰基因同时存在可变剪接事件。最后，本研究对与次生生长相关的纤维素、半纤维素及木质素相关基因开展了分析，发现这些基因均携带不同类型的DNA修饰。

本研究获得的高质量木麻黄基因组序列与注释信息，为深化我们对树木多样性状的理解提供了宝贵的研究资源。