《Agave hybrid NO.11648 的染色体级基因组组装为 CAM 光合作用提供了新见解》

A chromosome-level genome assembly of Agave hybrid NO.11648 provides insights into the CAM photosynthesis

Abstract

The subfamily Agavoideae comprises crassulacean acid metabolism (CAM), C3, and C4 plants with a young age of speciation and slower mutation accumulation, making it a model crop for studying CAM evolution. However, the genetic mechanism underlying CAM evolution remains unclear because of lacking genomic information. This study assembled the genome of Agave hybrid NO.11648, a constitutive CAM plant belonging to subfamily Agavoideae, at the chromosome level using data generated from high-throughput chromosome conformation capture, Nanopore, and Illumina techniques, resulting in 30 pseudo-chromosomes with a size of 4.87 Gb and scaffold N50 of 186.42 Mb. The genome annotation revealed 58 841 protein-coding genes and 76.91% repetitive sequences, with the dominant repetitive sequences being the I-type repeats (Copia and Gypsy accounting for 18.34% and 13.5% of the genome, respectively). Our findings also provide support for a whole genome duplication event in the lineage leading to A. hybrid, which occurred after its divergence from subfamily Asparagoideae. Moreover, we identified a gene duplication event in the phosphoenolpyruvate carboxylase kinase (PEPCK) gene family and revealed that three PEPCK genes (PEPCK3, PEPCK5, and PEPCK12) were involved in the CAM pathway. More importantly, we identified transcription factors enriched in the circadian rhythm, MAPK signaling, and plant hormone signal pathway that regulate the PEPCK3 expression by analysing the transcriptome and using yeast one-hybrid assays. Our results shed light on CAM evolution and offer an essential resource for the molecular breeding program of Agave spp.

摘要
Agavoideae 亚科包括了具有景天酸代谢（CAM）、C3 和 C4 光合作用类型的植物，且其物种形成较年轻，突变积累较慢，使其成为研究 CAM 进化的典型作物。然而，由于缺乏基因组信息，CAM 进化的遗传机制仍不明确。本研究使用高通量染色体构象捕获、Nanopore 和 Illumina 技术生成的数据，对 Agave hybrid NO.11648（一种常规 CAM 植物，属于 Agavoideae 亚科）进行染色体级基因组组装，结果获得了 30 条假染色体，基因组大小为 4.87 Gb，scaffold N50 为 186.42 Mb。基因组注释结果揭示了 58,841 个蛋白质编码基因和 76.91% 的重复序列，其中主要的重复序列为 I 型重复（Copia 和 Gypsy 分别占基因组的 18.34% 和 13.5%）。我们的研究还支持了 A. hybrid 所在谱系发生了一次全基因组重复事件，该事件发生在其与 Asparagoideae 亚科的分化之后。此外，我们在磷酸烯醇丙酮酸羧化酶激酶（PEPCK）基因家族中识别到了一次基因重复事件，并揭示了三个 PEPCK 基因（PEPCK3、PEPCK5 和 PEPCK12）参与了 CAM 通路。更重要的是，通过分析转录组并使用酵母单杂交实验，我们发现了富集于昼夜节律、MAPK 信号通路和植物激素信号通路的转录因子，这些转录因子通过调控 PEPCK3 的表达参与 CAM 途径的调控。我们的研究为 CAM 进化提供了新的见解，并为 Agave 物种的分子育种项目提供了重要资源。

比较基因组分析  Comparative genomic analysis

We aligned the protein-coding genes of 14 species to identify orthologous genes and assign gene families. Considering the evolutionary position of species, the quality of genome data, and the methods of obtaining genome data, we selected basal angiosperms (Amborella trichopoda), dicots (Arabidopsis thaliana, Durio zibethinus, Spinacia oleracea, and Solanum lycopersicum), and monocots (Zostera marina, Asparagus officinalis, Asparagus setaceus, Phalaenopsis equestris, Phoenix dactylifera, Oryza sativa, and Musa schizocarpa) for comparative genome analysis. Among these species, A. officinalis, A. setaceus, P. equestris (belong to Orchidaceae), and A. hybrid belong to the Asparagales, with A. officinalis, A. setaceus, and A. hybrid belonging to the family Asparagaceae, having the closest phylogenetic relationship. Our analysis revealed that out of the predicted 58 841 genes of A. hybrid, 48 315 genes were classified into 18 445 gene families, of which 2122 were found in 14 analysed species and 1332 were unique to A. hybrid (Fig. 2A;