Genome sequencing and CRISPR/Cas9 gene editing of an early flowering Mini‐Citrus (Fortunella hindsii)

First author: Chenqiao Zhu; Affiliations: Huazhong Agricultural University (华中农业大学): Wuhan, China

Corresponding author: Xiuxin Deng

Hongkong kumquat (Fortunella hindsii) is a wild citrus species characterized by dwarf plant height and early flowering. Here, we identified the monoembryonic F. hindsii (designated as ‘Mini‐Citrus’) for the first time and constructed its selfing lines. This germplasm constitutes an ideal model for the genetic and functional genomics studies of citrus, which have been severely hindered by the long juvenility and inherent apomixes of citrus. F. hindsii showed a very short juvenile period (~8 months) and stable monoembryonic phenotype under cultivation. We report the first de novo assembled 373.6 Mb genome sequences (Contig‐N50 2.2 Mb and Scaffold‐N50 5.2 Mb) for F. hindsii. In total, 32 257 protein‐coding genes were annotated, 96.9% of which had homologues in other eight Citrinae species. The phylogenomic analysis revealed a close relationship of F. hindsii with cultivated citrus varieties, especially with mandarin. Furthermore, the CRISPR/Cas9 system was demonstrated to be an efficient strategy to generate target mutagenesis on F. hindsii. The modifications of target genes in the CRISPR‐modified F. hindsii were predominantly 1‐bp insertions or small deletions. This genetic transformation system based on F. hindsii could shorten the whole process from explant to T1 mutant to about 15 months. Overall, due to its short juvenility, monoembryony, close genetic background to cultivated citrus and applicability of CRISPR, F. hindsii shows unprecedented potentials to be used as a model species for citrus research.

山金柑是野生柑橘类物种，特征是植株较矮并且早花。本文中，作者第一次鉴定了单胚山金柑，即小柑橘，并且构建了自交系。该种质资源只一个研究柑橘遗传和功能基因组学的理想模型，以往柑橘的研究由于幼年期较长，并且天然孤雌生殖导致研究停滞不前。山金柑的幼年期时间较短，只有大概8个月，并且栽培条件下具有稳定的单胚表型。作者报道了第一个山金柑的从头组装的基因组，大小约373.6 Mb，ContigN50约2.2 Mb，ScaffoldN50约5.2 Mb。作者注释了约32 257个蛋白编码基因，其中96.9%的基因在其它八个已报道的亚族物种中具有同源基因。系统演化分析显示山金柑与栽培柑橘品种非常近缘，尤其是橘子。此外，作者的研究显示CRISPR/Cas9系统是一套非常有效的策略用来产生山金柑靶向突变株系。该基于山金柑的遗传转化系统可以将从外植体到T1突变体整个过程缩短到15个月。综上，由于幼年期较短，单胚胎，并且与栽培种柑橘遗传关系较近，并且CRISPR系统的成功建立，山金柑可以用作于柑橘类物种研究的模式植物研究。

通讯：邓秀新 (http://chfs.hzau.edu.cn/info/1028/1143.htm)

个人简介：1978-1981年，湖南农业大学，学士；1982-1984年，华中农业大学，硕士；1985-1987年，华中农业大学，博士。

研究方向：1）种质资源挖掘与利用：野生柑橘资源的收集评价，芽变材料的筛选鉴定，新品种选育；2）种质创新与遗传改良：杂交群体创建，遗传连锁图谱构建，纯系创制，遗传转化及功能基因组平台构建；3）果实色泽形成机理与调控：红肉突变体性状形成机理解析，类胡萝卜素代谢调控关键基因挖掘，质体发育分化，类胡萝卜素代谢工程；4）柑橘生殖生物学研究：单/多胚性状形成机理解析；5）产业宏观问题研究：现代农业产业体系运行机制，产业安全及可持续发展研究。

doi: https://doi.org/10.1111/pbi.13132

Journal: Plant Biotechnology Journal

First Published: April 20, 2019