Puglia Giuseppe D, Prjibelski Andrey D, Vitale Domenico, Bushmanova Elena, Schmid Karl J, Raccuia Salvatore A
Institute for Plant Breeding, Seed Science and Population Genetics, University of Hohenheim, Fruwirthstrasse 21, 70599, Stuttgart, Germany.
Consiglio Nazionale delle Ricerche, Istituto per i Sistemi Agricoli e Forestali del Mediterraneo (CNR-ISAFOM) U.O.S. Catania, Via Empedocle, 58, 95128, Catania, Italy.
BMC Genomics. 2020 Aug 21;21(1):317. doi: 10.1186/s12864-020-6670-5.
The investigation of transcriptome profiles using short reads in non-model organisms, which lack of well-annotated genomes, is limited by partial gene reconstruction and isoform detection. In contrast, long-reads sequencing techniques revealed their potential to generate complete transcript assemblies even when a reference genome is lacking. Cynara cardunculus var. altilis (DC) (cultivated cardoon) is a perennial hardy crop adapted to dry environments with many industrial and nutraceutical applications due to the richness of secondary metabolites mostly produced in flower heads. The investigation of this species benefited from the recent release of a draft genome, but the transcriptome profile during the capitula formation still remains unexplored. In the present study we show a transcriptome analysis of vegetative and inflorescence organs of cultivated cardoon through a novel hybrid RNA-seq assembly approach utilizing both long and short RNA-seq reads.
The inclusion of a single Nanopore flow-cell output in a hybrid sequencing approach determined an increase of 15% complete assembled genes and 18% transcript isoforms respect to short reads alone. Among 25,463 assembled unigenes, we identified 578 new genes and updated 13,039 gene models, 11,169 of which were alternatively spliced isoforms. During capitulum development, 3424 genes were differentially expressed and approximately two-thirds were identified as transcription factors including bHLH, MYB, NAC, C2H2 and MADS-box which were highly expressed especially after capitulum opening. We also show the expression dynamics of key genes involved in the production of valuable secondary metabolites of which capitulum is rich such as phenylpropanoids, flavonoids and sesquiterpene lactones. Most of their biosynthetic genes were strongly transcribed in the flower heads with alternative isoforms exhibiting differentially expression levels across the tissues.
This novel hybrid sequencing approach allowed to improve the transcriptome assembly, to update more than half of annotated genes and to identify many novel genes and different alternatively spliced isoforms. This study provides new insights on the flowering cycle in an Asteraceae plant, a valuable resource for plant biology and breeding in Cynara and an effective method for improving gene annotation.
在缺乏注释完善基因组的非模式生物中，利用短读长进行转录组图谱研究受到部分基因重建和异构体检测的限制。相比之下，长读长测序技术显示出即使在缺乏参考基因组的情况下也有潜力生成完整的转录本组装。刺菜蓟（Cynara cardunculus var. altilis (DC)）（栽培刺苞菜）是一种多年生耐寒作物，适应干旱环境，因其花头中产生丰富的次生代谢产物而具有许多工业和营养保健应用。该物种的研究受益于最近发布的基因组草图，但头状花序形成过程中的转录组图谱仍未得到探索。在本研究中，我们展示了通过一种利用长读长和短读长RNA测序读数的新型混合RNA-seq组装方法，对栽培刺苞菜的营养器官和花序器官进行转录组分析。
在混合测序方法中纳入单个纳米孔流动池输出数据，相对于仅使用短读长而言，完整组装基因增加了15%，转录异构体增加了18%。在25463个组装的单基因中，我们鉴定出578个新基因并更新了13039个基因模型，其中11169个是可变剪接异构体。在头状花序发育过程中，3424个基因差异表达，约三分之二被鉴定为转录因子，包括bHLH、MYB、NAC、C2H2和MADS-box，这些转录因子在头状花序开放后尤其高度表达。我们还展示了参与头状花序中丰富的有价值次生代谢产物（如苯丙烷类、黄酮类和倍半萜内酯）生产的关键基因的表达动态。它们的大多数生物合成基因在花头中强烈转录，可变异构体在不同组织中表现出不同的表达水平。
这种新型混合测序方法能够改进转录组组装、更新超过一半的注释基因，并鉴定出许多新基因和不同的可变剪接异构体。本研究为菊科植物的开花周期提供了新见解，是刺菜蓟植物生物学和育种的宝贵资源，也是改进基因注释的有效方法。
