Central University of Kerala, Periya, Kasaragod, Kerala, 671320, India.
ICAR-Central Plantation Crops Research Institute, Kasaragod, Kerala, 671124, India.
Planta. 2020 Mar 12;251(4):79. doi: 10.1007/s00425-020-03368-4.
Genome-wide analysis of small RNAs identifies somatic embryogenesis- specific miRNAs and their targets and provides novel insights into the mechanisms governing somatic embryogenesis in coconut, a highly in vitro recalcitrant species. Coconut, a major plantation crop of the tropics is recalcitrant to in vitro culture with a very low rate of somatic embryo turnover. Clonal propagation to enhance the production of high yielding, disease-free planting material in coconut has remained a distant reality. To better understand the molecular basis of this recalcitrance and to throw light on the complex regulatory network involved in the transition of coconut somatic cells to embryogenic calli, genome-wide profiling of small RNAs from embryogenic (EC) and non-embryogenic calli (NEC) was undertaken using Illumina Hiseq 2000 platform. We have identified a total of 110 conserved miRNAs (representing 46 known miRNA families) in both types of calli. In addition, 97 novel miRNAs (48 specific to EC, 21 specific to NEC and 28 common to both the libraries) were also identified. Among the conserved miRNAs, 10 were found to be differentially expressed between NEC and EC libraries with a log2 fold change > 2 following RPM-based normalization. miR156f, miR167c, miR169a, miR319a, miR535a, and miR5179 are upregulated and miR160a, miR166a, miR171a, and miR319b are down-regulated in NEC. To confirm the differential expression pattern and their regulatory role in SE, the expression patterns of miRNAs and their putative targets were analyzed using qRT- PCR and most of the analyzed miRNA-target pairs showed inverse correlation during somatic embryogenesis. Selected targets were further validated by RNA ligase mediated rapid amplification of 5' cDNA ends (5'RLM-RACE). Our data suggest that a few conserved miRNAs and species-specific miRNAs act in concert to regulate the process of somatic embryogenesis in coconut. The results of this study provide the first overview into the regulatory landscape of somatic embryogenesis in coconut and possible strategies for fine-tuning or reprogramming to enhance somatic embryo turn over in coconut.
对小 RNA 的全基因组分析鉴定了体细胞胚胎发生特异性 miRNA 及其靶标，并为控制椰子体细胞胚胎发生的机制提供了新的见解，椰子是热带地区的主要种植作物，其离体培养非常困难，体细胞胚胎的转化率非常低。克隆繁殖以提高高产、无病种植材料在椰子中的产量仍然是一个遥远的现实。为了更好地理解这种抗逆性的分子基础，并阐明涉及椰子体细胞向胚胎发生愈伤组织转化的复杂调控网络，我们使用 Illumina Hiseq 2000 平台对胚胎发生（EC）和非胚胎发生（NEC）愈伤组织中的小 RNA 进行了全基因组分析。我们在两种愈伤组织中总共鉴定了 110 个保守的 miRNA（代表 46 个已知的 miRNA 家族）。此外，还鉴定了 97 个新的 miRNA（48 个特异性 EC，21 个特异性 NEC，28 个在两个文库中都有）。在保守的 miRNA 中，有 10 个在 NEC 和 EC 文库之间差异表达，对数 2 倍变化（RPM 归一化后>2）。miR156f、miR167c、miR169a、miR319a、miR535a 和 miR5179 在 NEC 中上调，miR160a、miR166a、miR171a 和 miR319b 在 NEC 中下调。为了验证差异表达模式及其在 SE 中的调节作用，使用 qRT-PCR 分析了 miRNA 和其假定靶标的表达模式，在体细胞胚胎发生过程中，大多数分析的 miRNA-靶对表现出相反的相关性。选择的靶标进一步通过 RNA 连接酶介导的 5' cDNA 末端快速扩增（5'RLM-RACE）进行验证。我们的数据表明，一些保守的 miRNA 和物种特异性 miRNA 协同作用，调节椰子体细胞胚胎发生的过程。这项研究的结果提供了椰子体细胞胚胎发生调控景观的第一个概述，并为微调或重新编程以提高椰子体细胞胚胎的转化率提供了可能的策略。
