Identification and characterization of miRNAome in root, stem, leaf and tuber developmental stages of potato (Solanum tuberosum L.) by high-throughput sequencing

Abstract

BackgroundMicroRNAs (miRNAs) are ubiquitous components of endogenous plant transcriptome. miRNAs are small, single-stranded and ~21 nt long RNAs which regulate gene expression at the post-transcriptional level and are known to play essential roles in various aspects of plant development and growth. Previously, a number of miRNAs have been identified in potato through in silico analysis and deep sequencing approach. However, identification of miRNAs through deep sequencing approach was limited to a few tissue types and developmental stages. This study reports the identification and characterization of potato miRNAs in three different vegetative tissues and four stages of tuber development by high throughput sequencing.ResultsSmall RNA libraries were constructed from leaf, stem, root and four early developmental stages of tuberization and subjected to deep sequencing, followed by bioinformatics analysis. A total of 89 conserved miRNAs (belonging to 33 families), 147 potato-specific miRNAs (with star sequence) and 112 candidate potato-specific miRNAs (without star sequence) were identified. The digital expression profiling based on TPM (Transcripts Per Million) and qRT-PCR analysis of conserved and potato-specific miRNAs revealed that some of the miRNAs showed tissue specific expression (leaf, stem and root) while a few demonstrated tuberization stage-specific expressions. Targets were predicted for identified conserved and potato-specific miRNAs, and predicted targets of four conserved miRNAs, miR160, miR164, miR172 and miR171, which are ARF16 (Auxin Response Factor 16), NAM (NO APICAL MERISTEM), RAP1 (Relative to APETALA2 1) and HAM (HAIRY MERISTEM) respectively, were experimentally validated using 5′ RLM-RACE (RNA ligase mediated rapid amplification of cDNA ends). Gene ontology (GO) analysis for potato-specific miRNAs was also performed to predict their potential biological functions.ConclusionsWe report a comprehensive study of potato miRNAs at genome-wide level by high-throughput sequencing and demonstrate that these miRNAs have tissue and/or developmental stage-specific expression profile. Also, predicted targets of conserved miRNAs were experimentally confirmed for the first time in potato. Our findings indicate the existence of extensive and complex small RNA population in this crop and suggest their important role in pathways involved in diverse biological processes, including tuber development.

Identification and Target Prediction of MicroRNAs in Ulmus pumila L. Seedling Roots under Salt Stress by High-Throughput Sequencing

“…These sizes and proportions are consistent with typical small RNA distributions in angiosperms such as cotton [7], Lycium chinense Mill. [28], Punica granatum L. [38], and potato [39]. Forests 2016, 7, 318 6 of 14 Figure 1.…”

Achievements and prospects of applying high-throughput sequencing techniques to potato genetics and breeding

“…В этом разделе суммированы данные 1 583 экспериментов, посвященных анализу транскриптома картофеля. Результаты транс криптомных экспериментов в БД GEO представлены в 30 публикациях, в число которых входят исследования по изменению транскриптома картофеля в ответ на за ражение вирусами Petek et al, 2014;Stare et al, 2015), на воздействие факторами абиотического стресса (Gálvez et al, 2016), различия в уровне экспрессии генов miRNA в зависимости от стадии развития (Lakhotia et al, 2014). Первичные данные секвенирования транс криптомов в экспериментах RNAseq представлены в базе данных NCBI SRA (Sequence Read Archive), основном мировом архиве результатов высокопроизводительного секвенирования нуклеотидных последовательностей.…”

“…Для секвенирования используют как экспериментальные подходы, связанные с высокопроизводительным секвени рованием (Zhang et al, 2013), так и биоинформатические подходы для предсказания структуры микроРНК по уже известным последовательностям (Zhang et al, 2009). Ис следования направлены на изучение микроРНК, которые регулируют экспрессию конкретных генов (Chi et al, 2015), участвующих в формировании стрессового ответа на засуху (Zhang et al, 2014) и играющих роль в процессе клубнеобразования (Martin et al, 2009;Bhogale et al, 2014;Lakhotia et al, 2014).…”

“…Для большинства из них были пред сказаны генымишени. В дальнейших исследованиях сравнительный анализ микроРНКома (совокупности всех микроРНК) выполнял ся в разрезе онтогенетического развития (Lakhotia et al, 2014) и ответа на засуху (Zhang et al, 2014). В результате в работе (Lakhotia et al, 2014) были выявлены микроРНК картофеля, мишенями для которых являются гены, коди рующие известные факторы, важные для развития рас тений (ARF16 -auxin response factor 16, NAM -no apical meristem, RAP1 -relative to APETALA2 1, HAM -hairy meristem), а в работе (Zhang et al, 2014) выявлены четыре микроРНК, участвующие в регуляции ответа на данный вид стресса, и предсказаны мишени для этих микроРНК.…”

Recent advances in potato genomics, transcriptomics, and transgenicsunder drought and heat stresses: a review

“…In this study, 48 potential miRNAs were identified in S. tuberosum by in silico comparisons of known miRNAs from other plants against potato EST, GSS, and nucleotide databases. This first study to identify potato miRNAs was followed by several other studies Xie et al, 2011;Zhang et al, 2013;Lakhotia et al, 2014;Ou et al, 2014;Zhang et al, 2014), in which the number of predicted miRNAs and their potential targets in potato increased significantly, suggesting that the prediction algorithms employed to identify new miRNAs and their targets have improved in recent years.…”