Expression of juvenility related microRNAs and target genes during micropropagation of silver birch (Betula pendula Roth.)

Abstract

Silver birch (Betula pendula Roth.) is an ecologically and economically important deciduous tree species in Northern Europe. In vitro culture initiation and shoot rejuvenation from birches in their reproductive phase is difficult and often unsuccessful. Currently, markers to evaluate the degree of juvenility of birch in vitro shoots, which could be used to track the rejuvenation process and to determine factors affecting it, have not been developed. The aims of this study were to assess expression of juvenility related microRNAs and target genes during micropropagation of silver birch, and to investigate factors affecting juvenility of birch genotypes with different in vitro morphogenic ability. Potential precursor sequences and target genes of the microRNAs miR156 and miR172 were determined by alignment to the birch unigene set. Ten potential miR156 and miR172 precursor sequences were identified, and were tested for the ability to form the required stem-loop structure. Based on precursor sequences, primers were designed for real time PCR analysis of precursor miRNA expression. Expression patterns of two miR156 family precursors (miR156_511 and miR156_789) and one miR172 precursor (miR172_1931) and two target genes (BpSPL1 and BpAP2) had the best correlation with juvenility/maturity in the analysed in vitro propagated silver birch samples. Expression patterns of these miRNA precursors and target genes were also investigated in samples cultured under different in vitro conditions. This study provides an initial survey of molecular markers for assessment of phase change in birch in vitro micropropagation.

Differentially Expressed Conserved Plant Vegetative Phase-Change-Related microRNAs in Mature and Rejuvenated Silver Birch In Vitro Propagated Tissues

“…As described above, precursor sequences were not identified for all differentially expressed miRNAs. Expression of these selected precursors, as well as precursors for the miR156 and miR172 families, was determined as described previously [ 29 ]. Relative expression between the sample group MAT, IVM, REV and JUV was determined for 11 precursors, each from a different miRNA family ( Supplementary File S5 ).…”

“…Analysis of expression of selected precursor miRNAs using qPCR was conducted as described previously [ 29 ]. Primers for selected pre-miRNA amplification were designed using Primer 3 version 0.4.0.…”

Expression of highly conserved developmental phase change regulating miR156 and miR172 microRNAs in silver birch using real-time PCR and high-throughput sequencing methodologies

“…Analysis of specific precursor miRNA expression patterns was done using real-time PCR (Krivmane et al 2022). Of seven miR156 precursors tested, three showed the expected transcript levels.…”

“…This is in accordance with the central role that the miR156 family plays in the transition from the juvenile phase to maturity in a number of plant species (Wu et al 2009;Wang et al 2011;Xing et al 2014;Jia et al 2017;Ahsan et al 2019a;Ahsan et al 2019b). Interestingly, real-time PCR analysis identified two miR172 family members that were highly expressed in mature control samples (Krivmane et al 2022). However, high-throughput sequencing did not identify any differentially expressed mature miR172 miRNAs between the mature and juvenile controls, and expression of miR172 miRNAs was very low in all samples analysed.…”

“…RNA extraction, PCR primers and real-time PCR methods are described in Krivmane et al (2022). Briefly, total RNA was extracted from samples using a standard phenol/chloroform/isoamyl alcohol protocol, and checked for DNA contamination by PCR.…”