Real-time quantitative PCR (RT-qPCR) has a high sensitivity and strong specificity, and is widely used in the analysis of gene expression. Selecting appropriate internal reference genes is the key to accurately analyzing the expression changes of target genes by RT-qPCR. To find out the most suitable internal reference genes for studying the gene expression in Broussonetia papyrifera under abiotic stresses (including drought, salt, and ZnSO₄ treatments), seven different tissues of B. papyrifera, as well as the roots, stems, and leaves of B. papyrifera under the abiotic stresses were used as test materials, and 15 candidate internal reference genes were screened based on the transcriptome data via RT-qPCR. Then, the expression stability of the candidate genes was comprehensively evaluated through the software geNorm (v3.5), NormFinder (v0.953), BestKeeper (v1.0), and RefFinder. The best internal reference genes and their combinations were screened out according to the analysis results. rRNA and Actin were the best reference genes under drought stress. Under salt stress, DOUB, HSP, NADH, and rRNA were the most stable reference genes. Under heavy metal stress, HSP and NADH were the most suitable reference genes. EIF3 and Actin were the most suitable internal reference genes in the different tissues of B. papyrifera. In addition, HSP, rRNA, NADH, and UBC were the most suitable internal reference genes for the abiotic stresses and the different tissues of B. papyrifera. The expression patterns of DREB and POD were analyzed by using the selected stable and unstable reference genes. This further verified the reliability of the screened internal reference genes. This study lays the foundation for the functional analysis and regulatory mechanism research of genes in B. papyrifera.

Euonymus microcarpus (Oliv.) Sprague, is a species of evergreen shrub of the genus Euonymus, family Celastraceae. Here, we extracted the genomic DNA from the leaves of E. microcarpus and constructed a paired-end library. The chloroplast genome of E. microcarpus was generated with the high-throughput sequencing by the illumina Hiseq X Ten platform and de novo assembly. The chloroplast genome had a quadripartite structure, containing a long single copy region with a size of 85,386 bp and a short single copy region with a size of 18,456 bp, separated by two inverted repeat regions of 26,850 bp. The chloroplast genome contained 133 genes identified in total, including 87 potential protein-coding genes, 38 transfer RNA genes, and eight ribosomal RNA genes. A total of 282 simple sequence repeats and 63 long repeats were found. Furthermore, the phylogenetic relationships inferred that E. microcarpus is sister to E. japonicus and E. schensianus. A comparison of the structure of the chloroplast genomes of eight Euonymus species suggests a nucleotide variability of the junction sites and a higher divergence of non-coding regions, compared to the coding regions. The original findings of the study serves as a good reference for chloroplast genome assembly and a valuable foundation for the genetic diversity and evolution of E. microcarpus.

The argonaute (AGO) protein, as an important member of the small RNA (sRNA) regulatory pathway gene-silencing complex (RNA-induced silencing complex, RISC), is a key protein that mediates gene silencing and plays a key role in the recruitment of sRNAs. In this study, bioinformatics was used to identify the AGO gene family in poplar and study its expression in various tissues and in response to abiotic stress treatments. A total of 15 PtAGO genes were identified in poplar, which were unevenly distributed in 9 chromosomes. Most proteins were predicted to be located in the nucleus and chloroplast. The PtAGOs had similar motif structures and conserved motifs, except for PtAGO3. All the PtAGO genes could be clustered into 3 groups, and Group II, including PtAGO2/3/7, had the smallest number of exons, while the others had more than 20 exons. Cis-regulatory elements involved in light response, growth and development, abiotic stress and hormone-induced responses were found in the promoters of PtAGO members. Further expression analysis found that the PtAGO genes had tissue-specific expression patterns. For example, PtAGO7 and PtAGO10b were mainly expressed in the xylem and might be involved in secondary xylem development. Furthermore, abiotic stress tests, including heat, ABA and PEG treatments, showed that most PtAGO genes could respond quickly to ABA treatment, and multiple PtAGO genes were constantly regulated under heat-shock stress. These results provide a basis for the elucidation mechanism of PtAGO genes and further molecular breeding in poplar.

The main objective of this study was to reveal the molecular mechanism of the albinism in Schima superba and to identify the related functional genes to provide theoretical support for the optimization of S. superba seedling nursery technology. Combining third-generation SMRT sequencing with second-generation high-throughput sequencing technology, the transcriptomes of normal seedlings and albinism seedlings of S. superba were analyzed and the sequencing data were functionally annotated and deeply resolved. The results showed that 270 differentially expressed transcripts were screened by analyzing second-generation sequencing data. KEGG enrichment analysis of the annotation information revealed that, among the photosynthesis-antenna protein-related pathways, the expression of LHCA3 and LHCB6 was found to be down-regulated in S. superba albinism seedlings, suggesting that the down-regulation of photosynthesis-related proteins may affect the development of chloroplasts in leaves. Down-regulated expression of VDE in the carotenoid biosynthesis leads to impaired chlorophyll cycling. In addition, transcription factors (TFs), such as bHLH, MYB, GLK and NAC, were closely associated with chloroplast development in S. superba seedlings. In summary, the present study systematically explored the transcriptomic features of S. superba albinism seedlings, screened out key genes with significant differential expression and provide a reference for further localization and cloning of the key genes for S. superba albinism, in addition to laying an essential theoretical foundation for an in-depth understanding of the molecular mechanism of the S. superba albinism. The genes identified in this study that are associated with S. superba albinism will be important targets for genetic modification or molecular marker development, which is essential for improving the cultivation efficiency of S. superba.