The precision and reliability of reverse transcription quantitative polymerase chain reaction (RT-qPCR) depend mainly on suitable reference genes; however, reference genes have not yet been identified for Liriodendron chinense (Hemsl.) Sarg. In this study, the expression stability of 15 candidate reference genes, ACT7, ACT97, UBQ1, eIF2, eIF3, HIS, BIG, AGD11, EFG, GAPDH, CYP, RPL25, UBC, RPB1, and TUB, was tested across multiple organs of L. chinense using four algorithms, geNorm, NormFinder, BestKeeper, and RefFinder. To understand the difference between the selected reference genes and the unsuitable candidate reference genes, the expression level of a target gene, LcPAT7, was normalized across various plant samples. ACT97 and eIF3 represented the best combination across all samples tested, while AGD11 and UBQ1 were unsuitable for normalization in this case. In the vegetative organ subset, ACT97, ACT7, and GAPDH showed the highest expression stability. For floral organs, UBC and eIF3 were the most stable reference genes. Unsuitable reference genes underestimated the expression levels of a target gene, LcPAT7. This study identified two reference genes (ACT97 and eIF3) for the precise and reliable normalization of L. chinense RT-qPCR data across various organs. Our work provides an effective framework for quantifying gene expression in L. chinense.

The leaf and the flower are vital plant organs owing to their roles in photosynthesis and reproduction. Long non-coding RNAs (lncRNAs), microRNAs (miRNAs), and transcription factors (TFs) are very important to the development of these organs. Liriodendron chinense is a common ornamental tree species in southern China with an unusual leaf shape and tulip-like flowers. The genetic mechanisms underlying leaf and flower development in L. chinense and the miRNA-lncRNA-TF regulatory networks are poorly studied. Through the integration and analysis of different types of sequencing data, we identified the miRNA-lncRNA-TF regulatory networks that were related to leaf and flower development. These networks contained 105 miRNAs, 258 lncRNAs, 393 TFs, and 22 endogenous target mimics. Notably, lch-lnc7374-miR156h-SPL3 and lch-lnc7374-miR156j-SPL9 were potential regulators of stamen and pistil development in L. chinense, respectively. miRNA-lncRNA-mRNA regulatory networks were shown to impact anther development, male and female fertility, and petal color by regulating the biosynthesis of phenylpropanoid metabolites. Phenylpropanoid metabolite biosynthesis genes and TFs that were targeted by miRNAs and lncRNAs were differentially expressed in the leaf and flower. Moreover, RT-qPCR analysis confirmed 22 differentially expressed miRNAs, among which most of them showed obvious leaf or flower specificity; miR157a-SPL and miR160a-ARF module were verified by using RLM-RACE, and these two modules were related to leaf and flower development. These findings provide insight into the roles of miRNA-lncRNA-mRNA regulatory networks in organ development and function in L. chinense, and will facilitate further investigation into the regulatory mechanisms of leaf and flower development in L. chinense.

Alternative splicing (AS) plays pivotal roles in regulating plant growth and development, flowering, biological rhythms, signal transduction, and stress responses. However, no studies on AS have been performed in Liriodendron chinense, a deciduous tree species that has high economic and ecological value. In this study, we used multiple tools and algorithms to analyze transcriptome data derived from seven tissues via hybrid sequencing. Although only 17.56% (8,503/48,408) of genes in L. chinense were alternatively spliced, these AS genes occurred in 37,844 AS events. Among these events, intron retention was the most frequent AS event, producing 1,656 PTCcontaining and 3,310 non-PTC-containing transcripts. Moreover, 183 long noncoding RNAs (lncRNAs) also underwent AS events. Furthermore, weighted gene coexpression network analysis (WGCNA) revealed that there were great differences in the activities of transcription and post-transcriptional regulation between pistils and leaves, and AS had an impact on many physiological and biochemical processes in L. chinense, such as photosynthesis, sphingolipid metabolism, fatty acid biosynthesis and metabolism. Moreover, our analysis showed that the features of genes may affect AS, as AS genes and non-AS genes had differences in the exon/intron length, transcript length, and number of exons/introns. In addition, the structure of AS genes may impact the frequencies and types of AS because AS genes with more exons or introns tended to exhibit more AS events, and shorter introns tended to be retained, whereas shorter exons tended to be skipped. Furthermore, eight AS genes were verified, and the results were consistent with our analysis. Overall, this study reveals that AS and gene interaction are mutual-on one hand, AS can affect gene expression and translation, while on the other hand, the structural characteristics of the gene can also affect AS. This work is the first to comprehensively report on AS in L. chinense, and it can provide a reference for further research on AS in L. chinense.

Liriodendron chinense is an economically and ecologically important deciduous tree species. Although the reference genome has been revealed, alternative polyadenylation (APA), transcription factors (TFs), long non-coding RNAs (lncRNAs), and co-expression networks of tissue-specific genes remain incompletely annotated. In this study, we used the bracts, petals, sepals, stamens, pistils, leaves, and shoot apex of L. chinense as materials for hybrid sequencing. On the one hand, we improved the annotation of the genome. We detected 13,139 novel genes, 7,527 lncRNAs, 1,791 TFs, and 6,721 genes with APA sites. On the other hand, we found that tissue-specific genes play a significant role in maintaining tissue characteristics. In total, 2,040 tissue-specific genes were identified, among which 9.2% of tissue-specific genes were affected by APA, and 1,809 tissue-specific genes were represented in seven specific co-expression modules. We also found that bract-specific hub genes were associated plant defense, leaf-specific hub genes were involved in energy metabolism. Moreover, we also found that a stamen-specific hub TF Lchi25777 may be involved in the determination of stamen identity, and a shoot-apex-specific hub TF Lchi05072 may participate in maintaining meristem characteristic. Our study provides a landscape of APA, lncRNAs, TFs, and tissue-specific gene co-expression networks in L. chinense that will improve genome annotation, strengthen our understanding of transcriptome complexity, and drive further research into the regulatory mechanisms of tissue-specific genes.

Hoffmann & Sgro, 2011). Indeed, due to their sessile nature and limited dispersal distance of pollen/seeds, it is impossible for plants to avoid disadvantageous environments by rapid migration, as observed in animals. Therefore, ecological adaptive differentiation is