Background: The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow. Results: Here, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR) for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs). These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis. Conclusions: The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures.

SQUAMOSA PROMOTER BINDING PROTEIN (SBP)-box genes encode a family of plant-specific transcription factors (TFs) that play important roles in many growth and development processes including phase transition, leaf initiation, shoot and inflorescence branching, fruit development and ripening etc. The SBP-box gene family has been identified and characterized in many species, but has not been well studied in Petunia, an important ornamental genus. We identified 21 putative SPL genes of Petunia axillaris and P. inflata from the reference genome of P. axillaris N and P. inflata S6, respectively, which were supported by the transcriptome data. For further confirmation, all the 21 genes were also cloned from P. hybrida line W115 (Mitchel diploid). Phylogenetic analysis based on the highly conserved SBP domains arranged PhSPLs in eight groups, analogous to those from Arabidopsis and tomato. Furthermore, the Petunia SPL genes had similar exon-intron structure and the deduced proteins contained very similar conserved motifs within the same subgroup. Out of 21 PhSPL genes, fourteen were predicted to be potential targets of PhmiR156/157, and the putative miR156/157 response elements (MREs) were located in the coding region of group IV, V, VII and VIII genes, but in the 3’-UTR regions of group VI genes. SPL genes were also identified from another two wild Petunia species, P. integrifolia and P. exserta, based on their transcriptome databases to investigate the origin of PhSPLs. Phylogenetic analysis and multiple alignments of the coding sequences of PhSPLs and their orthologs from wild species indicated that PhSPLs were originated mainly from P. axillaris. qRT-PCR analysis demonstrated differential spatiotemperal expression patterns of PhSPL genes in petunia and many were expressed predominantly in the axillary buds and/or inflorescences. In addition, overexpression of PhSPL9a and PhSPL9b in Arabidopsis suggested that these genes play a conserved role in promoting the vegetative-to-reproductive phase transition. Petunia genome contains at least 21 SPL genes, and most of the genes are expressed in different tissues. The PhSPL genes may play conserved and diverse roles in plant growth and development, including flowering regulation, leaf initiation, axillary bud and inflorescence development. This work provides a comprehensive understanding of the SBP-box gene family in Petunia and lays a significant foundation for future studies on the function and evolution of SPL genes in petunia.

Somatic embryogenesis is a notable illustration of cell totipotency, by which somatic cells undergo dedifferentiation and then differentiate into somatic embryos. Our previous work demonstrated that pretreatment of immature zygotic embryos with 0.5 M sucrose solution for 72 h efficiently induced somatic embryo initiation in camphor tree. To better understand the molecular basis of somatic embryogenesis induced by osmotic stress, de novo transcriptome sequencing of three tissues of camphor tree (immature zygotic embryos, sucrose-pretreated immature zygotic embryos, and somatic embryos induced from sucrose-pretreated zygotic embryos) were conducted using Illumina Hiseq 2000 platform. A total of 30.70 G high quality clean reads were obtained from cDNA libraries of the three samples. The overall de novo assembly of cDNA sequence data generated 205592 transcripts, with an average length of 998 bp. 114229 unigenes (55.56 % of all transcripts) with an average length of 680 bp were annotated with gene descriptions, gene ontology terms or metabolic pathways based on Blastx search against Nr, Nt, Swissprot, GO, COG/KOG, and KEGG databases. CEGMA software identified 237 out of 248 ultra-conserved core proteins as ‘complete’ in the transcriptome assembly, showing a completeness of 95.6 %. A total of 897 genes previously annotated to be potentially involved in somatic embryogenesis were identified. Comparative transcriptome analysis showed that a total of 3335 genes were differentially expressed in the three samples. The differentially expressed genes were divided into six groups based on K-means clustering. Expression level analysis of 52 somatic embryogenesis-related genes indicated a high correlation between RNA-seq and qRT-PCR data. Gene enrichment analysis showed significantly differential expression of genes responding to stress and stimulus. The present work reported a de novo transcriptome assembly and global analysis focused on gene expression changes during initiation and formation of somatic embryos in camphor tree. Differential expression of somatic embryogenesis-related genes indicates that sucrose induced somatic embryogenesis may share or partly share the mechanisms of somatic embryogenesis induced by plant hormones. This study provides comprehensive transcript information and gene expression data for camphor tree. It could also serve as an important platform resource for further functional studies in plant embryogenesis.

Roses are significant botanical species with both ornamental and economic value, displaying diverse floral traits, particularly an extensive array of petal colors. The red pigmentation of rose petals is predominantly attributed to anthocyanin accumulation. However, the underlying regulatory mechanism of anthocyanin biosynthesis in roses remains elusive. This study presents a novel light-responsive regulatory module governing anthocyanin biosynthesis in rose petals, which involves the transcription factors RhHY5, RhMYB114a, and RhMYB3b. Under light conditions, RhHY5 represses RhMYB3b expression, and induces RhMYB114a expression that positively regulates anthocyanin biosynthesis in rose petals through the direct activation of anthocyanin structural genes via the MYB114a-bHLH3-WD40 complex. Notably, this function likely involves an interaction and synergy between RhHY5 and the MYB114a-bHLH3-WD40 complex. Additionally, RhMYB3b is activated by RhMYB114a to prevent excessive accumulation of anthocyanin. Conversely, in low light conditions, the degradation of RhHY5 leads to down-regulation of RhMYB114a and up-regulation of RhMYB3b, which in turn inhibits the expression of both RhMYB114a and anthocyanin structural genes. Additionally, RhMYB3b competes with RhMYB114a for binding to RhbHLH3 and the promoters of anthocyanin-related structural genes. Overall, our study uncovers a complex light-mediated regulatory network that governs anthocyanin biosynthesis in the rose, thereby advancing our understanding of the underlying molecular mechanisms of anthocyanin biosynthesis in rose flowers.

The C2H2 zinc finger proteins (ZFPs) play essential roles in regulating cold stress responses. Similarly, raffinose accumulation contributes to freezing stress tolerance. However, the relationship between C2H2 functions and raffinose synthesis in cold tolerance remains uncertain. Here, we report the characterization of the cold-induced C2H2-type zinc finger protein PhZFP1 in Petunia hybrida. PhZFP1 was found to be predominantly localized in the nucleus. Overexpression of PhZFP1 conferred enhanced cold tolerance in transgenic petunia lines. In contrast, RNAi mediated suppression of PhZFP1 led to increased cold susceptibility. PhZFP1 regulated the expression of a range of abiotic stress responsive-genes including genes encoding proteins involved in reactive oxygen species (ROS) scavenging and raffinose metabolism. The accumulation of galactinol and raffinose, and the levels of PhGolS1-1 transcripts, were significantly increased in PhZFP1-overexpressing plants and decreased in PhZFP1-RNAi plants under cold stress. Moreover, the galactinol synthase (GolS)-encoding gene PhGolS1-1 was identified as a direct target of PhZFP1. Taken together, these results demonstrate that PhZFP1 functions in cold stress tolerance by modulation of galactinol synthesis via regulation of PhGolS1-1. This study also provides new insights into the mechanisms underlying C2H2 zinc finger protein-mediated cold stress tolerance, and has identified a candidate gene for improving cold stress tolerance.