Shade effects on growth, photosynthesis and chlorophyll fluorescence parameters of three Paeonia species.

Insufficient light intensity inhibits the growth of cultivated herbaceous peony and decreases its economic value. Owing to the increased demand for shade-tolerant herbaceous peony, the selection of appropriate parents for hybridization is essential. Paeonia anomala, Paeonia intermedia and Paeonia veitchii can grow under shade conditions in their natural habitats; however, their photosynthetic capacities under shade have not been studied. In this study, we simulated low light intensity (30% sunlight) and evaluated the morphological, photosynthetic and chlorophyll fluorescence parameters of these three species. Moreover, the shade tolerance of these species as well as two common cultivars (Paeonia lactiflora 'Da Fugui', which is suitable for solar greenhouse cultivation, and P. lactiflora 'Qiao Ling', which is not suitable for solar greenhouse cultivation) was evaluated. The results showed that under shade, the leaf area of P. anomala and P. intermedia increased, the single flowering period of P. intermedia and P. veitchii was prolonged, and the flower color of P. veitchii faded. With respect to P. anomala, P. intermedia and P. veitchii, shade eliminated the photosynthetic 'lunch break' phenomenon and decreased photoinhibition at midday. Furthermore, the maximum photochemical efficiency (Fv/Fm) and maximum primary photochemical yield (Fv/Fo) of photosystem II (PSII) in the three species improved significantly, and their changes in light dissipation were different. The shade tolerance of the tested accessions was in the order P. veitchii > P. intermedia > P. anomala > 'Da Fugui' > 'Qiao Ling', showing that the three wild species were better adapted to low light intensity than the cultivars. Thus, P. anomala, P. intermedia and P. veitchii could potentially be used in the development of shade-tolerant herbaceous peony cultivars.

Characteristics of Microsatellites Mined from Transcriptome Data and the Development of Novel Markers in Paeonia lactiflora.

The insufficient number of available simple sequence repeats (SSRs) inhibits genetic research on and molecular breeding of Paeonia lactiflora, a flowering crop with great economic value. The objective of this study was to develop SSRs for P. lactiflora with Illumina RNA sequencing and assess the role of SSRs in gene regulation. The results showed that dinucleotides with AG/CT repeats were the most abundant type of repeat motif in P. lactiflora and were preferentially distributed in untranslated regions. Significant differences in SSR size were observed among motif types and locations. A large number of unigenes containing SSRs participated in catalytic activity, metabolic processes and cellular processes, and 28.16% of all transcription factors and 21.74% of hub genes for inflorescence stem straightness were found to contain SSRs. Successful amplification was achieved with 89.05% of 960 pairs of SSR primers, 55.83% of which were polymorphic, and most of the 46 tested primers had a high level of transferability to the genus Paeonia. Principal component and cluster dendrogram analyses produced results consistent with known genealogical relationships. This study provides a set of SSRs with abundant information for future accession identification, marker-trait association and molecular assisted breeding in P. lactiflora.

Transcriptome and weighted correlation network analyses provide insights into inflorescence stem straightness in Paeonia lactiflora.

KEY MESSAGE: Lack of structural components results in inflorescence stem bending. Differentially expressed genes involved in lignin and hemicellulose biosynthesis are vital; genes involved in cellulose and glycan biosynthesis are also relevant. An erect inflorescence stem is essential for high-quality cut herbaceous peony flowers. To explore the factors underlying inflorescence stem bending, major cell walls contents were measured, and stem structure was observed in two herbaceous peony varieties with contrasting stem straightness traits ('Da Fugui', upright; 'Chui Touhong', bending). In addition, Illumina sequencing was performed and weighted correlation network analysis (WGCNA) was used to analyze the results. The results showed significant differences in lignin, hemicellulose and soluble sugar contents, sclerenchyma and xylem areas and thickening in cell walls in pith at stage S3, when bending begins. In addition, 44,182 significantly differentially expressed genes (DEGs) were found, and these DEGs were mainly enriched in 36 pathways. Among the DEGs, hub genes involved in lignin, cellulose, and xylan biosynthesis and transcription factors that regulated these process were identified by WGCNA. These results suggested that the contents of compounds that provided cell wall rigidity were vital factors affecting inflorescence stem straightness in herbaceous peony. Genes involved in or regulating the biosynthesis of these compounds are thus important; lignin and hemicellulose are of great interest, and cellulose and glycan should not be ignored. This paper lays a foundation for developing new herbaceous peony varieties suitable for cut flowers by molecular-assisted breeding.

Selection and validation of reference genes of Paeonia lactiflora in growth development and light stress.

The stem of Paeonia lactiflora will bend when it grows in greenhouse at a low light intensity. It is important to explore causes of morphological changes of peony to improve its quality. Gene expression can be evaluated by quantitative real-time PCR, based on reference gene. However, systematic selection of reference genes under weak lighting for herbaceous peony is lacking. To address this problem, we first selected 10 candidate reference genes based on a coefficient of variation of gene expression from peony stem transcriptome data. Then, geNorm, NormFinder and BestKeeper were applied to assess the stability of the genes, and RankAggreg was used to give a comprehensive ranking. The results show that there are some differences in optimal reference genes among samples from different organs and under the two lighting conditions, and the optimal number of suitable reference genes is distinct. Two selected suitable reference genes were then used to normalize target genes, and the results were compared with transcriptome data. Consistent gene expression trends were obtained, indicating the reliability of the method. To the best of our knowledge, this is the first time reference genes for herbaceous peony were selected in different organs, developmental stages and under two kinds of lighting conditions. The findings can provide a practical method for selecting reference genes for peony under these conditions and demonstrate a useful combination of reference genes.