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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.

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.

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. Keywords Herbaceous peony Á Reference gene Á Light stress Á F-box Á GAPDH Abbreviations DFG Paeonia lactiflora 'Da Fugui' CTH Paeonia lactiflora 'Chui Touhong' qPCR Quantitative real-time PCR CV Coefficient of variation F-box F-box protein GAPDH Glyceraldehyde-3-phosphate dehydrogenase UBQ10 Polyubiquitin CYP Cyclophilin ACT Actin/actin-like conserved site-containing protein EF-a Elongation factor-1 alpha 3 AQU Probable aquaporin PIP1-2 ETI Eukaryotic translation initiation factor 5A-2 E3 E3 ubiquitin protein ligase RIE1 pp2A Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform-like Tm The melting temperature Cq Quantification cycle Electronic supplementary material The online version of this article (

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