The Genome Sizes of Fourteen Wild Paeonia Species Were Systematically Determined for the First Time

The Genome Sizes of Fourteen Wild Paeonia Species Were Systematically Determined for the First Time | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Genome Sizes of Fourteen Wild Paeonia Species Were Systematically Determined for the First Time Le Chen, Qihang Chen, Li Wang, Yong Yang, Miao Sun, Shaocai Zhu, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7705305/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract China is recognized as one of the global distribution centers for the genus Paeonia . However, among wild peony species, only Paeonia lactiflora has been relatively well-studied and utilized, while other wild peony resources remain under-investigated. In particular, the genome sizes of wild Chinese peonies have not been clearly defined. Among these, P. mairei , an endemic species in China, is classified as Near Threatened on the 2004 China Species Red List due to its limited and valuable wild populations. Nevertheless, its ploidy level and genome size remain unresolved, significantly hindering the further development and application of wild peony resources. In this study, eight populations of P. mairei and 13 additional wild peony species were analyzed using flow cytometry to determine ploidy levels and estimate genome sizes. All eight populations of P. mairei were confirmed as tetraploid, with no diploid types detected. The genome sizes of P. mairei ranged from 21.11 to 25.27 Gb, this study examined the relationship between pronounced variation in genome size among geographically distinct populations and their habitat conditions. The genome sizes of the 13 wild peony species were determined as follows: P. tenuifolia : 9.21 Gb; P. anomala : 9.91 Gb; P. emodi : 10.02 Gb; P. intermedia : 10.75 Gb; P. mlokosewitschii : 11.49 Gb; P. daurica : 11.77 Gb; P. lactiflora : 11.86 Gb; P. veitchii : 12.24 Gb; P. parnassica : 12.72 Gb; P. obovata : 13.51 Gb; P. sterniana : 13.24 Gb; P. officinalis : 18.66 Gb; P. arietina : 23.24 Gb. The study provides the first comprehensive measurements of genome sizes across 14 wild peony species, thereby enriching the genomic repository of the genus Paeonia and establishing a critical foundation for future research in genomics and molecular cytogenetics within this taxon. Paeonia flow cytometry P. mairei wild peony genome size Figures Figure 1 Figure 2 Figure 3 1. Introduction China is recognized as both the center of origin of the genus Paeonia and one of the distribution centers for wild resources of the subgenus Paeonia (Zhou, 2020). However, current analyses of their conservation status and studies on their potential for resource development remain limited, which hinders the effective conservation and utilization of wild peony resources in China. The subgenus Paeonia exhibits rich germplasm resources and is widely distributed across central and eastern Asia, the Himalayas, and the Mediterranean region. It is claimed to comprise three ploidy levels: diploid, triploid, and tetraploid (Hong, 2011 ). The diverse habitats of Paeonia species have contributed to high genetic variability (Halda & Waddick, 2004 ). Wild peonies display a wide range of ornamental traits. For instance, Paeonia tenuifolia is characterized by its finely dissected leaflets, while Paeonia veitchii , Paeonia anomala , and Paeonia sterniana exhibit deeply lobed leaves. Paeonia obovata is admired for its attractive flowers, and Paeonia mairei blooms early. The direct domestication or breeding of wild peonies with such diverse ornamental traits would significantly enrich the horticultural characteristics of cultivated varieties in China (Yang, 2020; Zhou, 2021). However, traditional Chinese peony cultivars have a relatively narrow genetic base, primarily derived from the domestication or hybridization of Paeonia lactiflora , with limited utilization of wild Paeonia resources. In contrast, international peony breeding programs extensively incorporate wild species, whereas China’s peony breeding efforts remain largely confined to P. lactiflora (Ferguson, 2001 ; Hong, 2010). This not only restricts the development of novel and diverse cultivars but also represents a substantial underutilization of China’s wild peony resources. Therefore, the conservation and exploitation of wild peonies are issues of great significance to the ornamental industry. P. mairei , an endemic Chinese species within the genus Paeonia , is classified as Near Threatened on the China Species Red List (2004) and represents a valuable wild genetic resource. It is characterized by its magenta-red flowers, early flowering period, and the horticulturally significant trait of a single stem bearing a solitary bloom. The species is primarily distributed across Shaanxi, Sichuan, and Yunnan provinces (Chen, 2019). P. mairei was first described by Léveillé in 1915 based on a specimen collected from Yunnan. The species has a narrow distribution and is often indiscriminately harvested as a substitute for P. veitchii , leading to the depletion of its already limited wild resources (Chen, 2023). The current lack of research on P. mairei hinders its effective conservation and utilization as a Chinese endemic species, highlighting an urgent need for studies on its resource status. Ploidy is a fundamental biological characteristic and an essential criterion for breeding applications (Dolezel, 2005 ). Early karyotypic analyses by La Lour (1952) and Leeper ( 1968 ) suggested that P. mairei is diploid. However, the provenance of La Lour’s plant material is unknown, and Leeper’s material was obtained from a garden in Germany, raising doubts about its taxonomic authenticity. In contrast, Hong (1988) conducted karyotypic analyses on three wild populations from Sichuan and Shaanxi, revealing that P. mairei is tetraploid. Since these populations were located in the northwestern and northern parts of the species’ distribution range, Hong proposed that diploid populations might exist in the southern and southeastern regions and recommended extensive sampling to determine whether variation in ploidy is correlated with geographical distribution. Based on previous research (Hong, 1988), it became established that at least a tetraploid cytotype exists in P. mairei , which is likely of allopolyploid origin. Allopolyploids represent important materials for investigating polyploidization, a major driver of plant evolution that leads to profound changes at the chromosomal level (Bottani, 2018). As an allopolyploid species endemic to China, P. mairei has not been sufficiently studied in terms of genome size, chromosomal composition, and genetic background. Understanding the structural variations in its chromosomal architecture and the consequent cytogenetic alterations is critical for elucidating the genomic mechanisms underlying polyploid speciation. Thus, determining the ploidy level and genome size of P. mairei is of fundamental significance. Ploidy and genome size represent fundamental biological characteristics in plants. The analysis of genomic features serves as a critical indicator for estimating the nuclear DNA content in haploid cells, forming an essential basis for both the exploitation of plant genetic resources and the investigation of molecular mechanisms. Determining genome size provides theoretical support for genomic studies, population-level genomic diversity analyses, and research in evolutionary genetics (Dan, 2023). To date, the genome sizes of wild species in section Paeonia have not been systematically determined. Mahelka and Krahulec ( 2018 ) used flow cytometry (FCM) to distinguish between closely related Elytrigia species and their interspecific hybrids by comparing their genome sizes. Ozkan et al. (2021) measured the genome sizes of 376 individuals from 41 diploid and tetraploid wild wheat accessions and correlated these data with geographical and climatic variables. Their results revealed significant differences in genome size between diploid and tetraploid wheat, substantial interspecific variation, and correlations with environmental factors. FCM has been widely applied in determining ploidy and genome size in genera (Bourge et al., 2018 ; Dolezel et al., 2007 ), it serves as a vital tool for investigating evolutionary divergence, gene-environment interactions, and phylogenetic relationships, thereby offering important insights into plant evolution. In this study, FCM was employed to determine the ploidy and genome size of eight populations of P. mairei and 13 other wild peony species. The obtained fundamental characteristics were used to investigate the influence of geographical and environmental factors on the ploidy and genome size of P. mairei , as well as to assess genomic dimensions across wild peony taxa. These findings provide a foundation for future research on phylogenetic evolution and genetic resource utilization in the genus Paeonia . 2. Materials and Reagents 2.1 Plant Materials Leaf samples were collected from eight populations of P. mairei and 13 wild peony species. The collection sites for P. mairei are listed in Table 1 and Fig. 1 , Leaves of P. ostii with a known genome size of 12.28 Gb (Yuan, 2022), and used as the reference control in this study. The thirteen wild peony species and P. ostii , excluding P. mairei , were all sourced from and are cultivated at the Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences. Table 1 Locations of populations from which P. mairei test material was sampled Population Location in China Longitude, latitude P1 Dongchuan District, Kunming City, Yunnan Province 102°56′30′′E, 26°16′30′′N P2 Yaoshan Nature Reserve, Qiaojia County, Yunnan Province 103°07′29′′E, 27°11′58′′N P3 Xiling Snow Mountain, Dayi County, Sichuan Province 103°12′36′′E, 30°41′50′′N P4 Baoxing County, Ya'an City, Sichuan Province 102°49′12′′E, 30°22′12′′N P5 Hanshuiyuan, Ningqiang County, Shaanxi Province 106°07′29′′E, 32°46′25′′N P6 Maiping, Maiji District, Gansu Province 106°28′20′′E, 34°18′25′′N P7 Foping County, Shaanxi Province 107°53′33′′E, 33°30′36′′N P8 Zhenba County, Shaanxi Province 107°53′24′′E, 32°32′14′′N 2.2 Reagents Cell lysis buffer and DAPI (4',6-diamidino-2-phenylindole) staining solution were purchased from Sysmex Parte GmbH (Görlitz, Germany). 2.3 Instruments The following instruments and reagents were used in this study: a CyFlow Cube flow cytometer (Sysmex Partec GmbH, Görlitz, Germany); Petri dishes; and double-sided razor blades (Gillette, Boston, Massachusetts, USA). 2.4 Experimental Procedure Diploid P. ostii was used as the reference standard. All leaf samples were collected in the nursery, and fresh leaf tissues were transported to the laboratory in ice-cooled containers. Leaf samples (approximately 0.05 cm²) were collected from P. ostii , eight populations of P. mairei , and 13 wild peony species. Each sample was placed in 300 µL of lysis buffer, ensuring the leaf tissue was fully immersed. Using a sharp double-sided blade, the tissue was finely and uniformly chopped within the buffer until no visible fragments remained. The homogenate was filtered through a 400-mesh nylon sieve into a 10 mL centrifuge tube to obtain a nuclear suspension. Then, 1200 µL of DAPI staining solution was added (lysis buffer to stain ratio = 1:4). After verifying stable power supply, the flow cytometer (CyFlow Cube) was initialized and flushed with pure water. A low flow rate was applied initially to stabilize signal acquisition. For DAPI excitation, a 532 nm laser was used. Stained nuclear suspensions were analyzed immediately after preparation. A total of 2000 nuclei per sample were analyzed and three independent replications were conducted for each tested sample. The average CV values closer to 5% were considered to be reliable results. Each species or population was measured as three replicates. 2.5 Data Analysis Data were processed using FCS Express software (Version V3; De Novo Software, Pasadena, CA, USA). Genome size was calculated as follows: DNA content of sample = (DNA content of reference × Mean fluorescence of reference) / Mean fluorescence of sample (Goodin, 2008). 3. Results 3.1 Ploidy Identification and Genome Size Analysis of Different P. mairei Populations The DNA content of eight populations of P. mairei was determined using diploid P. ostii as the reference standard. Flow cytometric analysis showed that the peak fluorescence intensity (mean value) of P. ostii occurred at 12,929. The fluorescence intensities of the eight P. mairei populations were 1.93, 2.07, 2.06, 1.86, 1.98, 1.97, 1.72, and 1.78 times greater than that of the diploid reference, P1-P8 respectively, confirming that all populations are tetraploid. Distinct differences in peak positions were observed among the populations, and the peaks of both the reference and samples were well-separated and clearly identifiable (Fig. 2 ), demonstrating the suitability of FCM for ploidy and genome size analysis in P. mairei . Based on the fluorescence intensity ratios and the known genome size of P. ostii (12.28 Gb), the genome sizes of the eight P. mairei populations were calculated (Table 2 ). The mean fluorescence values for P1 to P8 were 24,915, 26,816, 26,580, 24,086, 25,658, 25,412, 22,226, and 22,998, respectively (Table 2 ). The corresponding estimated genome sizes were 23.66, 25.27, 25.25, 22.87, 24.56, 24.14, 21.11, and 22.80 Gb. Among these, the populations from Foping County (P7) and Zhenba County (P8) in Shaanxi Province exhibited the smallest genome sizes (21.11 and 22.80 Gb, respectively), while the populations from the Yaoshan Nature Reserve in Qiaojia County, Yunnan Province (P2) and the Xiling Snow Mountain in Dayi County, Sichuan Province (P3) possessed the largest genomes (25.27 and 25.25 Gb, respectively). Table 2 Genome size of eight P. mairei populations Population Ploidy Mean fluorescence intensity Genome size (Gb) P. ostii 2X 12,929 12.28 P1 4X 24,915 23.66 P2 4X 26,816 25.27 P3 4X 26,580 25.25 P4 4X 24,086 22.87 P5 4X 25,658 24.56 P6 4X 25,412 24.14 P7 4X 22,226 21.11 P8 4X 22,998 22.80 3.2 Ploidy Identification and Genome Size Analysis of 13 Wild Peony Species The DNA content of 13 wild peony species (section Paeonia ) was determined using diploid P. ostii as the reference standard. Flow cytometric analysis detected a clear peak for P. ostii at a mean fluorescence intensity of 12,929. Distinct peaks were also observed for P. anomala , P. arietina, P. daurica , P. emodi , P. intermedia , P. lactiflora , P. mlokosewitschii , P. officinalis , P. obovata , P. parnassica , P. sterniana and P. tenuifolia . The mean fluorescence intensity ratios of P. officinalis and P. arietina relative to the diploid reference were 1.52 and 1.89, respectively, consistent with tetraploidy, as noted previously (Hong, 2011 ). In contrast, the mean fluorescence intensity ratios for P. lactiflora , P. anomala , P. obovate , P. veitchii, P. sterniana , P. emodi , P. intermedia , P. parnassica , P. daurica , P. mlokosewitschii , and P. tenuifolia were 0.96, 0.81, 1.10, 0.98, 1.08, 0.82, 0.88, 1.04, 0.96, 0.94, and 0.75, respectively. All peaks were well-resolved and clearly distinguishable from the reference (Fig. 3 ), confirming the diploid nature of these 13 taxa. Based on the mean fluorescence intensity values of P. ostii and the 13 wild species, genome sizes were calculated using the known genome size of the reference (12.28 Gb). The mean fluorescence intensity values for each species were as follows: P. anomala : 10,430; P. arietina : 24,473; P. daurica : 12,391; P. emodi : 10,555; P. intermedia : 11,317; P. lactiflora : 12,430; P. mlokosewitschii : 12,099; P. obovata : 14,223; P. officinalis : 19,645; P. parnassica : 13,389; P. sterniana : 13,935; P. tenuifolia : 9,648; P. veitchii : 12,717 (Table 3 ). The corresponding estimated genome sizes were: P. anomala : 9.91 Gb; P. arietina : 23.24 Gb; P. daurica : 11.77 Gb; P. emodi : 10.02 Gb; P. intermedia : 10.75 Gb; P. lactiflora : 11.86 Gb; P. mlokosewitschii : 11.49 Gb; P. obovata : 13.51 Gb; P. officinalis : 18.66 Gb; P. parnassica : 12.72 Gb; P. sterniana : 13.24 Gb; P. tenuifolia : 9.21 Gb; P. veitchii : 12.08 Gb. Among these, P. arietina exhibited the largest genome size (23.24 Gb), while P. tenuifolia had the smallest (9.21 Gb) (Table 3 ). Table 3 Genome sizes of 13 wild peony species, plus P. ostii as the reference standard Species Ploidy Mean fluorescence intensity Genome size (Gb) P. ostii 2X 12,929 12.28 P. anomala 2X 10,430 9.91 P. arietina 4X 24,473 23.24 P. daurica 2X 12,391 11.77 P. emodi 2X 10,555 10.02 P. intermedia 2X 11,317 10.75 P. lactiflora 2X 12,430 11.86 P. mlokosewitschii 2X 12,099 11.49 P. obovata 2X 14,223 13.51 P. officinalis 4X 19,645 18.66 P. parnassica 2X 13,389 12.72 P. sterniana 2X 13,935 13.24 P. tenuifolia 2X 9,698 9.21 P. veitchii 2X 12,717 12.08 4. Discussion Ploidy analysis serves as a foundation for taxonomic studies, introduction, domestication, and hybrid breeding, particularly for endemic wild species in China. FCM, which is relatively unaffected by plant tissue type or developmental stage, allows for the rapid and sensitive determination of ploidy and genome size, making it a preferred method for such analyses (Li, 2022). P. mairei , an endemic Chinese species, has been confirmed to contain tetraploid individuals, but the presence of endopolyploidy or ploidy variation within populations remains unclear. In this study, P. ostii , a tree peony belonging to the same genus as section Paeonia species, was selected as the reference standard. The high degree of overlap between the main peak of the wild P. lactiflora and that of P. ostii in flow cytometric histograms indicates that P. ostii is a suitable reference for FCM in section Paeonia . The main peaks of all eight P. mairei populations had 1.72 to 2.07 times more mean fluorescence intensity than the P. ostii peak, with no additional major peaks observed, confirming that all sampled populations are tetraploid. The peak for population P7, with 1.72-fold more mean fluorescence intensity than the reference, may have been influenced by high levels of secondary metabolites in the leaves, which can interfere with accurate measurements (Loureiro, 2010). Additionally, the large genome size and thin leaves of P. mairei likely resulted in a low yield of intact nuclei during lysis and suboptimal DAPI staining, leading to an underestimation of genome size. Nevertheless, P7 is unequivocally tetraploid. All sampled P. mairei populations were uniformly tetraploid, with no evidence of endopolyploidy within populations or ploidy variation among populations. FCM results confirmed that P. lactiflora , P. anomala , P. veitchii , P. sterniana , P. intermedia , P. parnassica , P. mlokosewitschii , and P. tenuifolia are diploid, while P. officinalis is tetraploid, consistent with a previous report (Hong, 2011 ). Although P. obovata , P. emodi , and P. daurica were identified as diploid in this study, previous research recorded both diploid and tetraploid cytotypes in these species (Hong, 2021; Zhou, 2023). This discrepancy may be due to limited sampling or intrinsic genetic variation, highlighting the need for broader population-level sampling that accounts for environmental conditions to accurately assess ploidy and genome size in potentially variable species (Plaschil, 2022). Genome size is a fundamental trait in plant biology and provides essential data for genomic research. Technical challenges have limited such studies in Paeonia , a genus characterized by large genomes and widespread ploidy variation within section Paeonia , further complicating genome assembly (Yang, 2017). Amplification or deletion of chromosomal segments may also contribute to variation in genome size among populations of the same species (Sliwinska, 2018 ). This study represents the first comprehensive assessment of genome size in 13 wild species of section Paeonia using FCM. However, estimated genome sizes can be influenced by the choice of reference standard, lysis buffer composition, tissue type, staining protocol, and instrument settings. The genome sizes of P. lactiflora , P. veitchii , P. emodi , P. intermedia , P. daurica , P. mlokosewitschii , and P. tenuifolia were significantly smaller than 12.28 Gb. This may reflect genuine genomic variation driven by adaptation to different habitats or technical artifacts such as partial chromosome loss during lysis or insufficient staining. The tetraploid species P. arietina and P. officinalis had genome sizes of 23.24 and 18.66 Gb, respectively. The notably smaller genome of P. officinalis may be attributed to high levels of secondary metabolites affecting fluorescence measurement, as well as its distinct leaf morphology and texture compared to other wild peonies. This widespread species comprises both diploid and tetraploid cytotypes, and Hong (1988) proposed the existence of distinct geographic lineages, with northeastern Chinese populations differing significantly from those in central and southern regions, potentially explaining the observed genomic divergence. The large genome of P. arietina , an introduced accession, may result from coordinated genomic evolution in response to environmental change. The genome sizes of the eight P. mairei populations ranged from 21.11 to 25.27 Gb, with a mean of 23.71 Gb. Significant inter-population variation was observed, with populations north of the Qinling–Daba Mountains exhibiting smaller genomes than those to the south. Populations from Yunnan and Sichuan possessed larger genomes, possibly due to the retention of abundant repetitive sequences and limited post-speciation genomic evolution. Growing evidence suggests that genome size is closely linked to phenotypic traits, karyotypic characteristics, and environmental factors (Sliwinska, 2018 ). The distinct climates north (colder, drier) and south (warmer, more humid) of the Qinling–Daba divide may have driven genomic adaptation in P. mairei , with northern populations potentially deleting redundant sequences to reduce genome size, indicating a possible correlation between DNA content and altitude, latitude, precipitation, and temperature. Phenotypic data from field surveys revealed substantial genetic diversity and frequent gene flow with introgression among populations, suggesting that genomic variation in wild section Paeonia species may be driven by adaptive genetic changes or mutations affecting repetitive DNA. Ploidy diversity and polyploidization likely also contribute to the observed variation. In summary, genome size in section Paeonia is influenced by multiple factors whose precise mechanisms require further elucidation. FCM determines genome size by comparing the fluorescence intensity of an internal standard of known genome size to that of the sample. The ideal standard is conspecific, but the lack of such references in Paeonia makes the well-characterized diploid P. ostii a practical alternative for ploidy and genome size estimation. Under ideal conditions, peaks of tested samples should appear at 1×, 1.5×, or 2× the position of the reference peak, indicating diploidy, triploidy, or tetraploidy, respectively, with clear separation between peaks. However, evolutionary divergence, variation in nuclear size among species, and suboptimal staining can cause peaks to shift left or right of these theoretical positions (Plaschil et al., 2020 ). Species rich in secondary metabolites often generate high debris noise, reducing flow rate and optimizing instrument settings can improve data accuracy in these cases (Huang, 2022). Declarations Ethics approval and consent to participate Not Applicable. Consent for publication Not Applicable. Data availability All data are available from the corresponding author upon reasonable request. Conflict of interest The authors declare that they have no competing interests. Funding This research was funded by the National Natural Science Foundation of China (Grant No. 32071817). Authors’ contributions Xiaonan Yu designed and supervised the project. Le Chen conducted the experiments and performed the computational analysis with assistance from Qihang Chen , Li Wang , Yong Yang , Miao Sun , Shaocai Zhu , Jaime A. Teixeira da Silva . All the authors read the paper and agreed with the final version. References Bock DG, Cai Z, Elphinstone C, González-Segovia E, Hirabayashi K, Huang KC, Keais GL, Kim A, Owens GL, Rieseberg LH. Genomics of plant speciation. Plant Commun. 2023;4(5):100599. Bottani S, Zabet NR, Wendel JF, et al. Gene Expression dominance in allopolyploids: Hypotheses and models. Trends Plant Sci. 2018;23:393–402. Bourge M, Brown SC, Siljak-Yakovlev S. Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment. Genet Appl. 2018;2:1–12. Chen QH, Chen L, Teixeira da Silva JA, Yu XN. The plastome reveals new insights into the evolutionary and domestication history of peonies in East Asia. BMC Plant Biol. 2023;23:243. Chen QH, Yin YJ, Zhao R, Yang Y, Teixeira da Silva JA, Yu XN. Incorporating local adaptation into species distribution modeling of Paeonia mairei , an endemic plant to China. Front Plant Sci. 2019;10:1717–31. Dolezel J, Bartos J. Plant DNA flow cytometry and estimation of nuclear genome size. Ann Botany. 2005;95:99–110. Dolezel J, Greilhuber J, Suda J. Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc. 2007;2:2233–44. Ferguson D, Sang T. Speciation through homoploid hybridization between allotetraploids in peonies ( Paeonia ). Proc Natl Acad Sci USA. 2001;98:3915–9. Goodin MM, Zaitlin D, Naidu RA, Lommel SA. Nicotiana benthamiana: Its history and future as a model for plant-pathogen interactions. Mol Plant Microbe Interact. 2008;21:1015–26. Halda J, Waddick JW. The genus Paeonia . Timber; 2004. Hong DY, Zhang ZK, Zhu XY. The study of Paeonia (1)—Karyotypic study of several wild species in Paeonia . J Syst Evol. 1988;26:33–43. (in Chinese). Hong DY. Peonies of the World, Taxonomy and Phytogeography. Published by Kew Publishing, 2010. London and Missouri Botanic Garden, St. Louis. Hong DY. Peonies of the World: Phylogeny and Evolution. Published by Kew Publishing, 2021. London and Missouri Botanic Garden, St. Louis. Hong DY. Peonies of the world, Polymorphism and diversity. Published by Kew Publishing, 2011. London and Missouri Botanic Garden, St. Louis. Huang GY, Cao JM, Chen C, Wang M, Liu ZG, Gao FY, Yi MM, Chen G, Lu MX. Genome survey of Misgurnus anguillicaudatus to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome. Mol Biol Rep. 2022;49(3):2185–96. La Cour LF. 1952. Chromosome counts of species and varieties of garden plants. Journal of the Annual Report of the John Innes Horticultural Institution. 23: 83–110. Leeper L. UberPaeadichlorbenzol als Vorbehandlungsmittel in der Chromosomentechnik. Wiss. Z. Friedrich-Schiller-Univ. Jena, Math.-Natur-Wiss. Reihe. 1968;17:375–9. Li HY, Niu JH, Sun LP, Li Y, Leng QY, Chen JH, Zhang JR, Yanan YN, Ma, Lopez HA. Ploidy levels and DNA contents of Bougainvillea accessions determined by flow cytometry analysis. HortScience. 2022;57:12. Loureiro J, Trawniceek P, Rauchova J, Urfus T, Vit P, Stech M, Castro S, Suda J. The use of flow cytometry in the biosystematics, ecology and population biology of homoploid plants. Preslia. 2010;82:3–21. Léveillé AAH. Paeonia mairei. Bull Acad Int Geogr Bot. 1915;25:42–3. Mahelka V, Krahulec F. Hybridization in Elytrigia at Two ploidy levels: parents, progeny, chromosome numbers and genome size. Czech J Genet Plant Breed. 2018;41:97. Ozkan H, Levy AA, Feldman M. Allopolyploidy induced rapid genome evolution in the wheat ( Aegilops Triticum ) group. Plant Cell. 2001;13(8):1735–47. Plaschil S, Abel S, Klocke E. Flow cytometric investigations on Pelargonium × crispum : an estimation of nuclear DNA contents with two different internal standards. J für Kulturpflanzen. 2020;72:236–42. Plaschil S, Abel S, Klocke E. The variability of nuclear DNA content of different Pelargonium species estimated by flow cytometry. PLoS ONE. 2022;17:4. Sliwinska E. Flow cytometry: A modern method for exploring genome size and nuclear DNA synthesis in horticultural and medicinal plant species. Folia Horticulturae. 2018;30:103–28. Yang Y, Sun M, Li SS, Chen QH, Teixeira da Silva JA, Wang AJ, Yu XN, Wang LS. Germplasm resources and genetic breeding of Paeonia : a systematic review. Hortic Res. 2020;7(1):107–25. Yang Y, Zeng XL, Zhang SS, Zhang JJ, Yu XN. Geographical distribution and resource characteristics of 5 wild peonies in southwest China. J Sichuan Agric Univ. 2017;1:7. Yuan J, Jiang S, Jian J, Liu MY, Yue Z, Xu JB, Li J, Xu CY, Lin LH, Jing Y, Zhang XX, Chen HX, Zhang LJ, Fu T, Yu SY, Wu ZY, Zhang Y, Wang CZ, Zhang X, Huang LB, Wang HQ, Hong DY, Chen XY, Hu YH. Genomic basis of the giga-chromosomes and giga-genome of tree peony Paeonia ostii . Nat Commun. 2022;13:7328. Zhou P, Zhang Q, Li J, Li F, Huang J, Zhang M. A first insight into the genomic background of Ilex pubescens (Aquifoliaceae) by flow cytometry and genome survey sequencing. BMC Genomics. 2023;24:270. Zhou SL, Xu C, Liu J, Yu Y, Wu P, Cheng T, Hong DY. Out of the Pan-Himalaya: Evolutionary history of the Paeoniaceae revealed by phylogenomics. J Syst Evol. 2021;59:1170–82. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7705305","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":560236823,"identity":"9b562455-e6dc-45d1-bd14-239b58466a07","order_by":0,"name":"Le Chen","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Le","middleName":"","lastName":"Chen","suffix":""},{"id":560236825,"identity":"dd4f9cae-a01d-4b22-80b7-3ef39a5a557b","order_by":1,"name":"Qihang Chen","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Qihang","middleName":"","lastName":"Chen","suffix":""},{"id":560236827,"identity":"937780f3-49aa-4529-8760-62a7807c25c6","order_by":2,"name":"Li Wang","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Wang","suffix":""},{"id":560236828,"identity":"e5620403-f697-490f-a294-2625b5b6efd3","order_by":3,"name":"Yong Yang","email":"","orcid":"","institution":"Chinese Academy of Sciences","correspondingAuthor":false,"prefix":"","firstName":"Yong","middleName":"","lastName":"Yang","suffix":""},{"id":560236829,"identity":"b6ac2edf-53b6-497c-a558-b83d7b5c618a","order_by":4,"name":"Miao Sun","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Miao","middleName":"","lastName":"Sun","suffix":""},{"id":560236830,"identity":"e1bb1ecf-976c-4156-8e66-3ae914222b33","order_by":5,"name":"Shaocai Zhu","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Shaocai","middleName":"","lastName":"Zhu","suffix":""},{"id":560236831,"identity":"23c22f30-66f3-4b33-b1c3-8bcf5cf04874","order_by":6,"name":"Gang Li","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"prefix":"","firstName":"Gang","middleName":"","lastName":"Li","suffix":""},{"id":560236832,"identity":"17c4e7fd-cbe3-43e2-91c0-c2d282dcb89f","order_by":7,"name":"Jaime A. Teixeira Silva","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jaime","middleName":"A. Teixeira","lastName":"Silva","suffix":""},{"id":560236833,"identity":"dfffb6d6-402c-47b5-958b-a6ba5c0cbb34","order_by":8,"name":"Xiaonan Yu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5klEQVRIiWNgGAWjYJCCA2CSvfnggw8GNnIkaOE5lmw4oyDNmAS7JHzMhHk+HE4kqNDgRo7hgR8Vd+TNJRjMmG0MmBMY2A8f3YBfS1rCwZ4zzwx3zm5Ie5xjwJbHwJOWdgO/luQDB3jbDjNuuHPguHGOAU8xgwSPGQEtiQ0H/7Ydtt9wI7FN2sJAIrGBsJbkA4eBtiRuuJHMJs1gYEBYi+SZZwmHZc4cTt5w5hizYY9BgjEbIb/wHc8x/vim4rDthuP9Hx/8+PNfjp/98DG8WhQOoIuw4VMOAvINhFSMglEwCkbBKAAAu8VVpHocnu8AAAAASUVORK5CYII=","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":true,"prefix":"","firstName":"Xiaonan","middleName":"","lastName":"Yu","suffix":""}],"badges":[],"createdAt":"2025-09-24 15:38:15","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7705305/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7705305/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":98390722,"identity":"4c721564-3f34-4dc7-b1a3-44d1de5eb33d","added_by":"auto","created_at":"2025-12-17 09:27:06","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":959815,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript.docx","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/191d8577a8e57b1be0532820.docx"},{"id":98441449,"identity":"a3e244aa-3e48-4a9f-a96c-d03dbb54ba9b","added_by":"auto","created_at":"2025-12-17 17:05:25","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":9675,"visible":true,"origin":"","legend":"","description":"","filename":"e02114f5f5be45b9a833403bfaba617e.json","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/60e7945e194f7b97b9407477.json"},{"id":98439910,"identity":"cd0a1ba8-1411-42f0-9874-bc562ac8fb33","added_by":"auto","created_at":"2025-12-17 17:03:03","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":92636,"visible":true,"origin":"","legend":"","description":"","filename":"e02114f5f5be45b9a833403bfaba617e1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/03592aac677d785626328bb3.xml"},{"id":98390728,"identity":"d68a0530-bf44-4069-8b6c-6a7ae7c7065b","added_by":"auto","created_at":"2025-12-17 09:27:06","extension":"png","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":91936,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/bddd223632b9dee17f97348c.png"},{"id":98441305,"identity":"e6f1a7f6-4c95-4095-a427-b641347a86fc","added_by":"auto","created_at":"2025-12-17 17:05:09","extension":"png","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":40017,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/592178985306ada67e934280.png"},{"id":98441559,"identity":"26ded976-4d98-46d6-862d-3653b95a6598","added_by":"auto","created_at":"2025-12-17 17:05:35","extension":"png","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":58605,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/4a4faa0006b158e5391c68dc.png"},{"id":98440676,"identity":"008a1dd1-2b93-456a-b23a-e25daf6c485a","added_by":"auto","created_at":"2025-12-17 17:04:11","extension":"xml","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":90260,"visible":true,"origin":"","legend":"","description":"","filename":"e02114f5f5be45b9a833403bfaba617e1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/7bd66c29df05d96456ca87b9.xml"},{"id":98390732,"identity":"1d524bcf-f409-4f1f-b6c8-ea43ef4a4aa1","added_by":"auto","created_at":"2025-12-17 09:27:06","extension":"html","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":103709,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/5d32cf78d071d31e29acb9a3.html"},{"id":98441565,"identity":"321d74fc-4ed4-4b42-8fc5-b81151fd2e2d","added_by":"auto","created_at":"2025-12-17 17:05:36","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":606462,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution map of the locations of \u003cem\u003eP. mairei\u003c/em\u003e populations (see Table 1)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/774bde72b4272d67ff266a7e.png"},{"id":98440702,"identity":"f6da49b7-60ef-422b-9520-e614f39422ef","added_by":"auto","created_at":"2025-12-17 17:04:13","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":123596,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA ploidy estimation by flow cytometry analysis for eight \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eP. mairei \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003epopulations\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/9df63b8a35ffbd08dc45a8dc.jpeg"},{"id":98439873,"identity":"b7824335-4479-4f2e-abc2-34068584b059","added_by":"auto","created_at":"2025-12-17 17:03:02","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180347,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDNA ploidy estimation by flow cytometry analysis for 13 wild peony species\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/461f5c61c5873b3e105d037a.jpeg"},{"id":102143788,"identity":"88e70edb-ddf5-473b-8dc8-076836ac4708","added_by":"auto","created_at":"2026-02-08 12:39:36","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1685781,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7705305/v1/d6ce5b98-332d-4544-a9ac-00bbdccd0f1d.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Genome Sizes of Fourteen Wild Paeonia Species Were Systematically Determined for the First Time","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eChina is recognized as both the center of origin of the genus \u003cem\u003ePaeonia\u003c/em\u003e and one of the distribution centers for wild resources of the subgenus \u003cem\u003ePaeonia\u003c/em\u003e (Zhou, 2020). However, current analyses of their conservation status and studies on their potential for resource development remain limited, which hinders the effective conservation and utilization of wild peony resources in China. The subgenus \u003cem\u003ePaeonia\u003c/em\u003e exhibits rich germplasm resources and is widely distributed across central and eastern Asia, the Himalayas, and the Mediterranean region. It is claimed to comprise three ploidy levels: diploid, triploid, and tetraploid (Hong, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). The diverse habitats of \u003cem\u003ePaeonia\u003c/em\u003e species have contributed to high genetic variability (Halda \u0026amp; Waddick, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Wild peonies display a wide range of ornamental traits. For instance, \u003cem\u003ePaeonia tenuifolia\u003c/em\u003e is characterized by its finely dissected leaflets, while \u003cem\u003ePaeonia veitchii\u003c/em\u003e, \u003cem\u003ePaeonia anomala\u003c/em\u003e, and \u003cem\u003ePaeonia sterniana\u003c/em\u003e exhibit deeply lobed leaves. \u003cem\u003ePaeonia obovata\u003c/em\u003e is admired for its attractive flowers, and \u003cem\u003ePaeonia mairei\u003c/em\u003e blooms early. The direct domestication or breeding of wild peonies with such diverse ornamental traits would significantly enrich the horticultural characteristics of cultivated varieties in China (Yang, 2020; Zhou, 2021). However, traditional Chinese peony cultivars have a relatively narrow genetic base, primarily derived from the domestication or hybridization of \u003cem\u003ePaeonia lactiflora\u003c/em\u003e, with limited utilization of wild \u003cem\u003ePaeonia\u003c/em\u003e resources. In contrast, international peony breeding programs extensively incorporate wild species, whereas China\u0026rsquo;s peony breeding efforts remain largely confined to \u003cem\u003eP. lactiflora\u003c/em\u003e (Ferguson, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Hong, 2010). This not only restricts the development of novel and diverse cultivars but also represents a substantial underutilization of China\u0026rsquo;s wild peony resources. Therefore, the conservation and exploitation of wild peonies are issues of great significance to the ornamental industry.\u003c/p\u003e \u003cp\u003e \u003cem\u003eP. mairei\u003c/em\u003e, an endemic Chinese species within the genus \u003cem\u003ePaeonia\u003c/em\u003e, is classified as Near Threatened on the China Species Red List (2004) and represents a valuable wild genetic resource. It is characterized by its magenta-red flowers, early flowering period, and the horticulturally significant trait of a single stem bearing a solitary bloom. The species is primarily distributed across Shaanxi, Sichuan, and Yunnan provinces (Chen, 2019). \u003cem\u003eP. mairei\u003c/em\u003e was first described by L\u0026eacute;veill\u0026eacute; in 1915 based on a specimen collected from Yunnan. The species has a narrow distribution and is often indiscriminately harvested as a substitute for \u003cem\u003eP. veitchii\u003c/em\u003e, leading to the depletion of its already limited wild resources (Chen, 2023). The current lack of research on \u003cem\u003eP. mairei\u003c/em\u003e hinders its effective conservation and utilization as a Chinese endemic species, highlighting an urgent need for studies on its resource status. Ploidy is a fundamental biological characteristic and an essential criterion for breeding applications (Dolezel, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). Early karyotypic analyses by La Lour (1952) and Leeper (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1968\u003c/span\u003e) suggested that \u003cem\u003eP. mairei\u003c/em\u003e is diploid. However, the provenance of La Lour\u0026rsquo;s plant material is unknown, and Leeper\u0026rsquo;s material was obtained from a garden in Germany, raising doubts about its taxonomic authenticity. In contrast, Hong (1988) conducted karyotypic analyses on three wild populations from Sichuan and Shaanxi, revealing that \u003cem\u003eP. mairei\u003c/em\u003e is tetraploid. Since these populations were located in the northwestern and northern parts of the species\u0026rsquo; distribution range, Hong proposed that diploid populations might exist in the southern and southeastern regions and recommended extensive sampling to determine whether variation in ploidy is correlated with geographical distribution. Based on previous research (Hong, 1988), it became established that at least a tetraploid cytotype exists in \u003cem\u003eP. mairei\u003c/em\u003e, which is likely of allopolyploid origin. Allopolyploids represent important materials for investigating polyploidization, a major driver of plant evolution that leads to profound changes at the chromosomal level (Bottani, 2018). As an allopolyploid species endemic to China, \u003cem\u003eP. mairei\u003c/em\u003e has not been sufficiently studied in terms of genome size, chromosomal composition, and genetic background. Understanding the structural variations in its chromosomal architecture and the consequent cytogenetic alterations is critical for elucidating the genomic mechanisms underlying polyploid speciation. Thus, determining the ploidy level and genome size of \u003cem\u003eP. mairei\u003c/em\u003e is of fundamental significance.\u003c/p\u003e \u003cp\u003ePloidy and genome size represent fundamental biological characteristics in plants. The analysis of genomic features serves as a critical indicator for estimating the nuclear DNA content in haploid cells, forming an essential basis for both the exploitation of plant genetic resources and the investigation of molecular mechanisms. Determining genome size provides theoretical support for genomic studies, population-level genomic diversity analyses, and research in evolutionary genetics (Dan, 2023). To date, the genome sizes of wild species in section \u003cem\u003ePaeonia\u003c/em\u003e have not been systematically determined. Mahelka and Krahulec (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) used flow cytometry (FCM) to distinguish between closely related \u003cem\u003eElytrigia\u003c/em\u003e species and their interspecific hybrids by comparing their genome sizes. Ozkan et al. (2021) measured the genome sizes of 376 individuals from 41 diploid and tetraploid wild wheat accessions and correlated these data with geographical and climatic variables. Their results revealed significant differences in genome size between diploid and tetraploid wheat, substantial interspecific variation, and correlations with environmental factors. FCM has been widely applied in determining ploidy and genome size in genera (Bourge et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Dolezel et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2007\u003c/span\u003e), it serves as a vital tool for investigating evolutionary divergence, gene-environment interactions, and phylogenetic relationships, thereby offering important insights into plant evolution.\u003c/p\u003e \u003cp\u003eIn this study, FCM was employed to determine the ploidy and genome size of eight populations of \u003cem\u003eP. mairei\u003c/em\u003e and 13 other wild peony species. The obtained fundamental characteristics were used to investigate the influence of geographical and environmental factors on the ploidy and genome size of \u003cem\u003eP. mairei\u003c/em\u003e, as well as to assess genomic dimensions across wild peony taxa. These findings provide a foundation for future research on phylogenetic evolution and genetic resource utilization in the genus \u003cem\u003ePaeonia\u003c/em\u003e.\u003c/p\u003e"},{"header":"2. Materials and Reagents","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Plant Materials\u003c/h2\u003e \u003cp\u003eLeaf samples were collected from eight populations of \u003cem\u003eP. mairei\u003c/em\u003e and 13 wild peony species. The collection sites for P. mairei are listed in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Leaves of \u003cem\u003eP. ostii\u003c/em\u003e with a known genome size of 12.28 Gb (Yuan, 2022), and used as the reference control in this study. The thirteen wild peony species and \u003cem\u003eP. ostii\u003c/em\u003e, excluding \u003cem\u003eP. mairei\u003c/em\u003e, were all sourced from and are cultivated at the Key Laboratory of Plant Resources, Institute of Botany, Chinese Academy of Sciences.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eLocations of populations from which \u003cem\u003eP. mairei\u003c/em\u003e test material was sampled\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLocation in China\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eLongitude, latitude\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDongchuan District, Kunming City, Yunnan Province\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e102\u0026deg;56\u0026prime;30\u0026prime;\u0026prime;E, 26\u0026deg;16\u0026prime;30\u0026prime;\u0026prime;N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eYaoshan Nature Reserve, Qiaojia County, Yunnan Province\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103\u0026deg;07\u0026prime;29\u0026prime;\u0026prime;E, 27\u0026deg;11\u0026prime;58\u0026prime;\u0026prime;N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eXiling Snow Mountain, Dayi County, Sichuan Province\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e103\u0026deg;12\u0026prime;36\u0026prime;\u0026prime;E, 30\u0026deg;41\u0026prime;50\u0026prime;\u0026prime;N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaoxing County, Ya'an City, Sichuan Province\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e102\u0026deg;49\u0026prime;12\u0026prime;\u0026prime;E, 30\u0026deg;22\u0026prime;12\u0026prime;\u0026prime;N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHanshuiyuan, Ningqiang County, Shaanxi Province\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e106\u0026deg;07\u0026prime;29\u0026prime;\u0026prime;E, 32\u0026deg;46\u0026prime;25\u0026prime;\u0026prime;N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eMaiping, Maiji District, Gansu Province\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e106\u0026deg;28\u0026prime;20\u0026prime;\u0026prime;E, 34\u0026deg;18\u0026prime;25\u0026prime;\u0026prime;N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eFoping County, Shaanxi Province\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e107\u0026deg;53\u0026prime;33\u0026prime;\u0026prime;E, 33\u0026deg;30\u0026prime;36\u0026prime;\u0026prime;N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eZhenba County, Shaanxi Province\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e107\u0026deg;53\u0026prime;24\u0026prime;\u0026prime;E, 32\u0026deg;32\u0026prime;14\u0026prime;\u0026prime;N\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Reagents\u003c/h2\u003e \u003cp\u003eCell lysis buffer and DAPI (4',6-diamidino-2-phenylindole) staining solution were purchased from Sysmex Parte GmbH (G\u0026ouml;rlitz, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Instruments\u003c/h2\u003e \u003cp\u003eThe following instruments and reagents were used in this study: a CyFlow Cube flow cytometer (Sysmex Partec GmbH, G\u0026ouml;rlitz, Germany); Petri dishes; and double-sided razor blades (Gillette, Boston, Massachusetts, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experimental Procedure\u003c/h2\u003e \u003cp\u003eDiploid \u003cem\u003eP. ostii\u003c/em\u003e was used as the reference standard. All leaf samples were collected in the nursery, and fresh leaf tissues were transported to the laboratory in ice-cooled containers. Leaf samples (approximately 0.05 cm\u0026sup2;) were collected from \u003cem\u003eP. ostii\u003c/em\u003e, eight populations of \u003cem\u003eP. mairei\u003c/em\u003e, and 13 wild peony species. Each sample was placed in 300 \u0026micro;L of lysis buffer, ensuring the leaf tissue was fully immersed. Using a sharp double-sided blade, the tissue was finely and uniformly chopped within the buffer until no visible fragments remained. The homogenate was filtered through a 400-mesh nylon sieve into a 10 mL centrifuge tube to obtain a nuclear suspension. Then, 1200 \u0026micro;L of DAPI staining solution was added (lysis buffer to stain ratio\u0026thinsp;=\u0026thinsp;1:4).\u003c/p\u003e \u003cp\u003eAfter verifying stable power supply, the flow cytometer (CyFlow Cube) was initialized and flushed with pure water. A low flow rate was applied initially to stabilize signal acquisition. For DAPI excitation, a 532 nm laser was used. Stained nuclear suspensions were analyzed immediately after preparation. A total of 2000 nuclei per sample were analyzed and three independent replications were conducted for each tested sample. The average CV values closer to 5% were considered to be reliable results. Each species or population was measured as three replicates.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Data Analysis\u003c/h2\u003e \u003cp\u003eData were processed using FCS Express software (Version V3; De Novo Software, Pasadena, CA, USA). Genome size was calculated as follows: DNA content of sample = (DNA content of reference \u0026times; Mean fluorescence of reference) / Mean fluorescence of sample (Goodin, 2008).\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Ploidy Identification and Genome Size Analysis of Different \u003cem\u003eP. mairei\u003c/em\u003e Populations\u003c/h2\u003e \u003cp\u003eThe DNA content of eight populations of \u003cem\u003eP. mairei\u003c/em\u003e was determined using diploid \u003cem\u003eP. ostii\u003c/em\u003e as the reference standard. Flow cytometric analysis showed that the peak fluorescence intensity (mean value) of \u003cem\u003eP. ostii\u003c/em\u003e occurred at 12,929. The fluorescence intensities of the eight \u003cem\u003eP. mairei\u003c/em\u003e populations were 1.93, 2.07, 2.06, 1.86, 1.98, 1.97, 1.72, and 1.78 times greater than that of the diploid reference, P1-P8 respectively, confirming that all populations are tetraploid. Distinct differences in peak positions were observed among the populations, and the peaks of both the reference and samples were well-separated and clearly identifiable (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), demonstrating the suitability of FCM for ploidy and genome size analysis in \u003cem\u003eP. mairei\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eBased on the fluorescence intensity ratios and the known genome size of \u003cem\u003eP. ostii\u003c/em\u003e (12.28 Gb), the genome sizes of the eight \u003cem\u003eP. mairei\u003c/em\u003e populations were calculated (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The mean fluorescence values for P1 to P8 were 24,915, 26,816, 26,580, 24,086, 25,658, 25,412, 22,226, and 22,998, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The corresponding estimated genome sizes were 23.66, 25.27, 25.25, 22.87, 24.56, 24.14, 21.11, and 22.80 Gb. Among these, the populations from Foping County (P7) and Zhenba County (P8) in Shaanxi Province exhibited the smallest genome sizes (21.11 and 22.80 Gb, respectively), while the populations from the Yaoshan Nature Reserve in Qiaojia County, Yunnan Province (P2) and the Xiling Snow Mountain in Dayi County, Sichuan Province (P3) possessed the largest genomes (25.27 and 25.25 Gb, respectively).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenome size of eight \u003cem\u003eP. mairei\u003c/em\u003e populations\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePopulation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePloidy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean fluorescence intensity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGenome size (Gb)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. ostii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12,929\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24,915\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26,816\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.27\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e26,580\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e25.25\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24,086\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22.87\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25,658\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.56\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e25,412\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e24.14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22,226\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e21.11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e22,998\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e22.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Ploidy Identification and Genome Size Analysis of 13 Wild Peony Species\u003c/h2\u003e \u003cp\u003eThe DNA content of 13 wild peony species (section \u003cem\u003ePaeonia\u003c/em\u003e) was determined using diploid \u003cem\u003eP. ostii\u003c/em\u003e as the reference standard. Flow cytometric analysis detected a clear peak for \u003cem\u003eP. ostii\u003c/em\u003e at a mean fluorescence intensity of 12,929. Distinct peaks were also observed for \u003cem\u003eP. anomala\u003c/em\u003e, \u003cem\u003eP. arietina, P. daurica\u003c/em\u003e, \u003cem\u003eP. emodi\u003c/em\u003e, \u003cem\u003eP. intermedia\u003c/em\u003e, \u003cem\u003eP. lactiflora\u003c/em\u003e, \u003cem\u003eP. mlokosewitschii\u003c/em\u003e, \u003cem\u003eP. officinalis\u003c/em\u003e, \u003cem\u003eP. obovata\u003c/em\u003e, \u003cem\u003eP. parnassica\u003c/em\u003e, \u003cem\u003eP. sterniana\u003c/em\u003e and \u003cem\u003eP. tenuifolia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe mean fluorescence intensity ratios of \u003cem\u003eP. officinalis\u003c/em\u003e and \u003cem\u003eP. arietina\u003c/em\u003e relative to the diploid reference were 1.52 and 1.89, respectively, consistent with tetraploidy, as noted previously (Hong, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). In contrast, the mean fluorescence intensity ratios for \u003cem\u003eP. lactiflora\u003c/em\u003e, \u003cem\u003eP. anomala\u003c/em\u003e, \u003cem\u003eP. obovate\u003c/em\u003e, \u003cem\u003eP. veitchii, P. sterniana\u003c/em\u003e, \u003cem\u003eP. emodi\u003c/em\u003e, \u003cem\u003eP. intermedia\u003c/em\u003e, \u003cem\u003eP. parnassica\u003c/em\u003e, \u003cem\u003eP. daurica\u003c/em\u003e, \u003cem\u003eP. mlokosewitschii\u003c/em\u003e, and \u003cem\u003eP. tenuifolia\u003c/em\u003e were 0.96, 0.81, 1.10, 0.98, 1.08, 0.82, 0.88, 1.04, 0.96, 0.94, and 0.75, respectively. All peaks were well-resolved and clearly distinguishable from the reference (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), confirming the diploid nature of these 13 taxa.\u003c/p\u003e \u003cp\u003eBased on the mean fluorescence intensity values of \u003cem\u003eP. ostii\u003c/em\u003e and the 13 wild species, genome sizes were calculated using the known genome size of the reference (12.28 Gb). The mean fluorescence intensity values for each species were as follows: \u003cem\u003eP. anomala\u003c/em\u003e: 10,430; \u003cem\u003eP. arietina\u003c/em\u003e: 24,473; \u003cem\u003eP. daurica\u003c/em\u003e: 12,391; \u003cem\u003eP. emodi\u003c/em\u003e: 10,555; \u003cem\u003eP. intermedia\u003c/em\u003e: 11,317; \u003cem\u003eP. lactiflora\u003c/em\u003e: 12,430; \u003cem\u003eP. mlokosewitschii\u003c/em\u003e: 12,099; \u003cem\u003eP. obovata\u003c/em\u003e: 14,223; \u003cem\u003eP. officinalis\u003c/em\u003e: 19,645; \u003cem\u003eP. parnassica\u003c/em\u003e: 13,389; \u003cem\u003eP. sterniana\u003c/em\u003e: 13,935; \u003cem\u003eP. tenuifolia\u003c/em\u003e: 9,648; \u003cem\u003eP. veitchii\u003c/em\u003e: 12,717 (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe corresponding estimated genome sizes were: \u003cem\u003eP. anomala\u003c/em\u003e: 9.91 Gb; \u003cem\u003eP. arietina\u003c/em\u003e: 23.24 Gb; \u003cem\u003eP. daurica\u003c/em\u003e: 11.77 Gb; \u003cem\u003eP. emodi\u003c/em\u003e: 10.02 Gb; \u003cem\u003eP. intermedia\u003c/em\u003e: 10.75 Gb; \u003cem\u003eP. lactiflora\u003c/em\u003e: 11.86 Gb; \u003cem\u003eP. mlokosewitschii\u003c/em\u003e: 11.49 Gb; \u003cem\u003eP. obovata\u003c/em\u003e: 13.51 Gb; \u003cem\u003eP. officinalis\u003c/em\u003e: 18.66 Gb; \u003cem\u003eP. parnassica\u003c/em\u003e: 12.72 Gb; \u003cem\u003eP. sterniana\u003c/em\u003e: 13.24 Gb; \u003cem\u003eP. tenuifolia\u003c/em\u003e: 9.21 Gb; \u003cem\u003eP. veitchii\u003c/em\u003e: 12.08 Gb. Among these, \u003cem\u003eP. arietina\u003c/em\u003e exhibited the largest genome size (23.24 Gb), while \u003cem\u003eP. tenuifolia\u003c/em\u003e had the smallest (9.21 Gb) (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGenome sizes of 13 wild peony species, plus \u003cem\u003eP. ostii\u003c/em\u003e as the reference standard\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePloidy\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMean fluorescence intensity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGenome size (Gb)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. ostii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12,929\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.28\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. anomala\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10,430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.91\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. arietina\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e24,473\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e23.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. daurica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12,391\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.77\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. emodi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e10,555\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. intermedia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e11,317\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e10.75\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. lactiflora\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12,430\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.86\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. mlokosewitschii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12,099\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e11.49\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. obovata\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e14,223\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.51\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. officinalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e19,645\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e18.66\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. parnassica\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13,389\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.72\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. sterniana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e13,935\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e13.24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. tenuifolia\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9,698\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e9.21\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eP. veitchii\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2X\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12,717\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e12.08\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003ePloidy analysis serves as a foundation for taxonomic studies, introduction, domestication, and hybrid breeding, particularly for endemic wild species in China. FCM, which is relatively unaffected by plant tissue type or developmental stage, allows for the rapid and sensitive determination of ploidy and genome size, making it a preferred method for such analyses (Li, 2022). \u003cem\u003eP. mairei\u003c/em\u003e, an endemic Chinese species, has been confirmed to contain tetraploid individuals, but the presence of endopolyploidy or ploidy variation within populations remains unclear. In this study, \u003cem\u003eP. ostii\u003c/em\u003e, a tree peony belonging to the same genus as section \u003cem\u003ePaeonia\u003c/em\u003e species, was selected as the reference standard. The high degree of overlap between the main peak of the wild \u003cem\u003eP. lactiflora\u003c/em\u003e and that of \u003cem\u003eP. ostii\u003c/em\u003e in flow cytometric histograms indicates that \u003cem\u003eP. ostii\u003c/em\u003e is a suitable reference for FCM in section \u003cem\u003ePaeonia\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe main peaks of all eight \u003cem\u003eP. mairei\u003c/em\u003e populations had 1.72 to 2.07 times more mean fluorescence intensity than the \u003cem\u003eP. ostii\u003c/em\u003e peak, with no additional major peaks observed, confirming that all sampled populations are tetraploid. The peak for population P7, with 1.72-fold more mean fluorescence intensity than the reference, may have been influenced by high levels of secondary metabolites in the leaves, which can interfere with accurate measurements (Loureiro, 2010). Additionally, the large genome size and thin leaves of \u003cem\u003eP. mairei\u003c/em\u003e likely resulted in a low yield of intact nuclei during lysis and suboptimal DAPI staining, leading to an underestimation of genome size. Nevertheless, P7 is unequivocally tetraploid. All sampled \u003cem\u003eP. mairei\u003c/em\u003e populations were uniformly tetraploid, with no evidence of endopolyploidy within populations or ploidy variation among populations. FCM results confirmed that \u003cem\u003eP. lactiflora\u003c/em\u003e, \u003cem\u003eP. anomala\u003c/em\u003e, \u003cem\u003eP. veitchii\u003c/em\u003e, \u003cem\u003eP. sterniana\u003c/em\u003e, \u003cem\u003eP. intermedia\u003c/em\u003e, \u003cem\u003eP. parnassica\u003c/em\u003e, \u003cem\u003eP. mlokosewitschii\u003c/em\u003e, and \u003cem\u003eP. tenuifolia\u003c/em\u003e are diploid, while \u003cem\u003eP. officinalis\u003c/em\u003e is tetraploid, consistent with a previous report (Hong, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). Although \u003cem\u003eP. obovata\u003c/em\u003e, \u003cem\u003eP. emodi\u003c/em\u003e, and \u003cem\u003eP. daurica\u003c/em\u003e were identified as diploid in this study, previous research recorded both diploid and tetraploid cytotypes in these species (Hong, 2021; Zhou, 2023). This discrepancy may be due to limited sampling or intrinsic genetic variation, highlighting the need for broader population-level sampling that accounts for environmental conditions to accurately assess ploidy and genome size in potentially variable species (Plaschil, 2022).\u003c/p\u003e \u003cp\u003eGenome size is a fundamental trait in plant biology and provides essential data for genomic research. Technical challenges have limited such studies in \u003cem\u003ePaeonia\u003c/em\u003e, a genus characterized by large genomes and widespread ploidy variation within section \u003cem\u003ePaeonia\u003c/em\u003e, further complicating genome assembly (Yang, 2017). Amplification or deletion of chromosomal segments may also contribute to variation in genome size among populations of the same species (Sliwinska, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). This study represents the first comprehensive assessment of genome size in 13 wild species of section \u003cem\u003ePaeonia\u003c/em\u003e using FCM. However, estimated genome sizes can be influenced by the choice of reference standard, lysis buffer composition, tissue type, staining protocol, and instrument settings.\u003c/p\u003e \u003cp\u003eThe genome sizes of \u003cem\u003eP. lactiflora\u003c/em\u003e, \u003cem\u003eP. veitchii\u003c/em\u003e, \u003cem\u003eP. emodi\u003c/em\u003e, \u003cem\u003eP. intermedia\u003c/em\u003e, \u003cem\u003eP. daurica\u003c/em\u003e, \u003cem\u003eP. mlokosewitschii\u003c/em\u003e, and \u003cem\u003eP. tenuifolia\u003c/em\u003e were significantly smaller than 12.28 Gb. This may reflect genuine genomic variation driven by adaptation to different habitats or technical artifacts such as partial chromosome loss during lysis or insufficient staining. The tetraploid species \u003cem\u003eP. arietina\u003c/em\u003e and \u003cem\u003eP. officinalis\u003c/em\u003e had genome sizes of 23.24 and 18.66 Gb, respectively. The notably smaller genome of \u003cem\u003eP. officinalis\u003c/em\u003e may be attributed to high levels of secondary metabolites affecting fluorescence measurement, as well as its distinct leaf morphology and texture compared to other wild peonies. This widespread species comprises both diploid and tetraploid cytotypes, and Hong (1988) proposed the existence of distinct geographic lineages, with northeastern Chinese populations differing significantly from those in central and southern regions, potentially explaining the observed genomic divergence. The large genome of \u003cem\u003eP. arietina\u003c/em\u003e, an introduced accession, may result from coordinated genomic evolution in response to environmental change.\u003c/p\u003e \u003cp\u003eThe genome sizes of the eight \u003cem\u003eP. mairei\u003c/em\u003e populations ranged from 21.11 to 25.27 Gb, with a mean of 23.71 Gb. Significant inter-population variation was observed, with populations north of the Qinling\u0026ndash;Daba Mountains exhibiting smaller genomes than those to the south. Populations from Yunnan and Sichuan possessed larger genomes, possibly due to the retention of abundant repetitive sequences and limited post-speciation genomic evolution. Growing evidence suggests that genome size is closely linked to phenotypic traits, karyotypic characteristics, and environmental factors (Sliwinska, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). The distinct climates north (colder, drier) and south (warmer, more humid) of the Qinling\u0026ndash;Daba divide may have driven genomic adaptation in \u003cem\u003eP. mairei\u003c/em\u003e, with northern populations potentially deleting redundant sequences to reduce genome size, indicating a possible correlation between DNA content and altitude, latitude, precipitation, and temperature. Phenotypic data from field surveys revealed substantial genetic diversity and frequent gene flow with introgression among populations, suggesting that genomic variation in wild section \u003cem\u003ePaeonia\u003c/em\u003e species may be driven by adaptive genetic changes or mutations affecting repetitive DNA. Ploidy diversity and polyploidization likely also contribute to the observed variation. In summary, genome size in section \u003cem\u003ePaeonia\u003c/em\u003e is influenced by multiple factors whose precise mechanisms require further elucidation.\u003c/p\u003e \u003cp\u003eFCM determines genome size by comparing the fluorescence intensity of an internal standard of known genome size to that of the sample. The ideal standard is conspecific, but the lack of such references in \u003cem\u003ePaeonia\u003c/em\u003e makes the well-characterized diploid \u003cem\u003eP. ostii\u003c/em\u003e a practical alternative for ploidy and genome size estimation. Under ideal conditions, peaks of tested samples should appear at 1\u0026times;, 1.5\u0026times;, or 2\u0026times; the position of the reference peak, indicating diploidy, triploidy, or tetraploidy, respectively, with clear separation between peaks. However, evolutionary divergence, variation in nuclear size among species, and suboptimal staining can cause peaks to shift left or right of these theoretical positions (Plaschil et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Species rich in secondary metabolites often generate high debris noise, reducing flow rate and optimizing instrument settings can improve data accuracy in these cases (Huang, 2022).\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot Applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (Grant No. 32071817).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eXiaonan Yu\u003c/strong\u003e designed and supervised the project. \u003cstrong\u003eLe Chen\u003c/strong\u003e conducted the experiments and performed the computational analysis with assistance from\u003cstrong\u003eQihang Chen\u003c/strong\u003e, \u003cstrong\u003eLi Wang\u003c/strong\u003e, \u003cstrong\u003eYong Yang\u003c/strong\u003e, \u003cstrong\u003eMiao Sun\u003c/strong\u003e, \u003cstrong\u003eShaocai Zhu\u003c/strong\u003e, \u003cstrong\u003eJaime A. Teixeira da Silva\u003c/strong\u003e. All the authors read the paper and agreed with the final version.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBock DG, Cai Z, Elphinstone C, Gonz\u0026aacute;lez-Segovia E, Hirabayashi K, Huang KC, Keais GL, Kim A, Owens GL, Rieseberg LH. Genomics of plant speciation. Plant Commun. 2023;4(5):100599.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBottani S, Zabet NR, Wendel JF, et al. Gene Expression dominance in allopolyploids: Hypotheses and models. Trends Plant Sci. 2018;23:393\u0026ndash;402.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBourge M, Brown SC, Siljak-Yakovlev S. Flow cytometry as tool in plant sciences, with emphasis on genome size and ploidy level assessment. Genet Appl. 2018;2:1\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen QH, Chen L, Teixeira da Silva JA, Yu XN. The plastome reveals new insights into the evolutionary and domestication history of peonies in East Asia. BMC Plant Biol. 2023;23:243.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen QH, Yin YJ, Zhao R, Yang Y, Teixeira da Silva JA, Yu XN. Incorporating local adaptation into species distribution modeling of \u003cem\u003ePaeonia mairei\u003c/em\u003e, an endemic plant to China. Front Plant Sci. 2019;10:1717\u0026ndash;31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDolezel J, Bartos J. Plant DNA flow cytometry and estimation of nuclear genome size. Ann Botany. 2005;95:99\u0026ndash;110.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDolezel J, Greilhuber J, Suda J. Estimation of nuclear DNA content in plants using flow cytometry. Nat Protoc. 2007;2:2233\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFerguson D, Sang T. Speciation through homoploid hybridization between allotetraploids in peonies (\u003cem\u003ePaeonia\u003c/em\u003e). Proc Natl Acad Sci USA. 2001;98:3915\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodin MM, Zaitlin D, Naidu RA, Lommel SA. Nicotiana benthamiana: Its history and future as a model for plant-pathogen interactions. Mol Plant Microbe Interact. 2008;21:1015\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHalda J, Waddick JW. The genus \u003cem\u003ePaeonia\u003c/em\u003e. Timber; 2004.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong DY, Zhang ZK, Zhu XY. The study of \u003cem\u003ePaeonia\u003c/em\u003e (1)\u0026mdash;Karyotypic study of several wild species in \u003cem\u003ePaeonia\u003c/em\u003e. J Syst Evol. 1988;26:33\u0026ndash;43. (in Chinese).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong DY. Peonies of the World, Taxonomy and Phytogeography. Published by Kew Publishing, 2010. London and Missouri Botanic Garden, St. Louis.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong DY. Peonies of the World: Phylogeny and Evolution. Published by Kew Publishing, 2021. London and Missouri Botanic Garden, St. Louis.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHong DY. Peonies of the world, Polymorphism and diversity. Published by Kew Publishing, 2011. London and Missouri Botanic Garden, St. Louis.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang GY, Cao JM, Chen C, Wang M, Liu ZG, Gao FY, Yi MM, Chen G, Lu MX. Genome survey of \u003cem\u003eMisgurnus anguillicaudatus\u003c/em\u003e to identify genomic information, simple sequence repeat (SSR) markers, and mitochondrial genome. Mol Biol Rep. 2022;49(3):2185\u0026ndash;96.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLa Cour LF. 1952. Chromosome counts of species and varieties of garden plants. Journal of the Annual Report of the John Innes Horticultural Institution. 23: 83\u0026ndash;110.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLeeper L. UberPaeadichlorbenzol als Vorbehandlungsmittel in der Chromosomentechnik. Wiss. Z. Friedrich-Schiller-Univ. Jena, Math.-Natur-Wiss. Reihe. 1968;17:375\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi HY, Niu JH, Sun LP, Li Y, Leng QY, Chen JH, Zhang JR, Yanan YN, Ma, Lopez HA. Ploidy levels and DNA contents of \u003cem\u003eBougainvillea\u003c/em\u003e accessions determined by flow cytometry analysis. HortScience. 2022;57:12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLoureiro J, Trawniceek P, Rauchova J, Urfus T, Vit P, Stech M, Castro S, Suda J. The use of flow cytometry in the biosystematics, ecology and population biology of homoploid plants. Preslia. 2010;82:3\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eL\u0026eacute;veill\u0026eacute; AAH. Paeonia mairei. Bull Acad Int Geogr Bot. 1915;25:42\u0026ndash;3.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahelka V, Krahulec F. Hybridization in \u003cem\u003eElytrigia\u003c/em\u003e at Two ploidy levels: parents, progeny, chromosome numbers and genome size. Czech J Genet Plant Breed. 2018;41:97.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzkan H, Levy AA, Feldman M. Allopolyploidy induced rapid genome evolution in the wheat (\u003cem\u003eAegilops Triticum\u003c/em\u003e) group. Plant Cell. 2001;13(8):1735\u0026ndash;47.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlaschil S, Abel S, Klocke E. Flow cytometric investigations on \u003cem\u003ePelargonium\u003c/em\u003e \u0026times; \u003cem\u003ecrispum\u003c/em\u003e: an estimation of nuclear DNA contents with two different internal standards. J f\u0026uuml;r Kulturpflanzen. 2020;72:236\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePlaschil S, Abel S, Klocke E. The variability of nuclear DNA content of different \u003cem\u003ePelargonium\u003c/em\u003e species estimated by flow cytometry. PLoS ONE. 2022;17:4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSliwinska E. Flow cytometry: A modern method for exploring genome size and nuclear DNA synthesis in horticultural and medicinal plant species. Folia Horticulturae. 2018;30:103\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Sun M, Li SS, Chen QH, Teixeira da Silva JA, Wang AJ, Yu XN, Wang LS. Germplasm resources and genetic breeding of \u003cem\u003ePaeonia\u003c/em\u003e: a systematic review. Hortic Res. 2020;7(1):107\u0026ndash;25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Zeng XL, Zhang SS, Zhang JJ, Yu XN. Geographical distribution and resource characteristics of 5 wild peonies in southwest China. J Sichuan Agric Univ. 2017;1:7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYuan J, Jiang S, Jian J, Liu MY, Yue Z, Xu JB, Li J, Xu CY, Lin LH, Jing Y, Zhang XX, Chen HX, Zhang LJ, Fu T, Yu SY, Wu ZY, Zhang Y, Wang CZ, Zhang X, Huang LB, Wang HQ, Hong DY, Chen XY, Hu YH. Genomic basis of the giga-chromosomes and giga-genome of tree peony \u003cem\u003ePaeonia ostii\u003c/em\u003e. Nat Commun. 2022;13:7328.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou P, Zhang Q, Li J, Li F, Huang J, Zhang M. A first insight into the genomic background of \u003cem\u003eIlex pubescens\u003c/em\u003e (Aquifoliaceae) by flow cytometry and genome survey sequencing. BMC Genomics. 2023;24:270.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhou SL, Xu C, Liu J, Yu Y, Wu P, Cheng T, Hong DY. Out of the Pan-Himalaya: Evolutionary history of the Paeoniaceae revealed by phylogenomics. J Syst Evol. 2021;59:1170\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Paeonia, flow cytometry, P. mairei, wild peony, genome size","lastPublishedDoi":"10.21203/rs.3.rs-7705305/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7705305/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eChina is recognized as one of the global distribution centers for the genus \u003cem\u003ePaeonia\u003c/em\u003e. However, among wild peony species, only \u003cem\u003ePaeonia lactiflora\u003c/em\u003e has been relatively well-studied and utilized, while other wild peony resources remain under-investigated. In particular, the genome sizes of wild Chinese peonies have not been clearly defined. Among these, \u003cem\u003eP. mairei\u003c/em\u003e, an endemic species in China, is classified as Near Threatened on the 2004 China Species Red List due to its limited and valuable wild populations. Nevertheless, its ploidy level and genome size remain unresolved, significantly hindering the further development and application of wild peony resources. In this study, eight populations of \u003cem\u003eP. mairei\u003c/em\u003e and 13 additional wild peony species were analyzed using flow cytometry to determine ploidy levels and estimate genome sizes. All eight populations of \u003cem\u003eP. mairei\u003c/em\u003e were confirmed as tetraploid, with no diploid types detected. The genome sizes of \u003cem\u003eP. mairei\u003c/em\u003e ranged from 21.11 to 25.27 Gb, this study examined the relationship between pronounced variation in genome size among geographically distinct populations and their habitat conditions. The genome sizes of the 13 wild peony species were determined as follows: \u003cem\u003eP. tenuifolia\u003c/em\u003e: 9.21 Gb; \u003cem\u003eP. anomala\u003c/em\u003e: 9.91 Gb; \u003cem\u003eP. emodi\u003c/em\u003e: 10.02 Gb; \u003cem\u003eP. intermedia\u003c/em\u003e: 10.75 Gb; \u003cem\u003eP. mlokosewitschii\u003c/em\u003e: 11.49 Gb; \u003cem\u003eP. daurica\u003c/em\u003e: 11.77 Gb; \u003cem\u003eP. lactiflora\u003c/em\u003e: 11.86 Gb; \u003cem\u003eP. veitchii\u003c/em\u003e: 12.24 Gb; \u003cem\u003eP. parnassica\u003c/em\u003e: 12.72 Gb; \u003cem\u003eP. obovata\u003c/em\u003e: 13.51 Gb; \u003cem\u003eP. sterniana\u003c/em\u003e: 13.24 Gb; \u003cem\u003eP. officinalis\u003c/em\u003e: 18.66 Gb; \u003cem\u003eP. arietina\u003c/em\u003e: 23.24 Gb. The study provides the first comprehensive measurements of genome sizes across 14 wild peony species, thereby enriching the genomic repository of the genus \u003cem\u003ePaeonia\u003c/em\u003e and establishing a critical foundation for future research in genomics and molecular cytogenetics within this taxon.\u003c/p\u003e","manuscriptTitle":"The Genome Sizes of Fourteen Wild Paeonia Species Were Systematically Determined for the First Time","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-17 09:27:01","doi":"10.21203/rs.3.rs-7705305/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"419ab4cf-fa72-40f9-84b6-542cd17f7aeb","owner":[],"postedDate":"December 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-08T12:39:04+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-17 09:27:01","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7705305","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7705305","identity":"rs-7705305","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}