American Journal of Botany: e16–e18. 2011.

AJB Primer Notes & Protocols in the Sciences

M ICROSATELLITE MARKERS FOR THE CHINESE HERBACEOUS (PAEONIACEAE) 1

Lin Li 2 , Fang-yun Cheng3,4 , and Qi-xiang Zhang2

2 National Flower Engineering Research Center, Key Laboratory for the Genetics and Breeding of Forest Trees and Ornamental , Beijing 100083, China; 3 Landscape Architecture College of Beijing Forestry University, Beijing 100083, China

• Premise of the study : Microsatellite (simple sequence repeat, SSR) primers were developed for the Chinese peony, P. lactifl ora , to investigate the diversity within Chinese peony germplasm resources. • Methods and Results: Using an SSR-enriched genomic library, a set of 10 unique polymorphic genomic SSRs was developed and characterized. The primers amplifi ed 61 alleles in all 10 loci, including di-, tri-, and tetranucleotide repeats. The primers were also effective for P. veitchii and P. obovata . • Conclusions : The new primers will be useful for genetic research of the Chinese peony and extend the repertoire of SSR markers available to study the herbaceous taxa in Paeonia .

Key words: enriched library; microsatellite; Paeonia lactifl ora ; primer.

Paeonia (Peoniaceae) includes woody and herbaceous spe- because of its wide ecological adaptation, strong resistance to cies, respectively called Mudan and Shaoyao in China. The stress environments, and the variability in fl ower color and germplasm is important to the fl oral and medicinal industries. form from the offspring of seeds. Therefore, suitable molecular Although herbaceous traditionally have not been as de- markers are greatly needed to identify, assess, conserve, and sirable and popular as endemic tree peonies in China, varieties use the germplasm of P. lactifl ora , especially those cultivars and hybrids of Chinese origin are increasingly grown around inherited from ancestors in China where repeated hybridiza- the world for cut-fl ower production and landscape use (Wister, tions over thousands of years and the fl uctuating habitats of 1995). Cultivars of herbaceous peonies that originated and were wild plants have created abundant genetic diversity in the spe- bred in China are nearly all from the species P. lactifl ora Pall., cies (Hong, 2001; Wister, 1995). For such an objective, micro- so even though other species are specifi c to China (e.g., P. satellites or simple sequence repeats (SSRs) have proven to be veitchii , P.obovata , P. mairei , P. emodii ) ( Hong, 2001 ), P. lac- effective and useful for the study of genetic diversity in crops tifl ora and all its cultivars comprise the plants commonly called (Yves et al., 2005) because of their codominance and hyper- the Chinese peony ( Wister, 1995 ). As one of the most ancient variability. In Paeonia , however, available SSR markers are crops originating from China, P. lactifl ora was introduced to relatively limited (Wang et al., 2009 ; Yuan et al., 2010 ; Andreas gardens during the Xia dynasty (1936– 1909 B.C.) about 4 000 et al., 2010). In this paper, we report the fi rst development of 10 yr ago and become well known before the tree peony became polymorphic microsatellite primers and their characteristics for rapidly popularized during the Tang and Song dynasties (619– the Chinese peony, P. lactifl ora . 1279 A.D.) (Cheng, 2007). As an ancient economic crop with splendid fl oral beauty and an irreplaceable role in Chinese med- icine, P. lactifl ora has been sustainably developed and selected METHODS AND RESULTS for desirable traits from generation to generation in China. To- day, thousands of its cultivars are widely grown in temperate Fresh of P. lactifl ora ‘ Hong PanTuo Jin ’ ( Fig. 1 ) , a cultivar with climates of the world while the species still grows in the wild purple and semi-double fl owers and normally developed androecia and gynoe- cia, were collected in late spring in Heze, Shandong Province, China. Genomic ( Hong, 2001 ; Wister, 1995 ). DNA (200 ng/ μ l) was extracted with the CTAB method ( Doyle and Doyle, The germplasm resources of P. lactifl ora have brought great 1987 ), and purifi ed with a Wizard DNA Clean-up System Kit (Promega). An changes to peony breeding since being introduced to Europe SSR-enriched genomic library was developed according to Glenn and Schable and America in the 19th century (Wister, 1995). Undoubtedly, (2005), and this method is comprised of the following steps. First, the genomic P. lactifl ora will play a key role in future peony breeding efforts DNA was digested into segments using Rsa I and Xmn I (New England Biolabs, Beijing Branch, China) and ligated to double-strand Super SNX-24 (synthe- sized by Sangon Biotech, Shanghai, China) linker (forward: 5 ′-GTT- 1 Manuscript received 15 October 2010; revision accepted 6 November 2010. TAAGGCCTAGCTAGCAGAATC-3 ′, reverse 5 ′-pGATTCTGCTAGCTAG The authors thank Ms. Beth Hazen at Willows End for language GCCT TAA ACAAA-3 ′). Second, the ligated DNA was denatured and hybrid- improvement. This project is supported by the National Science and ized with 10 biotinylated microsatellite probes, including (AG) , (AT) , Technology Support Program of China (2006BAD01A1801), Key Project 12 12 (CG)12 , (GT)12 , (ACG)12 , (ACT)12 , (CCA)8 , (AACT)8 , (AAGT)8 , and (AGAT)8 . for Forestry Science Research (2006-40), and Co-constructive Project of The hybridized DNA was captured by streptavidin-coated paramagnetic beads Beijing Education Committee (2009). Dynabeads M-280 (Dynal Biotech, Oslo, Norway) and collected by a magnetic 4 Author for correspondence: [email protected] particle collecting unit Dynal MPC-S (Dynal Biotech). Third, the captured DNA was amplifi ed by PCR reaction using the Super SNX-24 linker-forward doi:10.3732/ajb.1000410 as primer. The 20 µ L PCR contained 1 × HiFi Taq PCR SuperMIX (TransGen

American Journal of Botany: e16–e18, 2011; http://www.amjbot.org/ © 2011 Botanical Society of America e16 February 2011] AJB Primer Notes & Protocols — Microsatellite markers for Chinese herbaceous peony e17

University, Nanjing, China), and primers were designed with the program Primer premier 5.0 (Premier Biosoft International, Palo Alto, California, USA). Primer pairs developed were assessed using 173 samples (from 30 wild plants and 143 cultivars) by PCR amplifi cation. In this case, the PCR reaction was 10 µ L, containing 1 × HiFi Taq PCR superMIX (TransGen Biotech, Beijing, China), 0.25 µ mol/L of each designed primer, 25 ng genome DNA, and the PCR profi le was started at 95 ° C for 3 min; followed by 28 cycles of 30 s at 94 ° C, 30 s at the annealing temperature, 1 min at 72 ° C; with a fi nal extension at 72 ° C for 10 min. Number of alleles per loci (N a ), observed heterozyosity ( Ho ), and polymorphism index content (PIC) were calculated using the software PowerMarker v3.23 ( Liu and Muse, 2005 ). Of 140 positive clones that were sequenced, 40 (28.57%) were unique and found to have microsatellites (SSRs), in which bi, tri, and tetranucleotide re- peats were detected. From the 40 sequences, 53 SSRs in total were found, among which 46 (86.79%) were binucleotide repeats, 6 (11.32%) were trinucle- otide repeats, and 1 (1.89%) was a tetranucleotide repeat. Among the 46 binu- cleotide repeats, 14 (30.4%) were (GA/CA) n, 27 (58.7%) were (CA/GT) n , 3 (6.5%) were (TA/AT)n , and 2 (4.3%) were (GC/CG)n , respectively. Of the 6 trinucleotide repeats, half were (GGT/CCA)n , and the other half were (GTG/ CAC) n . The tetranucleotide repeat was (AGAT)n . Among the detected se- quences, 21 were suitable for primer designing because they have long enough sequences (> 20 bp) on the upper and lower sides of the repeats. Therefore, 21 Fig. 1. Paeonia lactifl ora ‘ Hong PanTuo Jin,’ a traditional Chinese pairs of primers were designed to amplify 21 SSR loci, and 10 of them were peony cultivar grown in Heze, Shangdong, China. The plant was intro- found to be polymorphic (Table 1) . Of these polymorphic loci, 61 alleles were duced for conservation in the Peony Collection of the JiuFeng Forestry detected, N a ranged from 3 to 14 with an average of 6.1, H o ranged from 0.0058 Experiment Station at the Beijing Forestry University. A voucher specimen to 1.0000 with an average of 0.3574, and the PIC ranged from 0.1847 to 0.6800 (number 00070537) is deposited in the Beijing Forestry University Her- with an average of 0.4714 (Table 2) . When we used these 10 primers with two barium (BFUH). other herbaceous species of Paeonia endemic to China, P. veitchii and P. obo- vata , the primers cross-amplifi ed in the two species, but whether they are poly- morphic needs further detection. Thus, these primer pairs may be useful in Biotech, Beijing, China), 0.4 µ mol/L primer, and 2 µ L enriched DNA frag- other herbaceous species of Paeonia such as P. anomala , P. emodii, and P. ments. PCR profi le started with 95 ° C for 4 min; followed by 30 cycles of 95 ° C sterniana , but their use necessitates further study. 20 s, 60 ° C 20 s, 72 ° C for 1.5 min; and fi nal extension in 72 ° C for 10 min. Fourth, the PCR product was introduced into the vector of pGEM-T easy (Pro- mega, Beijing Branch, China), and then transferred into Top10 competent cells CONCLUSIONS of E. coli (TransGen Biotech, Beijing, China), spread on plates with IPTG (iso- propyl β - d -1-thiogalactopyranoside), X-gal (5-bromo-4-chloro-3-indolyl X-d - Our set of 10 SSRs for the Chinese peony P. lactifl ora en- galactopyranoside). After white-Blue selection, 140 positive clones were picked out and sequenced on ABI 3730 DNA analyzer (Applied Biotech, San Diego, larges the bank of molecular markers available for the herba- California, USA). Finally, sequences were analyzed using the program Se- ceous species of Paeonia and should be useful for evaluating qencher 4.6 (Gene Codes Corp., Ann Arbor, Michigan, USA), SSR loci were genetic diversity, analyzing parentage, identifying hybrids, detected with the program SSRHunter 1.3.0 (Qiang Li, Nanjing Agricultural conserving germplasm, and breeding desired traits.

Table 1. Characteristics of 10 polymorphic microsatellite primers developed for Paeonia lactifl ora . For each primer pair, forward (F) and reverse (R) sequences (Sequence), repeat type (Repeat), size of original fragment (Size), optional annealing temperature (T a ), and GenBank accession (GenBank) are given.

GenBank Accession Primer Sequence (5 ’ – 3 ’ ) Repeat Size (bp)T a ( ° C) No.

P2 F: TTACGTACGCGAGCTATTCAT (TG) 16 89 51.9 HQ170522 R: ATGAATAGCTCGCGTACG

P5 F: CTGCCATCTCTACTTGT (AG)15 195 47.8 HQ170525 R: ACCATCAGTTATGGAAATGC

P7 F: GTGAGTATAGGGCCAAGT (GT)11 171 47 HQ188673 R: ACCAACTATCTCCCACAG

P8 F: CTACAGTACATATCTTCCCACA (AGAT)6 (AG)14 195 50.5 HQ188674 R: TCCATCCGAATACCCTAA

P10 F: TTT CGC ATC ATA ACA GTG TG (GT) 8 121 50.9 HQ170531 R: CTA GAA CAC GAG TAG CGA G

P12 F: TTC AAC CAT TTT CTT GTA AC (GA)5 (AG)12 225 48.3 HQ170535 R: TCT CCA AGA ACA TGA TGT AT

P14 F: TTG CAC CTG ATC CTC (AC) 15 (CA)8 158 47. 8HQ170536 R: CAC TCA CTA AAC CAC ATA TC

P19 F: TAC TTT ACT GAG AAG CAC TA (TG) 6 (GA)8 122 47.1 HQ188687 R: GAC ATC CAA GCT ACA ACA

P20 F: CTG AGA AGC ACT ATG TTC AT (TC) 9 (CA)6 100 48.3 HQ188688 R: ACA CCA AAA CCA TTA CAC A

P21 F: GCA GCG GCC ATC GCT ACA TA (TGG)6 132 54 HQ170542 R: GTA CAT GAG GAT GAG CAG AC e18 American Journal of Botany [Vol. 0

Table 2. Results of initial primer screening using 173 accessions of Chinese Doyle , J. J. , and J. L. Doyle . 1987 . A rapid DNA isolation procedure herbaceous peony germplasm including 30 accessions of landrace for small quantities of tissue. Phytochemistry Bulletin 19 : 11 – 15 . samples and 143 accessions of cultivated varieties. Parameters shown Glenn , T. C. , and N. A. Schable . 2005 . Isolation microsatellite DNA for each pair of primer are the numbers of alleles (N a ), observed loci. Methods in Enzymology 395 : 202 – 222 . heterozygosity (H o ), and polymorphic index content (PIC). Homolka , A. , M. Berenyi , K. Burg , D. Kopecky , and S. Fluch . 2010 . Microsatellite markers in the tree peony Paeonia suffruticosa Primer Na Ho PIC (Paeoniaceae). American Journal of Botany 97 : e42 – e44 . Hong , D. Y. 2001 . Flora of China, vol. 6. Science Press, Beijing; Missouri P2 4.0000 0.0058 0.1847 Botanical Garden Press, St. Louis, Missouri, USA. P5 4.0000 0.2616 0.2818 Liu, K., and S. V. Muse P7 7.0000 0.4393 0.4280 . 2005 . PowerMarker: Integrated analysis envi- P8 10.0000 0.1618 0.3529 ronment for genetic marker data. Bioinformatics (Oxford, England) P10 6.0000 0.1445 0.2695 21 : 2128 – 2129 . P12 8.0000 0.2601 0.6389 Wang , J. X. , T. Xia , J. M. Zhang , and S. L. Zhou . 2009 . Isolation and P14 4.0000 0.7977 0.3817 characterization of fourteen microsatellites from a tree peony (Paeonia P19 7.0000 0.1676 0.4429 suffruticosa ). Conservation Genetics 10 : 1029 – 1031 . P20 8.0000 0.3353 0.6800 Wister , J. C. 1995 . The peonies (2nd printing). American Peony Society, P21 3.0000 1.0000 0.4883 Hopkins, Minnesota, USA. Mean 6.1000 0.3574 0.4149 Yuan , J. H. , F. Y. Cheng , and S. Zhou . 2010 . Hybrid origin of Paeonia × yananensis revealed by microsatellite markers, chloroplast gene se- quences and morphological characteristics. International Journal of LITERATURE CITED Plant Sciences 171 : 409 – 420 . Yves , V. , S. Mitchell , Y. Matsuoka , M. Hamblin , S. Kresovich , Cheng , F. Y. 2007 . Advances in the breeding of tree peonies and a cul- C. S. Smith Jr ., J. Jaqueth , et al . 2005 . An analysis of genetic di- tivar system for the cultivar group. International Journal of Plant versity across the maize genome using microsatellites. Genetics 169 : Breeding 1 : 89 – 104 . 1617 – 1630 .

Appendix 1. Taxa used in this study. Paeonia lactifl ora ‘ Hong Pan Tuo Jin ’ was introduced under cultivation in the Peony Collection of the Beijing Forestry University at JiuFeng Forestry Experiment Station, Beijing, China. All specimens were deposited in the Beijing Forestry University Herbarium (BFUH).

Taxon — Voucher number; collection locale; Herbarium. Paeonia obovata subsp. obovata ; 00070536; Meixian, Shanxi, China; BFUH. Paeonia anomala subsp. veitchii; 00070535; Zhangxian, Gansu, China; Paeonia lactifl ora ‘ Hong Pan Tuo Jin ’ ; 00070537; Heze, Shandong, China; BFUH. BFUH.