Analysis of Genetic Diversity in Prunus Mira Koehne Ex Sargent Populations Using AFLP Markers

Plant Syst Evol (2014) 300:475–482 DOI 10.1007/s00606-013-0896-5 ORIGINAL ARTICLE Analysis of genetic diversity in Prunus mira Koehne ex Sargent populations using AFLP markers Tengfeng Li • Jiaren Liu • Yanan Xie • Qiuyu Wang • Fanjuan Meng Received: 13 June 2013 / Accepted: 7 August 2013 / Published online: 25 August 2013 Ó Springer-Verlag Wien 2013 Abstract Prunus mira Koehne ex Sargent (syn. Persica plateau about 2000 years ago. For its high tolerance to mira (Koehne) Kov. et Kostina), native to China, is an drought, cold and barren soil, it can be utilized for soil excellent fruit tree due to its high ecological and eco- erosion control, vegetation restoration, rootstocks, and as nomical value. However, there is limited knowledge on the useful genes pool in cultivated peach breeding (Wang et al. genetic information of P. mira. In this study, the genetic 1997; Fang et al. 2008; Hao et al. 2009). In addition, P. relationships of 83 P. mira accessions from five popula- mira fruits are traditionally used not only as delicious food, tions were assessed using amplified fragment length poly- but also as remedy for irregular menses, fracture and morphism (AFLP). The results showed that AFLP was a congestion (Dong 1991). It is usually used as an orna- powerful tool to detect levels of genetic diversity of natural mental plant with pink flowers (Zhong 2008). Therefore, P. populations in P. mira. The similarity coefficient between mira is an excellent fruit tree due to its high ecological and accessions ranged from 0.12 to 0.76, with an average 0.57. economical value. 83 accessions were clustered into two major clusters at In recent years, due to climate change as well as human similarity coefficient of 0.225. The highest values of Ne, interference of their natural habitats, P. mira species are H and I occurred in ML population. Most of the genetic now considered to be threatened (Fu 2002; Zhong 2008). variations occur within population. There is no close Thus, it is urgent to conserve this plant germplasm. To relationship between geographic distance and genetic dis- date, considerable efforts have focused on its ecological tance. At the same time, ex situ conservation needs to be (Fang et al. 2008), morphological (Geng et al. 2008) and established for P. mira. physiological (Wang et al. 1997; Hao et al. 2009) charac- teristics. However, knowledge is still too limited to Keywords Prunus mira Koehne Á Genetic diversity Á understand the genetic diversity and population genetic Population Á AFLP marker structure of P. mira. To evaluate the genetic diversity of plant species, the reliable, fast and cheap molecular marker techniques Introduction should be developed. Amplified fragment length poly- morphism (AFLP) is a high efficiency molecular marker Prunus mira Koehne ex Sargent (syn. Persica mira (Koe- for the identification of genetic diversity of plants. Com- hne) Kov. et Kostina), native to China, belongs to a deli- pared to other molecular markers, AFLP have many cious and wild fruit tree, called by ‘Guanghetao’ due to advantages such as reproducibility, higher resolution, sen- smooth fruit-stone (Zhong et al. 2010). It is widely dis- sitivity and no prior sequence information (Mueller and tributed at altitudes from 2,500 to 3,500 m in the Tibetan Wolfenbarger 1999; Meudt and Clarke 2007). Therefore, AFLP has been successfully used to elucidate genetic diversity in many plant species (Russell et al. 1997; Cao & T. Li Á J. Liu Á Y. Xie Á Q. Wang Á F. Meng ( ) et al. 2006; Christensen et al. 2011). In addition, previous College of Life Science, Northeast Forestry University, Harbin 150040, China studies showed that AFLP technique was an efficient sys- e-mail: [email protected] tem to assess the genetic diversity of cultivated peach 123 476 T. Li et al. species (Manubens et al. 1999; Hu et al. 2005; Da Silva Leaves (200 mg) were ground to fine powder in liquid Linge et al. 2011). However, there is limited knowledge on nitrogen with mortar and pestle and transferred to extrac- the evaluation of genetic diversity of P. mira. tion buffer (2% CTAB (w/v), 100 mM Tris-HCl, pH8.5, In this study, the genetic relationships of 83 P. mira 20 mM EDTA, 1.4 M NaCl, 0.5 % SDS, 2 % b-mercap- accessions from five populations were analyzed by AFLP toethanol (v/v), and 1 % PVP (w/v)), then incubated at technique. The objective was to investigate the pattern and 65 °C for 30 min. Subsequently, the mixture was centri- level of genetic diversity among five populations of P. mira fuged at 15,000g for 10 min at 4 °C. The supernatant was in the Linzhi region of the Tibetan plateau. washed with equal volume of chloroform: isoamyl alcohol (24:1) twice and was centrifuged at 15,000g for 10 min at 4 °C. After centrifugation, the remaining pellets were Materials and methods washed twice with ethanol (70 %), dried at room temper- ature, and dissolved in TE buffer (10 mM Tris-HCl, pH Plant materials 8.0, 1 mM EDTA, pH 8.0). DNA concentration and quality were estimated using spectrophotometer and 0.8 % agarose A total of 83 P. mira accessions from five natural popu- gel electrophoresis. lations including Langxian (LX), Gongbujiangda (GBJD), Milin (ML), Linzhi (LZ) and Bomi (BM) in the Linzhi AFLP analysis region of the Tibetan plateau were collected in spring of 2012 (Table 1; Fig. 1). In each population, individuals AFLP reactions were performed according to the method (from 16 to 18) were randomly selected exceeding 50 m of Vos et al. (1995) with some modifications. All primers interval. Young and healthy leaves sampled from individ- and adaptors are listed in Table 2. 150 ng DNA was uals were stored at -80 °C until DNA extraction. digested by 5 U EcoRI and 5 U MseI (Promega,Madison, Wisconsin, USA) in a total of 40 ll volume at 37 °C for DNA extraction 3 h, then incubated at 75 °C for 15 min. Digested DNA products were then ligated with 1 ll EcoRI adapter Total genomic DNA was extracted using the modified (50 lM) and 1 ll MseI adapter (5 lM) at 16 °C for 16 h. CTAB method described by Bouhadida et al. (2011). After ligation, the mixture was diluted 10 times with Table 1 List of 83 Prunus mira accessions from five populations Population code Symbol Source Size Longitude (E) Latitude (N) Altitude (m) LX (D) Langxian, Nyingchi, Tibetan, China 18 93°110 29°060 3,000–3,500 GBJD (d) Gongbo’gyamda, Nyingchi, Tibetan, China 17 93°250 29°920 3,000–3,500 ML (s) Milin, Nyingchi, Tibetan, China 16 94°130 29°180 2,500–3,000 LZ (j) Nyingchi, Nyingchi, Tibetan, China 16 94°370 29°680 3,000–3,100 BM (*) Bowo, Nyingchi, Tibetan, China 16 95°750 29°920 2,600–3,100 Fig. 1 Geographical distribution of five populations N of Prunus mira. A LX, B GBJD, C ML, D LZ, E BM B E D A C 50 Km 123 Analysis of genetic diversity in Prunus mira Koehne ex Sargent populations 477 Table 2 Sequences of adapters Name Code 50-Sequence-30 and selective primers EcoRI forward adapter EcoRI- F CTC GTA GAC TGC GTA CC EcoRI reverse adapter EcoRI- R AAT TGG TAC GCA GTC TAC MseI forward adapter MseI- F GAC GAT GAG TCC TGA G MseI reverse adapter MseI- R TAC TCA GGA CTC AT Pre-selective primers (EcoRI) EcoRI- P GAC TGC GTA CCA ATT C Pre-selective primers (MseI) MseI- P GAT GAG TCC TGA GTA A Selective primers EcoRI?AA E11 GAC TGC GTA CCA ATT C–AA EcoRI?AC E12 GAC TGC GTA CCA ATT C–AC EcoRI?AG E13 GAC TGC GTA CCA ATT C–AG EcoRI?AT E14 GAC TGC GTA CCA ATT C–AT EcoRI?CA E21 GAC TGC GTA CCA ATT C–CA EcoRI?TA E41 GAC TGC GTA CCA ATT C–TA MseI?AA M11 GAT GAG TCC TGA GTA A–AA MseI?AC M12 GAT GAG TCC TGA GTA A–AC MseI?AG M13 GAT GAG TCC TGA GTA A–AG MseI?AT M14 GAT GAG TCC TGA GTA A–AT MseI?CA M21 GAT GAG TCC TGA GTA A–CA MseI?GC M32 GAT GAG TCC TGA GTA A–GC MseI?GG M33 GAT GAG TCC TGA GTA A–GG MseI?GT M34 GAT GAG TCC TGA GTA A–GT ddH2O. The pre-amplified reaction was performed using After electrophoresis, the gel was removed to acetic acid pre-selective primers. The pre-amplified mixture composed solution (1 %) for 15 min and then was washed in ddH2O, of 5 ll diluted solution, 10 9 PCR buffer, 2 mM dNTPs, finally was stained with silver nitrate solution (0.2 %) for 20 mM MgCl2, 20 mM pre-amplification primer and 1 U 20 min. The stained gel was washed twice in ddH2O and Taq DNA polymerase in 20 ll volume. The pre-amplified was placed in a developing solution (3 % sodium carbon- reaction was performed in the following conditions: ated and 0.06 % formaldehyde), the development was denaturation at 94 °C for 3 min, followed by 30 cycles of stopped with 1 % acetic acid (Bassam et al. 1991). After 94 °C for 30 s, 56 °C for 30 s, 72 °C for 1 min and a final rinsing with ddH2O, the gel was dried and was extension for 72 °C for 5 min. The amplified products were photographed. diluted 30 times with ddH2O and used as the template for selective amplification. Selective amplification reactions Data analysis were performed in 20 ll volume containing 5 ll template, 10 9 PCR buffer, 2 mM dNTPs, 25 mM MgCl2,20mM Only reproducible, clear and well-resolved AFLP frag- selective amplification primer and 1 U Taq DNA poly- ments were scored as presence (1) or absence (0) for each merase.

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