Biochem Genet (2008) 46:162–179 DOI 10.1007/s10528-007-9140-8
Molecular Characterization of Tree Peony Germplasm Using Sequence-Related Amplified Polymorphism Markers Xiao Yan Han Æ Liang Sheng Wang Æ Qing Yan Shu Æ Zheng An Liu Æ Su Xia Xu Æ Takuya Tetsumura
Received: 22 March 2007 / Accepted: 1 November 2007 / Published online: 26 January 2008 Ó Springer Science+Business Media, LLC 2008
Abstract This study examined 63 tree peony specimens, consisting of 3 wild species and 63 cultivars, using sequence-related amplified polymorphism (SRAP) markers for the purpose of detecting genomic polymorphisms. Bulk DNA samples from each specimen were evaluated with 23 SRAP primer pairs. Among the 296 different amplicons, 262 were polymorphic. The maximum parsimony, neighborjoining, and unweighted pair-group method using arithmetic average trees were largely in congruence. In the three trees, the wild species Paeonia ludlowii and P. delavayi formed separate clusters with strong bootstrap support, and P. ostii was closely related to all cultivars. The cultivars were divided into groups with various corresponding bootstrap values. The genetic similarity among the genotypes ranged from 0.02 to 0.73. These results demonstrate that SRAP markers are effective in
Electronic supplementary material The online version of this article (doi:10.1007/s10528-007-9140-8) contains supplementary material, which is available to authorized users. X. Y. Han L. S. Wang (&) Q. Y. Shu (&) Z. A. Liu Beijing Botanical Garden, Institute of Botany, Chinese Academy of Sciences, 20 Nanxin Cun, Xiangshan, Haidian District, Beijing 100093, P.R. China e-mail:
[email protected] Q. Y. Shu e-mail:
[email protected] X. Y. Han Graduate School of the Chinese Academy of Sciences, Beijing 100049, P.R. China S. X. Xu Key Laboratory of Molecular Physiology and Biochemistry, Institute of Subtropical Botany, Xiamen, Fujian 361006, P.R. China T. Tetsumura Faculty of Agriculture, University of Miyazaki, Miyazaki 889-2192, Japan
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detecting genomic polymorphisms in the tree peony and should be useful for linkage map construction and molecular marker assisted selection breeding. Keywords Tree peony SRAP marker Genomic polymorphism MAS breeding
Introduction The tree peony belongs to the section Moutan in the genus Paeonia, Paeoniaceae. It is one of the earliest and most famous horticultural plants in the world. In China, tree peonies have been used as ornamental plants and have been cultivated since the Dongjin Dynasty 1600 years ago. The root bark of the tree peony, known as dan pi, is widely grown and is an important ingredient in Chinese traditional medicine. Tree peony was introduced to Japan early in 724–749 (Mega 1983) and brought to Europe in 1787 (Li 1999) from China, the center of cultivation. In China, all wild species are widely dispersed, and more than 1,500 cultivars have been planted. The section is divided into eight species with highly polymorphic phenotypes and wide distribution. As there are so many cultivars, some ornamental characters, such as color and flower forms, have been described for classification and practical utilization. Based on flower color, cultivars were simply classified as red, purple,
Fig. 1 Examples of tree peony flower form and flower color. A, Single form, reddish black. B, Single form, white. C, Anemone form, yellow. D, Chrysanthemum form, red. E, Rose form, pink. F, Crown form, yellow. G, Crown form, pink. H, Globular form, purple. I, Crown-proliferation form, purple
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yellow, white, and secondary colors. They were also characterized by flower form: single, lotus, chrysanthemum, rose, crown, globular, and others (Fig. 1). There are also four cultivar groups based on geographic locations in China: Zhongyuan, Xibei, Xinan, and Jiangnan. In addition, there are three cultivar groups around the world known as Japanese, European, and American (Li 2005). As the number of tree peony cultivars increases, it is difficult to classify them according to ornamental characters only. The sequence-related amplified polymorphism (SRAP) technique preferentially amplifies open reading frames based on twoprimer amplification. The forward and reverse primers are 17 and 18 nucleotides long and start at the 50 end. The forward primer consists of a filler sequence 10 bases long, followed by CCGG; the reverse primer has a filler sequence 11 bases long, followed by AATT. Both sequences are followed by three selective nucleotides at the 30 end (Li and Quiros 2001). With these unique primer designations, SRAP markers were more reproducible, stable, and less complex, compared with other molecular markers. SRAP has been used in the study of Brassica (Li and Quiros 2001), Cucurbita (Ferriol et al. 2003), buffalograss [Buchloe dactyloides (Nutt.) Englem] (Budak et al. 2004a), and Nelumbo (Liu et al. 2006b). In the case of buffalograss, Budak et al. (2004b) showed SRAP to be more powerful for genetic diversity among closely related cultivars than SSR, ISSR, and RAPD. The purposes of this study were to characterize the genetic variability of the tree peony and to use SRAP markers to enhance our knowledge of the genetic basis of its agronomic characteristics. SRAP markers are expected to assist in establishing a new classification system of cultivars, shortening their breeding course, and investigating the relationship among cultivars. Moreover, this study will lay a foundation for further usage of SRAP marker systems in the breeding of other ornamental plants.
Materials and Methods Plant Materials Three wild species and 63 cultivars from the Resources Garden, Institute of Botany, Chinese Academy of Sciences, representing a wide range of adaptation, were employed in this study (Table 1, Fig. 1). The wild species are Paeonia ludlowii (Stern and Talor) Hong, P. delavayi Franchet, Bull (with yellow flowers), and P. ostii T. Hong. The cultivars included 2 American cultivars (‘Jin Dao’ = ‘Golden Isles’ and ‘Hai Huang’ = ‘High Noon’), 3 French cultivars (‘Jin Huang’ = ‘Alice Harding’, ‘Jin Ge’ = ‘Souvenir de Maxime Cornu’, and ‘Jin Zhi’ = ‘Chromatella’), 12 Japanese cultivars, and 46 Chinese cultivars (30 from the Zhongyuan cultivar group, 9 from the Xibei cultivar group, 2 from the Xinan cultivar group, and 5 from the Jangnan cultivar group). The samples covered six main color series (white, pink, yellow, red, reddish black, and purple) and seven main flower forms (single, lotus, crown, chrysanthemum, rose, globular, and crown-proliferation). The indices of flower color were measured in the spring when the plants blossom. The color of fresh petals in the middle portion was measured using the Royal Horticultural Society Color
123
Chrysanthemum form
Globular form
Single form
Crown form
Anemone form
Anemone form
Chrysanthemum form
Rose form
Single form
Crown form
Crown form
10 Xue Zhong Song Tan
11 Yu Ban Xiu Qiu
12 Bai He Liang Chi
13 Yin Bai He
14 Yao Chi Jiu Nu
15 Jiu Zui Yang Fei
16 Ru Hua Si Yu
17 Shao Nu Qun
18 Bai Yuan Chun
19 Zhao Fen
20 Zui Xi Shi
Chrysanthemum form
Single form
9 P. ostii
Rose form
Crown form
8 Feng Wei
23 Hua Jing (Hanakisoi)
Anemone form
7 Wu Da Zhou (Godaishu¯)
22 Han Ying Shi Zi (Kanzakurajishi)
Anemone form
6 Bai Wang Shi Zi (Hakuo¯jishi)
Single form
Crown form
5 Jing Yu
21 Zhong Chuan Fen
Chrysanthemum form
Single form
Anemone form
2 Yu Ban Bai
4 Bai Yu Bing
Single form
1 Feng Dan
3 Yu Pan Tuo Jin
Flower form
Cultivar
Table 1 Characteristics of 66 tree peonies in this study
Japanese
Japanese
Xibei
Zhongyuan
Zhongyuan
Zhongyuan
Zhongyuan
Zhongyuan
Zhongyuan
Xibei
Xibei
Xibei
Xibei
Xibei
Wild
Jiangnan
Japanese
Japanese
Zhongyuan
Zhongyuan
Zhongyuan
Zhongyuan
Jiangnan
Cultivar-group
Pink
Pink
Pink
Pink
Pink
Pink
Pink
Pink
Pink
White (spot)
White (spot)
White (spot)
White (spot)
White (spot)
White
White
White
White
White
White
White
White
White
Color
65D
56B
72B
63D
69C
62D
75B
84C
69A
56D
56D
56D
56D
56D
56D
56C
11D
4D
56D
69C
56A
56D
56D
RHSCCCa
36.99
71.18
48.10
65.69
77.14
72.52
61.72
66.69
68.15
70.15
66.43
69.65
67.92
66.62
83.44
83.76
78.75
77.16
75.75
79.91
68.70
78.26
75.74
L*
15.14
2.45
39.23
25.78
3.90
4.12
26.48
8.54
21.04
17.72
22.97
19.37
22.32
22.93
2.25
8.51
5.60
5.76
7.36
6.53
7.54
6.46
4.50
C*
CIELch coordinatesb
67.76
53.97
-19.45
-2.56
75.47
21.65
-21.07
-21.37
-22.82
-24.97
-24.13
-24.06
-23.75
-17.93
-26.91
117.65
120.00
126.67
109.76
106.83
11.16
115.22
97.66
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123
123
Anemone form
Single form
Chrysanthemum form
Anemone form
Rose form
Chrysanthemum form
42 Chu Wu (hatsugarasu)
43 Yu Yi Huang
44 Huang Hua Kui
45 Jin Dao2 (Golden Isles)
46 Hai Huang (High Noon)
Crown form
41 Shu Hua Zi
Crown form
Anemone form
35 Zhu Sha Lei
40 Yan Long Zi
Anemone form
34 Fang Ji (Ho¯ki)
39 Guan Shi Mo Yu
Chrysanthemum form
33 Qi Bao Dian (shichiho¯den)
Rose form
Single form
32 Hu Chuan Han (Togawakan)
38 Hua Wang (Kao¯)
Crown form
31 Hong Zhu Nu
Chrysanthemum form
Crown form
30 Fu Gui Hong
Chrysanthemum form
Crown form
29 Shan Hua Lan Man
37 Tai Yang (Taiyo¯)
Single form
28 Huo Lian Jin Dan
36 Chun Hong Jiao Yan
Anemone form
Crown form
27 Qing Chun
25 Gui Fei Cha Cui
26 Hong Lian
Chrysanthemum form
Crown-proliferation form
24 Jin Dao1 (Nishikijima)
Flower form
Cultivar
Table 1 continued
American
American
Zhongyuan
Zhongyuan
Japanese
Zhongyuan
Zhongyuan
Zhongyuan
Japanese
Japanese
Zhongyuan
Zhongyuan
Japanese
Japanese
Japanese
Zhongyuan
Zhongyuan
Zhongyuan
Zhongyuan
Xibei
Xibei
Zhongyuan
Japanese
Cultivar-group
Yellow
Yellow
Yellow
Yellow
Reddish black
Reddish black
Reddish black
Reddish black
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Red
Pink
Pink
Color
2B
5B
11D
11D
59A
59B
59A
79B
67A
52A
58B
80C
45B
63B
45B
64C
72B
58C
66C
71A
82C
62B
63A
RHSCCCa
76.60
77.91
72.92
73.95
18.80
24.11
20.77
17.41
40.09
37.28
59.17
60.73
34.52
40.36
35.51
39.45
33.01
49.15
48.58
28.25
47.82
63.45
36.15
L*
43.07
53.13
19.21
31.55
15.43
29.69
22.25
13.54
43.30
49.05
37.62
31.27
44.35
46.93
50.63
45.41
47.95
46.06
40.44
40.32
46.53
22.58
49.48
C*
CIELch coordinatesb
108.18
106.32
115.86
114.89
11.06
5.30
11.51
5.51
-5.13
17.08
-3.26
-16.88
17.68
3.22
18.51
-10.72
-15.16
5.89
-4.91
-12.78
-19.25
-9.56
10.34
[h]
166 Biochem Genet (2008) 46:162–179
Rose form
Rose form
Single form
Single form
Crown form
Crown form
Single form
Rose form
Anemone form
Single form
Chrysanthemum form
Crown-proliferation form
Globular form
Anemone form
Crown form
Rose form
Rose form
Chrysanthemum form
Rose form
48 Jin Huang (Alice Harding)
49 Jin Zhi (Chromatella)
50 P. ludlowii
51 P. delavayi
52 Yao Huang
53 Jin Yu Jiao Zhang
54 Tai Ping Hong Dan
55 Tai Ping Hong Chong
56 Hu lan
57 Pan Zhong Qu Guo
58 Ge Jin Zi
59 Jia Ge Jin Zi
60 Zi Guang Ge
61 Mu Ai
62 Shou An Hong
63 Luo Yang Hong
64 Dao Da Chen (Shimadaijin)
65 Qing Luo
66 Que Hao
b
L*, Lightness; C*, chromas (brightness); [h], hue angle (degree)
Royal Horticultural Society Color Chart
Crown form
47 Jin Ge (Souvenir de Maxime Cornu)
a
Flower form
Cultivar
Table 1 continued
Jiangnan
Jiangnan
Japanese
Zhongyuan
Zhongyuan
Xibei
Zhongyuan
Zhongyuan
Zhongyuan
Zhongyuan
Jiangnan
Xinan
Xinan
Zhongyuan
Zhongyuan
Wild
Wild
France
France
France
Cultivar-group
Purple
Purple
Purple
Purple
Purple
Purple
Purple
Purple
Purple
Purple
Purple
Purple
Purple
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Yellow
Color
80A
78A
72A
72B
77A
72A
72A
72B
67B
67C
76A
74B
74A
27D
2D
13A
14B
1C
1C
4B
RHSCCCa
40.20
34.18
28.79
37.00
34.93
29.73
27.27
25.92
27.29
26.24
67.34
66.84
67.92
67.62
74.71
73.24
75.48
75.50
75.93
75.28
L*
43.93
42.15
42.73
48.50
48.86
41.64
40.11
38.60
41.56
39.01
18.46
18.30
17.00
21.68
11.34
59.07
65.79
64.04
68.19
39.80
C*
CIELch coordinatesb
-3.88
-20.06
-22.41
-18.28
-22.31
-19.05
-13.26
-14.74
-11.52
-13.75
-24.38
-24.46
-23.65
111.39
117.24
102.74
99.26
99.18
98.76
106.33
[h]
Biochem Genet (2008) 46:162–179 167
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168
Biochem Genet (2008) 46:162–179
Chart (RHSCC 2001) and an NF333 spectrophotometer (Nippon Denshoku Industry Co., Tokyo).
DNA Extraction and PCR Reaction System Selection A modified version of the cetyltrimethyl ammonium bromide (CTAB) method was used to extract genomic DNA. Approximately 0.3 g fresh leaf tissue from a bulk of five plants was placed into liquid nitrogen, crushed rapidly in a mortar, and transferred to a 2.0 ml tube; 0.9 ml 2 9 CTAB buffer (0.5 M EDTA, 2 M Tris– HCl, pH 8, 5 M NaCl, 2% CTAB, and 0.1% b-mercaptoethanol) was added to the tubes, mixed, and incubated at 65°C for 60 min. After incubation, the samples were cooled to room temperature and centrifuged at 12,000 rpm for 10 min, followed by extraction with 0.5 ml chloroform-isoamyl alcohol (24:1) twice, and precipitation with 2.5 volumes ethanol at -20°C. The pellet was washed twice with 1 ml 75% ethanol. The DNA was then suspended in 0.1 9 TE (1 mM Tris–HCl, 0.1 mM EDTA, pH 8.0) buffer. DNA quality was detected in a 1% agarose gel stained with ethidium bromide (0.46 lg/ml) and quantified using a Nucleic Acid and Protein Analyzer (DU 640, Beckman Coulter, Calif.). The DNA was quantified to 30 ng/ll and then stored at -20°C for PCR templates. Based on our preliminary study, 25 SRAP primer pairs were assayed using five forward and reverse primers each on the 66 samples (Table 2). Primers were excluded from the study if their banding patterns were difficult to score or if they failed to amplify consistently in all lines. From the total 25 primer pairs, 23 primer combinations were selected for their consistent amplifications and clear banding patterns (Table 3). The PCR reaction mixture (2 ll) was composed of 90 ng genomic DNA, 200 lM dNTPs, 2.5 mM MgCl2, 0.3 lM primer, 10 9 buffer, and 1 U Taq DNA polymerase (Transgen Biotech, Beijing). The amplification was carried out in an Eppendorf Mastercycler Gradient (Type 5331, Eppendorf AG, Hamburg, Germany) using the following program: 3 min denaturing at 94°C, eight cycles of 30 s denaturing at 94°C, 30 s annealing at 37°C, and 90 s elongation at 72°C. In the following 32 cycles Table 2 Forward and reverse SRAP primers used in this study
123
Primer
Type
Sequence (50 –30 )
Me2
Forward
TGA GTC CAA ACC GGA GC
Me4
Forward
TGA GTC CAA ACC GGA CC
Me5
Forward
TGA GTC CAA ACC GGA AG
Me7
Forward
TGA GTC CAA ACC GGA CA
Me8
Forward
TGA GTC CAA ACC GGA AC
Em1
Reverse
GAC TGC GTA CGA ATT AAT
Em2
Reverse
GAC TGC GTA CGA ATT TGC
Em3
Reverse
GAC TGC GTA CGA ATT GAC
Em8
Reverse
GAC TGC GTA CGA ATT CTG
Em10
Reverse
GAC TGC GTA CGA ATT CAG
Me4 + Em2
Me4 + Em3
Me4 + Em8
Me4 + Em10
Me5 + Em1
Me5 + Em2
Me5 + Em3
Me5 + Em8
Me5 + Em10
Me7 + Em2
Me7 + Em3
Me7 + Em8
Me7 + Em10
7
8
9
10
11
12
13
14
15
16
17
18
19
Me8 + Em3
Me4 + Em1
6
Me8 + Em2
Me2 + Em10
5
22
Me2 + Em8
4
21
Me2 + Em3
3
Me8 + Em1
Me2 + Em2
2
20
Me2 + Em1
1
Combinations
11
11
12
19
11
15
6
11
15
16
6
12
10
13
10
12
10
12
11
20
16
21
Total fragments
10
10
10
18
9
15
5
9
14
16
5
10
8
9
9
11
9
10
9
18
14
21
pol
a
Total
90.9
90.9
83.3
94.7
81.8
100
83.3
81.8
93.3
100
83.3
83.3
80
69.2
90
91.7
90
83.3
81.8
90
87.5
100
%pol
b
9
8
10
18
7
11
6
9
11
14
4
10
9
10
10
6
9
9
6
17
11
18
Total
a
9
7
10
18
6
11
6
9
11
14
4
10
9
9
10
6
9
9
5
17
11
18
pol
Zhongyuan
Cultivar group
100
87.5
100
100
85.7
100
100
100
100
100
100
100
100
90
100
100
100
100
83.3
100
100
100
%pol
b
Table 3 Genetic diversity estimates of tree peony specimens using SRAP primer pairs
5
3
5
11
3
7
4
8
8
4
1
4
3
6
6
9
3
6
4
13
9
14
Total
Xibei
5
3
5
10
2
7
4
8
8
4
1
4
3
5
6
9
3
6
3
13
5
14
pol
a
100
100
100
90.9
66.7
100
100
100
100
100
100
100
100
83.3
100
100
100
100
75
100
55.6
100
%pol
b
2
1
2
3
3
3
1
2
1
1
1
3
3
3
0
0
0
1
3
0
2
0
Total
Xinan
2
0
2
2
1
3
1
1
1
1
1
3
3
3
0
0
0
1
1
0
2
0
pol
a
100
0
100
66.7
33.3
100
100
50
100
100
100
100
100
100
100
100
100
100
33.3
100
100
100
%pol
b
7
5
8
14
6
9
4
5
7
5
1
6
9
7
5
4
4
7
4
13
8
13
Total
Jiangnan
7
4
8
13
5
9
4
5
7
5
1
2
9
7
5
4
4
6
3
12
8
13
pola
100
80
100
92.9
83.3
100
100
100
100
100
100
33.3
100
100
100
100
100
85.7
75
92.3
100
100
%polb
Biochem Genet (2008) 46:162–179 169
123
123
4.2
66
SD
Num. of samples
Me2 + Em1
Me2 + Em2
Me2 + Em3
Me2 + Em8
Me2 + Em10
Me4 + Em1
Me4 + Em2
Me4 + Em3
Me4 + Em8
Me4 + Em10
Me5 + Em1
Me5 + Em2
2
3
4
5
6
7
8
9
10
11
12
6
12
10
13
10
12
10
12
11
20
16
21
Total fragments
12.9
Average
Combinations
16
296
Total
Total fragments
Me8 + Em10
1
23
Combinations
Table 3 continued
17.5
87.5
–
81.3
%pol
b
3
9
6
9
7
7
8
6
6
12
9
17
Total
Japan
3
9
6
9
7
7
8
6
5
12
9
17
pol
a
Cultivar group
4.2
11.4
262
13
pol
a
Total
30
100
100
100
100
100
100
100
100
10
b
3.8
9.9
228
83.3
100
100
100
a
pol
%pol
3.8
10.1
233
11
Total
Zhongyuan
Cultivar group
3
4
4
4
2
5
4
4
6
3
7
4
Total
France
18.3
97.3
–
90.9
%pol
b
3
4
4
2
2
5
4
4
3
3
6
4
pol
a
9
3.5
6.3
146
10
Total
Xibei
100
100
100
50
100
100
100
100
50
100
85.7
100
%pol
b
3.4
6
137
9
pol
a
1
1
2
2
1
3
0
5
4
5
3
4
Total
2
a
0
1
0
2
1
1
0
4
2
4
1
4
pol
1.3
1.5
35
0
Total
Xinan
America
19.8
94
–
90
%pol
b
0
100
0
100
100
33.3
100
80
50
80
33.3
100
b
27.3
86.2
–
100
b
%pol
%pol
1.2
1.2
28
0
pol
a
5
6
2
9
4
6
4
7
6
6
11
7
Total
Wild
5
3.2
6.9
159
8
Total
6.5
6.5
149
8
pola
4
6
0
9
4
6
4
6
6
6
9
6
pola
Jiangnan
80
100
0
100
100
100
100
85.7
100
100
81.8
85.7
%polb
93.2
93.2
–
100
%polb
170 Biochem Genet (2008) 46:162–179
11
18 Me7 + Em8
b
a
4.2
66
SD
Num. of samples
Percentage of polymorphic fragments
Number of polymorphic fragments
12.9
Average
16
296
23 Me8 + Em10
Total
11
11
21 Me8 + Em2
22 Me8 + Em3
19
15
17 Me7 + Em3
12
6
19 Me7 + Em10
11
15 Me5 + Em10
16 Me7 + Em2
20 Me8 + Em1
16
15
13 Me5 + Em3
Total fragments
14 Me5 + Em8
Combinations
Table 3 continued
12
3.8
8.2
189
8
8
4
8
17
5
9
3
10
11
7
Total
Japan
3.7
8.1
186
8
8
4
8
16
4
9
3
10
11
7
pol
a
Cultivar group
18.3
98.1
–
100
100
100
100
94.1
80
100
100
100
100
100
%pol
b
3
2.6
4.3
99
3
6
5
4
12
5
0
2
5
3
4
Total
France
2.3
3.9
90
3
5
5
3
12
4
0
2
5
3
4
pol
a
16.3
92.3
–
100
83.3
100
75
100
80
100
100
100
100
100
%pol
b
2
1.9
3
68
6
5
2
3
4
5
1
1
3
2
5
Total
America
1.5
2
46
4
4
2
2
3
3
1
1
1
1
4
pol
a
33.4
69.1
–
66.7
80
100
66.7
75
60
100
100
33.3
50
80
%pol
b
3
3
7
161
12
8
6
8
14
5
10
3
6
11
5
Total
Wild
2.9
6.4
147
11
8
5
8
12
3
9
3
6
11
5
pola
21.9
88.9
–
91.7
100
83.3
100
85.7
60
90
100
100
100
100
%polb
Biochem Genet (2008) 46:162–179 171
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Biochem Genet (2008) 46:162–179
the annealing temperature was increased to 50°C, with a final elongation step of 7 min at 72°C. Each PCR product (10 ll) was fractionated on 2% agarose gel and stained with ethidium bromide for 40 min. A 100 bp plus DNA Ladder Marker (Transgen Biotech) was used as the size marker. Electrophoresis was carried out at a constant 108 V for 1 h at room temperature. Gels were photographed using a White/ Ultraviolet Transilluminator (UVP, Spring Scientific, New York). Data Analysis Using ‘‘1’’ to represent the presence and ‘‘0’’ the absence of each SRAP fragment and thus a specific allele, genetic similarities between cultivars were measured by the Dice similarity coefficient based on the proportion of shared alleles (Dice 1945; Nei and Li 1979). The MP (maximum parsimony), NJ (neighbor-joining), and UPGMA (unweighted pair-group method with arithmetic averages) trees were constructed using the PAUP 4.0b10 computer program (Swofford 1998). Parsimony analysis was performed by a heuristic search with a tree bisection-reconnection branch-swapping algorithm. The wild species P. ludlowii and P. delavayi were used as outgroups in the NJ and MP trees based on previous phylogenetic hypotheses. Topological congruence with previous phylogenetic hypotheses was assessed with the Templeton test (Templeton 1983), as implemented in PAUP 4.0b10 (Swofford 1998). NJ and UPGMA trees were constructed using distance measures. The number of 1,000 replicates was used for all bootstrap tests. Results Performance of SRAP Marker All 23 SRAP primer pairs amplified products in all bulk DNA specimens (Table 3). In total 296 amplicons were observed, of which 34 (12.5%) were monomorphic and 262 (87.5%) were polymorphic. The number of amplicons produced by each primer set ranged from 6 (Me5/Em2; Me7/Em2) to 21 (Me2/Em1), with an average of 12.9 amplicons/primer set. The polymorphic amplicons ranged from 5 (Me7/Em2 and Me5/Em2) to 21 ((Me2/Em1), with an average of 11.4 amplicons/primer set. The percentage of polymorphic markers produced by each primer pair ranged from 69.2% (Me4/Em8) to 100% (Me2/Em1; Me5/Em3; Me7/Em3). The percentage of polymorphic markers produced by each primer pair was 97.3% for the Zhongyuan, 94.0% for Xibei, 86.2% for Xinan, 93.2% for Jiangnan, 98.1% for Japanese, 92.3% for French, 69.1% for American, and 88.9% for wild groups. Twelve Japanese cultivars shared the highest polymorphic percentage (98.1%), and the two American cultivars shared the lowest (69.1%). The limited number of cultivars along with the two primer combinations (Me4/Em10 and Me5/Em2) did not generate many polymorphic fragments in American cultivars, which may partly contribute to the low polymorphic percentage. An example of the amplification products of SRAP reactions is presented in Fig. 2 for Me8/Em10. The cultivars shared a common band (780 bp), and different cultivar
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3000
3000
1500
1500
1000
1000
500
500
300
300
Fig. 2 PCR amplification of tree peony genomic DNA from 23 specimens. Lane: 1 ‘Shou An Hong’, 2 ‘Zhao Fen’, 3 ‘Jing Yu’, 4 ‘Gui Fei Cha Cui’, 5 ‘Yu Yi Huang’, 6 ‘Ru Hua Si Yu’, 7 ‘Zhu Sha Lei’, 8 ‘Hong Zhu Nu’, 9 ‘Yin Bai He’, 10 ‘Zhong Chuan Fen’, 11 ‘Qing Chun’, 12 ‘Feng Dan’, 13 ‘Que Hao’, 14 ‘Qing Luo’, 15 P. ludowii, 16 P. delavayi, 17 P. ostii, 18 ‘Chu Wu’, 19 ‘Han Ying Shi Zi’, 20 ‘Dao Da Chen’, 21 ‘Jin Huang’, 22 ‘Jin Dao’, 23 ‘Hai Huang’. M: 100 bp plus DNA ladder marker (5,000, 3,000, 2,000, 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100; Transgen Biotech, Beijing). SRAP primer pair was Me8/Em10. The DNA samples were fractionated in 2% agarose gels stained with ethidium bromide
groups had different common bands. The Zhongyuan group (lanes 1–8) shared a band of 700 bp. The Xibei group (lanes 9–11) shared bands of 1,400 and 1,500 bp. The Jiangnan group (lanes 12–14) shared bands of 500, 550, and 600 bp. The wild species (lanes 15–17) shared the band of 780 bp. The Japanese group (lanes 18–20) shared bands of 520 and 1,400 bp. The French and American groups (lanes 21–23) shared bands of 450, 1,400, and 1,550 bp. However, the large number of polymorphic bands indicated abundant genetic differences among and within cultivar groups, especially between the wild species (P. ludlowii, lane 15; P. delavayi, lane 16) and the cultivars. Three wild species had only one band in common (780 bp), and P. ludlowii and P. delavayi shared another two bands (330 and 650 bp). P. ostii was more similar to the cultivars, compared with P. ludlowii and P. delavayi.
Genetic Similarity Genetic similarity among all specimens ranged from 0.02 to 0.73, with an average of 0.62 (Supplementary material S1). The most similar cultivars are ‘Yao Chi Jiu Nu’ and ‘Yin Bai He’, sharing 0.73 genetic similarity, both of which originated from the Xibei group and have a blackish purple blotch at the base of the white petals. The biggest difference between them is the flower form; ‘Yao Chi Jiu Nu’ has an anemone form with 3–4 whorls of petals, and ‘Yin Bai He’ has a crown form with many whorls of petals in which most of the stamens develop into petals. The least similar cultivars are ‘Dao Da Chen’ and ‘Zui Xi Shi’, sharing 0.02 genetic similarity. The flower of ‘Dao Da Chen’ is purple with a rose form; it originated from Japan. ‘Zui Xi Shi’ is pink with a crown flower form and is derived from the Zhongyuan group. Although most cultivars had the same ancestors, after developing and adapting to various environments over thousands of years, they now share moderate similarity.
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Topological Trees and Bootstrap Support of Different Clades The NJ, UPGMA, and one of the MP trees (obtained from 1,934 trees) with bootstrap support above 50% are shown in Fig. 3 and Supplementary materials S2 and S3. The wild species P. ludlowii and P. delavayi formed separate clusters from the cultivars tested in this study, with bootstraps of 93%, 100%, and 92% in NJ, UPGMA, and MP trees, respectively. P. ostii was more closely related to the cultivars. Since P. ludlowii and P. delavayi are wild species, their genetic diversity
Fig. 3 Neighbor-joining tree of 66 tree peony genotypes calculated on PAUP 4.0b10 by means of 23 SRAP primer pairs. Bootstrap values over 50 are indicated above the branch; based on 1,000 resamplings of the data set
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was different from that of the cultivars (Fig. 2). The two were very different in geographic distribution and morphology among wild species. P. ludlowii has yellow flowers with one whorl of petals (single form) and was distributed mainly in Tibet and Sichuan; P. delavayi, with yellow or red flowers and a single form, was found mainly in Yunnan province. Their floral disks are fleshy, but the floral disks of other wild species and all the cultivars are leathery. According to references, the two species did not contribute to the formation of Chinese cultivars because of geographic isolation, and the dendrograms support this hypothesis. P. ostii was found in Henan province and was considered to contribute to the formation of Chinese cultivars; indeed, it shares more genetic similarity and is distributed as a sister clade among cultivars of the Zhongyuan group. The NJ, UPGMA, and MP trees generally agree on the division of cultivars, and incongruence occurred in only a few branches. The NJ tree was divided into two clades. The clade including ‘Bai Yu Bing’, ‘Dao Da Chen’, ‘Ru Hua Si Yu’, and ‘Hu Lan’ was the most distinct clade of all the cultivars examined in this study (Fig. 3). In this clade, ‘Ru Hua Si Yu’ and ‘Hu Lan’ were distributed in a separate branch with 77% bootstrap value (82% in the UPGMA tree, 56% in the MP tree). The other clade was further divided into two branches, with branch I including all Xibei cultivars, most Japanese cultivars, and one French cultivar, and branch II including all American cultivars and most of the Zhongyuan and French cultivars. In cluster I, nine Xibei cultivars fell into one large branch and were concentrated on two small branches with 71% and 95% bootstrap values. In the first small branch, ‘Xue Zhong Song Tan’ and ‘Yu Ban Xiu Qiu’, with white flowers, were divided from the other three with red or pink flowers. In the UPGMA and MP trees, Xibei tree peonies are distributed on two branches. P. ostii, ‘Bai Wang Shi Zi’, and ‘Jing Yu’, with white flowers, are distributed in the same branch with a 69% bootstrap value. Four other white-flower cultivars formed another branch: ‘Yu Ban Bai’ and ‘Yu Pan Tuo Jin’, which came from the Zhongyuan group, showed a 99% bootstrap value (97% in UPGMA and MP trees) and formed a secondary branch, and ‘Feng Wei’ and ‘Feng Dan’, which came from the Jiangnan group, showed a 51% bootstrap value and formed the other secondary branch. The Japanese cultivar ‘Hua Wang’ and the French cultivar ‘Jin Huang’ were distributed in one branch with a 71% bootstrap value (67% UPGMA, 63% MP). Another Japanese cultivar, ‘Chu Wu’, and the Zhongyuan tree peony ‘Shu Hua Zi’, both with reddish black flowers, clustered together with a 97% bootstrap value (93% UPGMA, 90% MP). It can be concluded that flower color has some relation to genetic diversity. In cluster II, all non-Chinese cultivars with yellow flowers were grouped into one branch. They were from America (‘Jin Dao’ and ‘Hai Huang’) and France (‘Jin Ge’ and ‘Jin Zhi’). Two cultivars from the Chinese Zhongyuan group with light yellow flowers, ‘Yu Yi Huang’ and ‘Huang Hua Kui’, were also grouped in this branch. The female parent of ‘Jin Huang’, ‘Hai Huang’, ‘Jin Dao’, ‘Jin Ge’, and ‘Jin Zhi’ is P. lutea (= P. delavayi with yellow flowers). The male parent is unclear. The origin of Chinese cultivars with light yellow flowers such as ‘Yao Huang’, ‘Jin Yu Jiao Zhang’, ‘Huang Hua Kui’, and ‘Yu Yi Huang’ is not clear either. Generally, the color of Chinese cultivars with yellow flowers is thinner than that of American and French cultivars. Most cultivars from the Zhongyuan group were distributed nearby
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on the dendrogram with strong bootstrap support, indicating their closely consanguineous relationships. For example, ‘Gui Fei Cha Cui’ and ‘Shan Hua Lan Man’ formed a branch with bootstrap values of 97% (NJ), 89% (UPGMA), and 83% (MP). Unequivocally, further subgroups were based mainly on flower color. Three cultivars with red flowers, ‘Huo Lian Jin Dan’, ‘Zhu Sha Lei’, and’Yan Long Zi’, and a lot of cultivars with white flowers were grouped together. Considering the flower form, only ‘Fang Ji’ and ‘Wu Da Zhou’, with an anemone form, and ‘Tai Ping Hong Chong’, ‘Jin Dao 2’, and ‘Hai Huang’, with a rose form, were grouped together.
Discussion In this study, 23 primer pairs were used to distinguish 66 specimens, resulting in 262 polymorphic fragments, accounting for 87.5% of the total fragments amplified. Three large branches in close congruence with their geographic classification were grouped on the dendrogram. Some secondary branches had a certain degree of relationship to the flower color and form. This is the first attempt using SRAP molecular markers to characterize the genetic diversity of the tree peony and considering so many cultivars and wild species. SRAP markers were indicated to be effective for examining genetic diversity and genetic relationships among a wide range of diverse populations, and they show promise for developing molecular markers that could reliably determine the relative genetic contributions of specific populations to existing and newly developed cultivars. Some molecular marker techniques have recently been employed in tree peony studies, such as RAPD (Pei et al. 1995; Zou et al. 1999a, b), RFLP (Zhao et al. 2004), and ISSR (Suo et al. 2004, 2005). Most of the studies, however, focused on establishment of the phylogenetic relationships among wild species or interspecies. Until now only two molecular marker techniques have been applied to analyze tree peony cultivars: RAPD (Chen et al. 2001; Su et al. 2006) and AFLP (Liu et al. 2006a). Chen, Liu, and Su got materials from Shandong, Henan, and Gansu provinces, respectively. Cultivars from Shandong and Henan provinces belong to the Zhongyuan group; cultivars from Gansu province belong to the Xibei group. Because they used haploid materials, they found only small genetic distances. We share nine and eight cultivars with Chen and Liu’s study, respectively. The relative positions of those cultivars on the dendrogram are not different between our work and theirs. We share no common cultivars with Su’s work, but both of our studies demonstrate that there are some, but not obvious, relations between flower color, flower form, and genetic information. The information obtained from these studies is insufficient for characterizing so many cultivars. Until now, the genetic diversity of cultivars used in this research has not been explored. Finding specific markers related to ornamental characteristics, such as flower color and form, are our major concerns for tree peony breeding. Though these studies suggest that they are not closely related, further research may be needed to develop more molecular markers and include more cultivars, which can further demonstrate the relationship between markers and ornamental characteristics.
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In China, the tree peony has been domesticated for more than 1,600 years. Its cultivars were developed mainly by natural and artificial hybridization and preserved by nonsexual propagation. The great majority of the cultivars are synthetic populations developed from successive generations of random matings of wild species and their progeny. China is the world center of tree peony cultivation, and the Zhongyuan region is the cultivation center of China. Five wild species, P. ostii, P. rockii, P. jishanensis, P. qiui, and P. suffruticosa ssp. Yinpingmudan, are known as the progenitors of cultivars dispersed in the Zhongyuan region (Zhou et al. 2003). In the long breeding history, wild species and their hybrid descendants coexisted in the same area. In that case, it is likely that different lots of seeds of the same cultivar are genetically heterogeneous, because of the multiple cycles of random mating that occur in seed production fields due to the pollinators. Thus the cultivars of the Zhongyuan group have a broad parental base and a complex genetic background; they demonstrate wide genetic diversity, which is in accordance with the NJ tree. Among all the wild species in this study, P. ostii contributed more genetic information to the cultivars of this group, showing high similarity with these cultivars, so it failed to appear as an outgroup on the NJ and MP trees. Comparatively, the Xibei group is more congenetic and simpler. Although it is widely distributed in different areas, it is considered to stem from P. rockii and mostly shares the same morphological characteristics, such as a clear black–purple or purple–red blotch at the base of the petals. The cultivars from the Xinan and Jiangnan groups were shown to resemble closely the Zhongyuan cultivars. Many Zhongyuan cultivars were introduced to Xinan and Jiangnan, and they joined into a large hybridization circle there and formed new cultivars. Japanese tree peony cultivars were introduced from China early on and have been bred indigenously for more than 1,000 years. The Japanese cultivars were not pooled in a single branch but were dispersed in every large branch on the dendrogram, indicating their intimate pedigree with Chinese cultivars. Only a few cultivars have yellow flowers. ‘Jin Huang’, ‘Jin Ge’, ‘Jin Zhi’, ‘Hai Huang’, and ‘Jin Dao’ with yellow flowers are the crossbred offspring of P. lutea (= P. delavayi, with yellow flowers) and some uncertain cultivars that may come from China or Japan. The origin of Chinese cultivars with yellow flowers is not clear. We compared all 11 yellow flower samples in this study. Among all the fragments generated by 23 primer combinations, they shared one fragment with the same size (approximately 400 bp) produced by primer pair Me7/Em8. Non-Chinese cultivars with yellow flowers (‘Jin Huang’, ‘Jin Ge’, ‘Jin Zhi’, ‘Hai Huang’, and ‘Jin Dao 2’) shared two fragments 750 bp and 450 bp in length with P. lutea, by primer pairs Me2/Em2 and Me8/Em3, respectively. Chinese cultivars with yellow flowers (‘Jin Yu Jiao Zhang’, ‘Yu Yi Huang’, ‘Huang Hua Kui’, ‘Yao Huang’) shared another two fragments, about 200 bp and 400 bp in length, with P. lutea, by primer pairs Me7/Em8 and Me4/Em8, respectively. All cultivars with yellow flowers shared more fragments with P. lutea than with P. ludlowii. We deduced that the relation of cultivars with yellow flowers is closer to P. lutea than to P. ludlowii. The exact information of the shared bands needs to be characterized. The complexity and overlap of the origin of tree peony cultivars has resulted in abundant genetic diversity. The cultivars adapted to different environments and
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underwent genetic modification, which is reflected in the comb form of the UPGMA and MP trees. The NJ tree, however, can give us instructive information. Increasing the knowledge of the molecular diversity of a crop is essential for extending its genetic base, identifying cultivars, and selecting parental varieties for breeding programs. In this sense, tree peony cultivars are poorly characterized. Although many classification methods, including many molecular marker systems, have been applied to these species, the cultivars are still classified mainly by traditional phenetic characteristics such as flower color, flower form, and geographic distribution. Wang et al. (2001, 2004) classified the Chinese Zhongyuan and Xibei cultivars according to anthocyanidin phenotypes in the petals, in an attempt at chemical classification, but much confusion remains and many problems are unsolved. Unresolved questions include the identification of homonym and synonym, and changes in flower color and flower form of the same cultivar in different areas, etc. The descriptions of cultivars are changeable based on traditional classification; thus, SRAP markers may serve as a suitable tool to identify substantial differences and the intrinsic diversity among them. In breeding, we should choose parents with large genetic distance, and we can select hybrid offspring by SRAP in the seedling stage. Based on so many advantages, SRAP markers can also facilitate the construction of tree peony linkage maps, genetic fingerprinting, and core germplasm. Acknowledgments This work was supported by the National High Technology Research and Development Program of China (863 Program) (Grant No. 2006AA100109), the Knowledge Innovation Program of the Chinese Academy of Sciences (KSCX2–YW-Z-064), and the Pilot Research Program of the Institute of Botany, CAS. The authors thank Prof. Hong-Jie Li for his kind help. The authors also thank Xia Tao and Cheng Li-Bao for their technical instruction. The authors also thank the members of the Physiology and Genetic Breeding of Ornamental Plants Research Group, IBCAS, for their kind help.
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