关键词:古蔺牛皮茶; 种质资源; 遗传多态性; ISSR Genetic Diversity of Gulin-niupicha [Camellia sinensis (L.)O. Kuntze] Germplasm- Resources LIU Shao-Jie1,2, CHI Lin2, XIE Wen-Gang1, HAN Nan1, CHEN Ying-Hui2, HE Xin2, TANG Qian1,* 1College of Horticulture, Sichuan Agricultural University, Ya#cod#x02019;an 625014, China
2Department of Agriculture, Forestry and Water Conservancy, Chongqing Economic and Trade Vocational School, Chongqing 402100, China
AbstractGulin-niupicha (Camellia sinensis) is a tea landrace population specifically grown in Jiaozigou, Gulin County, Sichuan Province, China. To protect and utilize Gulin-niupicha resources effectively, we investigated the morphologic characteristics, biochemical components, and genetic diversity based on inter-simple sequence repeat (ISSR) markers by sampling 76 individuals from seven populations of Gulin-niupicha germplasm. The results showed that: (1) there were various biological characteristics among the seven populations. Importantly, a majority of Gulin-niupicha were shrubs or semi-arbor. Their mature leaves were medium in size, elliptic or oblong in shape, and in green or dark green color; while the young shoots showed more colors varying form whitish, yellow green, green or purple green. (2) In the seven populations, two had high contents of free amino acids (>4.5%) and two had both high contents of tea polyphenols (>27% in spring shoots and >31% in summer shoots) and catechins (>150 mg g-1 in spring shoots and >190 mg g-1 in summer shoots). The ratio of polyphenols-to-free amino acids ranged from 3.84 to 16.95 in the seven populations. The results of biochemical component measurements indicated that the Gulin-niupicha germplasm resources are of great value in tea tree breeding and tea processing. (3) A total of 135 fragments were amplified in the 76 accessions using 15 pairs of ISSR primers, in which 90.4% (122/135) were polymorphic bands. The genetic diversity of the Gulin-niupicha germplasm revealed by ISSR markers was low, with the Nei’s gene diversity index of 0.254 and Shannon’s information index of 0.392.
Keyword:Gulin-niupicha (Camellia sinensisL.); Germplasm resources; Genetic diversity; ISSR Show Figures Show Figures
表3 古蔺牛皮茶居群间8个生物学表型性状统计分析 Table 3 Statistical analysis of eight phenotypic traits among different Gulin-niupicha populations
居群 Population
树型 Tree type
树姿 Tree posture
树高 Tree height (cm)
叶片大小 Leaf size
叶形 Leaf shape
叶色 Leaf color
芽颜色泽 Bud color
芽叶茸毛 Bud hair
A
1.20±0.42 a
1.20±0.42 a
198.35±90.90 a
2.50±0.53 cd
1.40±0.70 b
1.60±0.52 a
2.10±0.32 bc
2.40±1.26 a
B
1.38±0.52 a
2.00±0.76 b
382.11±127.01 d
2.38±0.52 cd
1.25±0.46 b
1.75±0.71 ab
2.63±0.74 c
2.88±0.64 a
C
1.13±0.35 a
1.80±0.68 ab
308.72±84.61 cd
1.93±0.59 abc
1.33±0.49 b
1.87±0.35 ab
2.47±0.52 c
2.47±1.13 a
D
1.00±0.00 a
1.55±0.69 ab
190.74±69.92 a
1.82±0.60 ab
1.18±0.40 b
2.09±0.30 b
2.91±0.83 d
2.82±0.98 a
E
1.15±0.55 a
1.85±0.72 ab
180.81±80.24 a
1.54±0.52 a
1.38±0.51 b
1.85±0.55 ab
2.38±0.51 bc
3.08±0.86 a
F
1.20±0.42 a
1.80±0.63 ab
213.50±107.82 ab
2.10±0.57 bcd
1.30±0.48 b
1.80±0.63 ab
2.50±0.85 c
2.30±0.48 a
G
1.00±0 a
1.56±0.73 ab
279.61±79.36 bc
2.00±0 abc
1.33±0.71 b
2.00±0 ab
2.33±0.71 bc
2.22±0.44 a
CK
1.00±0 b
2.00±0 b
172.42±36.63 ab
2.14±0.21 bcd
1.00±0 a
3.00±0 d
1.30±0.15 a
3.00±0 a
Values followed by different letters are significantly different at 0.05 probability level. 同列标以不同小写字母的值在处理间差异在0.05的水平显著。
表3 古蔺牛皮茶居群间8个生物学表型性状统计分析 Table 3 Statistical analysis of eight phenotypic traits among different Gulin-niupicha populations
表4 Table 4 表4(Table 4)
表4 2012年古蔺牛皮茶群体春、夏梢主要生化成分 Table 4 Biochemical composition of Gulin-niupicha in spring and summer of 2012
居群 Population
季节 Season
茶多酚 Polyphenols (%)
儿茶素 Total amount of catechin (mg g-1)
游离氨基酸 Free amino acid (%)
咖啡碱 Caffeine (%)
酚氨比 TP/AA
A
春Spring
27.93±0.52 c
157.15±4.12 a
3.88±0.08 c
3.99±0.08 c
7.20±0.24 e
夏Summer
31.51±0.87 c
194.60±8.52 a
3.62±0.07 c
4.86±0.02 c
8.70±0.30 d
B
春Spring
30.17±0.62 a
155.19±3.79 a
2.48±0.04 h
4.81±0.11 a
12.17±0.07 b
夏Summer
36.57±0.57 a
191.69±1.80 a
2.16±0.06 g
5.22±0.04 a
16.93±0.65 a
C
春Spring
25.12±0.41 ef
138.35±1.96 c
4.63±0.10 b
4.79±0.09 a
5.42±0.25 f
夏Summer
28.10±0.61 de
164.53±2.06 cd
3.79±0.07 b
4.51±0.02 d
7.41±0.03 e
D
春Spring
22.57±0.57 g
128.70±3.99 de
4.84±0.03 a
3.32±0.05 f
4.66±0.11 g
夏Summer
26.82±1.52 e
135.27±1.12 f
3.97±0.07 a
4.03±0.05 g
6.76±0.33 e
E
春Spring
26.34±0.39 d
131.23±3.00 d
2.08±0.03 i
3.55±0.07 e
12.66±0.22 a
夏Summer
31.52±1.44 c
178.26±4.77 b
1.86±0.03 h
3.83±0.02 h
16.95±0.75 a
F
春Spring
28.97±0.21 b
126.52±4.03 de
3.56±0.05 d
4.28±0.05 b
8.14±0.05 d
夏Summer
33.45±1.30 b
158.79±4.24 d
3.13±0.11 d
4.96±0.05 b
10.69±0.81 c
G
春Spring
24.38±0.22 f
139.22±2.64 c
2.94±0.08 f
4.03±0.06 c
8.29±0.15 d
夏Summer
28.79±1.37 d
171.03±2.42 c
2.61±0.06 e
4.56±0.06 d
11.03±0.64 c
CK
春Spring
25.60±0.37 de
148.07±2.51 b
2.64±0.03 g
3.77±0.08 d
9.70±0.17 c
夏Summer
29.42±0.51 d
184.09±3.83 b
2.33±0.07 f
4.12±0.02 f
12.63±0.44 b
Values followed by different letters are significantly different at 0.05 probability level. AA: free amino acid; TP: tea polyphenols. 同列标以不同小写字母的值在处理间差异在0.05的水平显著。
表4 2012年古蔺牛皮茶群体春、夏梢主要生化成分 Table 4 Biochemical composition of Gulin-niupicha in spring and summer of 2012
表5 2013年古蔺牛皮茶群体春梢主要生化成分 Table 5 Biochemical composition of Gulin-niupicha in spring of 2013
居群 Population
茶多酚 Polyphenols (%)
游离氨基酸总量 Free amino acid (%)
酚/氨 TP/AA
儿茶素总量 Total amount of catechin
茶氨酸 Theanine (mg g-1)
咖啡碱 Caffeine (%)
A
29.79±0.29 a
4.17±0.08 b
7.62±0.09 e
175.01±0.95 a
17.48±0.80 c
3.78±0.04 c
B
29.56±0.63 a
2.55±0.02 e
12.38±0.17 a
167.80±4.60 b
12.34±0.25 f
3.94±0.07 b
C
25.19±0.19 c
4.78±0.04 a
5.27±0.03 f
140.37±3.30 c
20.51±0.66 b
4.28±0.02 a
D
19.04±0.42 f
4.96±0.11 a
3.84±0.02 h
104.73±3.34 g
24.13±0.34 a
3.58±0.07 d
E
24.11±0.13 d
2.27±0.02 e
10.64±0.08 b
121.76±2.80 e
10.98±0.55 g
3.36±0.04 f
F
26.51±0.45 b
3.43±0.08 c
7.72±0.26 d
139.83±4.04 c
15.49±0.32 d
3.62±0.06 d
G
23.31±0.43 e
3.37±0.08 c
6.92±0.22 e
131.73±1.23 d
16.26±0.47 d
3.57±0.07 de
CK
27.03±0.38 b
2.99±0.07 d
9.02±0.23 c
162.38±5.13 b
13.39±0.57 e
3.48±0.04 e
Values followed by different letters are significantly different at 0.05 probability level. AA: free amino acid; TP: tea polyphenols. 同列标以不同小写字母的值在处理间差异在0.05的水平显著。
表5 2013年古蔺牛皮茶群体春梢主要生化成分 Table 5 Biochemical composition of Gulin-niupicha in spring of 2013
表6 Table 6 表6(Table 6)
表6 不同居群古蔺牛皮茶春梢儿茶素组分分析(2013年) Table 6 Analysis of catechin components about the tea plants in spring of 2013
居群Population
表没食子儿茶素 EGC (mg g-1)
儿茶素 C (mg g-1)
表儿茶素 EC (m g g-1)
表没食子儿茶 素没食子酸酯 EGCG (mg g-1)
没食子儿茶素 没食子酸酯 GCG (mg g-1)
表儿茶素没 食子酸酯 ECG (mg g-1)
儿茶素总量 Total amount catechin (mg g-1)
A
16.23±0.05 a
4.31±0.16 a
8.18±0.10 a
69.54±0.54 c
36.22±0.28 a
20.20±0.14 d
175.01±0.95 a
B
11.74±0.04 c
2.29±0.10 e
6.12±0.05 c
82.80±1.05 a
27.08±0.11 e
28.01±0.12 a
167.80±4.60 b
C
10.85±0.12 d
2.54±0.06 d
5.25±0.04 f
63.07±0.33 e
27.64±0.07 d
19.89±0.14 e
140.37±3.30 c
D
6.71±0.04 f
1.60±0.11 g
5.24±0.03 f
45.75±0.06 h
18.27±0.07 i
18.36±0.09 g
104.73±3.34 g
E
8.22±0.08 e
4.11±0.08 b
4.97±0.06 h
50.13±0.38 g
24.05±0.20 g
20.82±0.08 c
121.76±2.80 e
F
10.70±1.50 d
1.93±0.04 f
5.44±0.06 e
74.75±0.18 b
22.94±0.13 h
16.30±0.03 h
139.83±4.04 c
G
11.04±0.18 cd
1.92±0.06 f
5.62±0.08 d
60.98±0.16 f
29.34±0.04 c
14.29±0.15 i
131.73±1.23 d
CK
14.24±0.03 b
2.24±0.04 e
7.10±0.04 b
67.72±0.29 d
31.84±0.19 b
21.46±0.15 b
162.38±5.13 b
Values followed by different letters are significantly different at 0.05 probability level. EGC: epigallocatechin; C: catechin; EC: epicatechin; EGCG: epigallocatechin gallate; GCG: gallocatechin gallate; ECG: epicatechin gallate. 同列标以不同小写字母的值在处理间差异在0.05的水平显著。
表6 不同居群古蔺牛皮茶春梢儿茶素组分分析(2013年) Table 6 Analysis of catechin components about the tea plants in spring of 2013
表7 15对ISSR引物对76株古蔺牛皮茶扩增的多态性 Table 7 Polymorphism detected with 15 ISSR primers in 76 Gulin-niupicha individuals
引物 Primer
引物序列 Primer sequence
扩增条带 Total bands
多态性条带 Polymorphic bands
多态性百分比 Percentage (%)
退火温度 Annealing temp. (℃)
产物大小 Product size (bp)
R1
(AG)8YA
12
11
91.7
56.5
400-1800
R2
(CTC)6
8
6
75.0
57.5
300-3000
R13
(GGAGA)3
8
7
87.5
49.0
450-1800
R4
(GACA)4T
12
12
100.0
51.5
250-3000
R5
(CTGA)4G
10
8
80.0
50.0
300-3000
R6
(GA)8CTT
10
9
90.0
58.0
200-2000
R7
(CTGA)4
10
10
100.0
54.0
300-3000
R8
(GGGTG)3
8
7
87.5
59.5
300-1500
R9
(AG)8YC
11
11
100.0
54.0
220-4000
R10
(CT)8RC
6
5
83.3
54.5
250-2800
R11
(GA)8YC
11
9
81.8
51.5
200-2000
R12
DBD(AC)7
6
5
83.3
51.5
200-1600
R13
(TC)8AGT
6
6
100.0
51.5
200-1500
R14
(AG)8CTT
8
7
87.5
57.5
200-3000
R15
(AG)8CTA
9
9
100.0
57.5
200-2500
总计Mean
135
122
90.4
平均Total
9
8.1
90.4
表7 15对ISSR引物对76株古蔺牛皮茶扩增的多态性 Table 7 Polymorphism detected with 15 ISSR primers in 76 Gulin-niupicha individuals
表8 Table 8 表8(Table 8)
表8 不同古蔺牛皮茶群体中的ISSR遗传多态性 Table 8 Genetic diversity of Gulin-niupicha distributed among different populations detected by ISSR
群体 Population
Nei’s多态性 指数 H
Shannon’s 指数 I
遗传分化 指数 Gst
每代迁移数 Nm
等位基因数 Na
有效等位 基因数 Ne
多态位点数 NPL
多态位点比率 PPL (%)
A
0.200
0.307
1.644
1.334
87
64.44
B
0.186
0.263
1.570
1.301
77
57.04
C
0.218
0.333
1.689
1.365
93
68.89
D
0.202
0.314
0.307
1.323
95
70.37
E
0.212
0.323
1.674
1.356
91
67.41
F
0.214
0.326
1.682
1.356
92
68.15
G
0.187
0.282
1.563
1.316
76
56.30
平均Mean
0.203
0.307
1.447
1.334
64.66
总计Total
0.254
0.392
0.198
2.022
1.867
1.417
64.66
H: Nei’s gene diversity; I: Shannon’s information index; Gst: coefficient of gene differentiation; Nm: estimate of gene flow from Gst; Na: observed number of alleles; Ne: effective number of alleles; NPL: number of polymorphic loci; PPL: percentage of polymorphic loci.
表8 不同古蔺牛皮茶群体中的ISSR遗传多态性 Table 8 Genetic diversity of Gulin-niupicha distributed among different populations detected by ISSR
4 结论古蔺牛皮茶在形态学特征和品质生化成分上呈现出多态性, 但居群的平均Nei’s基因多样性指数( H)为0.254, 平均Shannon’s信息指数( I)为0.392, 表明遗传多态性较低。7个居群的UPGMA遗传相似性聚类显示, 在相似系数为0.956处可将之分为3个组。居群间的遗传分化系数( Gst)是0.198, 证明古蔺牛皮茶居群的遗传多样性主要分布在自然居群内。 The authors have declared that no competing interests exist. 作者已声明无竞争性利益关系。
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