药学学报

Pop. ID	Sample No.	Voucher specimens	Locality
T1	1	Ni L.H.et al XZ201214-4	Dingqing, Tibet
2	Ni L.H.et al XZ201214-5	Dingqing, Tibet
3	Ni L.H.et al XZ201214-6	Dingqing, Tibet
T2	4	Yang Y.H. 2012XZ014-A	Dingqing, Tibet
5	Yang Y.H. 2012XZ014-B	Dingqing, Tibet
6	Yang Y.H. 2012XZ014-C	Dingqing, Tibet
K1	7	Ni L.H.et al GS201301-1	Maqu, Gansu
8	Ni L.H.et al GS201301-2	Maqu, Gansu
9	Ni L.H.et al GS201301-3	Maqu, Gansu
10	Ni L.H.et al GS201301-4	Maqu, Gansu
11	Ni L.H.et al GS201301-5	Maqu, Gansu
K2	12	Ni L.H. GS201601-1	Maqu, Gansu
13	Ni L.H. GS201601-2	Maqu, Gansu
14	Ni L.H. GS201601-3	Maqu, Gansu
15	Ni L.H. GS201601-4	Maqu, Gansu
16	Ni L.H. GS201601-5	Maqu, Gansu
17	Ni L.H. GS201601-6	Maqu, Gansu
18	Ni L.H. GS201601-7	Maqu, Gansu
19	Ni L.H. GS201601-8	Maqu, Gansu
20	Ni L.H. GS201601-9	Maqu, Gansu
21	Ni L.H. GS201601-10	Maqu, Gansu
22	Ni L.H. GS201601-11	Maqu, Gansu
23	Ni L.H. GS201601-12	Maqu, Gansu
24	Ni L.H. GS201601-13	Maqu, Gansu
25	Ni L.H. GS201601-14	Maqu, Gansu
26	Ni L.H. GS201601-15	Maqu, Gansu
G1	27	Ni L.H.et al GZ201601-1	Weining, Guizhou
28	Ni L.H.et al GZ201601-2	Weining, Guizhou
29	Ni L.H.et al GZ201601-3	Weining, Guizhou
30	Ni L.H.et al GZ201601-4	Weining, Guizhou
31	Ni L.H.et al GZ201601-5	Weining, Guizhou
32	Ni L.H.et al GZ201601-6	Weining, Guizhou
33	Ni L.H.et al GZ201601-7	Weining, Guizhou
34	Ni L.H.et al GZ201601-8	Weining, Guizhou
35	Ni L.H.et al GZ201601-9	Weining, Guizhou
36	Ni L.H.et al GZ201601-10	Weining, Guizhou
37	Ni L.H.et al GZ201601-11	Weining, Guizhou
38	Ni L.H.et al GZ201601-12	Weining, Guizhou
39	Ni L.H.et al GZ201601-13	Weining, Guizhou
40	Ni L.H.et al GZ201601-14	Weining, Guizhou
41	Ni L.H.et al GZ201601-15	Weining, Guizhou
G2	42	Ni L.H.et al GZ201602-1	Weining, Guizhou
43	Ni L.H.et al GZ201602-2	Weining, Guizhou
44	Ni L.H.et al GZ201602-3	Weining, Guizhou
45	Ni L.H.et al GZ201602-4	Weining, Guizhou
46	Ni L.H.et al GZ201602-5	Weining, Guizhou
47	Ni L.H.et al GZ201602-6	Weining, Guizhou
48	Ni L.H.et al GZ201602-7	Weining, Guizhou
49	Ni L.H.et al GZ201602-8	Weining, Guizhou
50	Ni L.H.et al GZ201602-9	Weining, Guizhou
51	Ni L.H.et al GZ201602-10	Weining, Guizhou
52	Ni L.H.et al GZ201601-11	Weining, Guizhou
53	Ni L.H.et al GZ201602-12	Weining, Guizhou
54	Ni L.H.et al GZ201602-13	Weining, Guizhou
55	Ni L.H.et al GZ201602-14	Weining, Guizhou
56	Ni L.H.et al GZ201602-15	Weining, Guizhou
T3	57	Ni L.H.et al 2016XZ008-1	Basu, Tibet
58	Ni L.H.et al 2016XZ008-2	Basu, Tibet
59	Ni L.H.et al 2016XZ008-3	Basu, Tibet
60	Ni L.H.et al 2016XZ008-4	Basu, Tibet
61	Ni L.H.et al 2016XZ008-5	Basu, Tibet
T4	62	Ni L.H.et al 2016XZ009-1	Basu, Tibet
63	Ni L.H.et al 2016XZ009-2	Basu, Tibet
64	Ni L.H.et al 2016XZ009-3	Basu, Tibet
T5	65	Ni L.H.et al 2016XZ011-1	Changdu, Tibet
66	Ni L.H.et al 2016XZ011-2	Changdu, Tibet
67	Ni L.H.et al 2016XZ011-3	Changdu, Tibet
68	Ni L.H.et al 2016XZ011-4	Changdu, Tibet
69	Ni L.H.et al 2016XZ011-5	Changdu, Tibet
70	Ni L.H.et al 2016XZ011-6	Changdu, Tibet
71	Ni L.H.et al 2016XZ011-7	Changdu, Tibet
72	Ni L.H.et al 2016XZ011-8	Changdu, Tibet
73	Ni L.H.et al 2016XZ011-9	Changdu, Tibet
74	Ni L.H.et al 2016XZ011-10	Changdu, Tibet
75	Ni L.H.et al 2016XZ011-11	Changdu, Tibet
76	Ni L.H.et al 2016XZ011-12	Changdu, Tibet
77	Ni L.H.et al 2016XZ011-13	Changdu, Tibet
78	Ni L.H.et al 2016XZ011-14	Changdu, Tibet
79	Ni L.H.et al 2016XZ011-15	Changdu, Tibet
S1	80	Ni L.H. SC201601-1	Kangding, Sichuan
81	Ni L.H. SC201601-2	Kangding, Sichuan
82	Ni L.H. SC201601-3	Kangding, Sichuan
83	Ni L.H. SC201601-4	Kangding, Sichuan
84	Ni L.H. SC201601-5	Kangding, Sichuan
85	Ni L.H. SC201601-6	Kangding, Sichuan
86	Ni L.H. SC201601-7	Kangding, Sichuan
87	Ni L.H. SC201601-8	Kangding, Sichuan
88	Ni L.H. SC201601-9	Kangding, Sichuan
89	Ni L.H. SC201601-10	Kangding, Sichuan
S2	90	Ni L.H. SC201602-1	Daofu, Sichuan
91	Ni L.H. SC201602-2	Daofu, Sichuan
92	Ni L.H. SC201602-3	Daofu, Sichuan
93	Ni L.H. SC201602-4	Daofu, Sichuan
94	Ni L.H. SC201602-5	Daofu, Sichuan
95	Ni L.H. SC201602-6	Daofu, Sichuan
96	Ni L.H. SC201602-7	Daofu, Sichuan
97	Ni L.H. SC201602-8	Daofu, Sichuan
98	Ni L.H. SC201602-9	Daofu, Sichuan
99	Ni L.H. SC201602-10	Daofu, Sichuan
100	Ni L.H. SC201602-11	Daofu, Sichuan
101	Ni L.H. SC201602-12	Daofu, Sichuan
102	Ni L.H. SC201602-13	Daofu, Sichuan
103	Ni L.H. SC201602-14	Daofu, Sichuan
104	Ni L.H. SC201602-15	Daofu, Sichuan
Y1	105	Ni L.H.et al YN201601-1	Lijiang, Yunnan
106	Ni L.H.et al YN201601-2	Lijiang, Yunnan
107	Ni L.H.et al YN201601-3	Lijiang, Yunnan
108	Ni L.H.et al YN201601-4	Lijiang, Yunnan
109	Ni L.H.et al YN201601-5	Lijiang, Yunnan
110	Ni L.H.et al YN201601-6	Lijiang, Yunnan
111	Ni L.H.et al YN201601-7	Lijiang, Yunnan
112	Ni L.H.et al YN201601-8	Lijiang, Yunnan
113	Ni L.H.et al YN201601-9	Lijiang, Yunnan
114	Ni L.H.et al YN201601-10	Lijiang, Yunnan
115	Ni L.H.et al YN201601-11	Lijiang, Yunnan
116	Ni L.H.et al YN201601-12	Lijiang, Yunnan
117	Ni L.H.et al YN201601-13	Lijiang, Yunnan
118	Ni L.H.et al YN201601-14	Lijiang, Yunnan
119	Ni L.H.et al YN201601-15	Lijiang, Yunnan
Y2	120	Ni L.H. YN201603-1	Lijiang, Yunnan
121	Ni L.H. YN201603-2	Lijiang, Yunnan
122	Ni L.H. YN201603-3	Lijiang, Yunnan
123	Ni L.H. YN201603-4	Lijiang, Yunnan
124	Ni L.H. YN201603-5	Lijiang, Yunnan
Y3^*	125	Ni L.H. YN201301-1	Lijiang, Yunnan
126	Ni L.H. YN201301-2	Lijiang, Yunnan
127	Ni L.H. YN201301-3	Lijiang, Yunnan
128	Ni L.H. YN201301-4	Lijiang, Yunnan
Q	129	Ni L.H.et al QH201405-1	Banma, Qinghai
130	Ni L.H.et al QH201405-2	Banma, Qinghai
131	Ni L.H.et al QH201405-3	Banma, Qinghai
T6	132	Zhao Z.L.et al 2017XZ006-1	Bomi, Tibet
133	Zhao Z.L.et al 2017XZ006-2	Bomi, Tibet
134	Zhao Z.L.et al 2017XZ006-3	Bomi, Tibet
135	Zhao Z.L.et al 2017XZ006-4	Bomi, Tibet
136	Zhao Z.L.et al 2017XZ006-5	Bomi, Tibet
137	Zhao Z.L.et al 2017XZ006-6	Bomi, Tibet
138	Zhao Z.L.et al 2017XZ006-7	Bomi, Tibet
139	Zhao Z.L.et al 2017XZ006-8	Bomi, Tibet
140	Zhao Z.L.et al 2017XZ006-9	Bomi, Tibet
141	Zhao Z.L.et al 2017XZ006-10	Bomi, Tibet
T7	142	Zhao Z.L.et al 2017XZ007-1	Basu, Tibet
143	Zhao Z.L.et al 2017XZ007-2	Basu, Tibet
144	Zhao Z.L.et al 2017XZ007-3	Basu, Tibet
145	Zhao Z.L.et al 2017XZ007-4	Basu, Tibet
146	Zhao Z.L.et al 2017XZ007-5	Basu, Tibet
147	Zhao Z.L.et al 2017XZ007-6	Basu, Tibet
148	Zhao Z.L.et al 2017XZ007-7	Basu, Tibet
149	Zhao Z.L.et al 2017XZ007-8	Basu, Tibet
T8	150	Zhao Z.L.et al 2017XZ008-1	Zuogong, Tibet
151	Zhao Z.L.et al 2017XZ008-2	Zuogong, Tibet
152	Zhao Z.L.et al 2017XZ008-3	Zuogong, Tibet
153	Zhao Z.L.et al 2017XZ008-4	Zuogong, Tibet
154	Zhao Z.L.et al 2017XZ008-5	Zuogong, Tibet
155	Zhao Z.L.et al 2017XZ008-6	Zuogong, Tibet
T9	156	Zhao Z.L.et al 2017XZ009-1	Zuogong, Tibet
157	Zhao Z.L.et al 2017XZ009-2	Zuogong, Tibet
158	Zhao Z.L.et al 2017XZ009-3	Zuogong, Tibet
159	Zhao Z.L.et al 2017XZ009-4	Zuogong, Tibet
160	Zhao Z.L.et al 2017XZ009-5	Zuogong, Tibet
Y4^*	161	Yang S.H. YN201701-1	Lijiang, Yunnan
162	Yang S.H. YN201701-2	Lijiang, Yunnan
163	Yang S.H. YN201701-3	Lijiang, Yunnan
M^**	164	Zhao Z.L.et al 2017XZ005-1	Bomi, Tibet
165	Zhao Z.L.et al 2017XZ005-2	Bomi, Tibet
H^**	166	Ni L.H.et al GZ201603-1	Weining, Guizhou
167	Ni L.H.et al GZ201603-2	Weining, Guizhou

Pop. ID	Sample No.	Voucher specimens	Locality
T1	1	Ni L.H.et al XZ201214-4	Dingqing, Tibet
2	Ni L.H.et al XZ201214-5	Dingqing, Tibet
3	Ni L.H.et al XZ201214-6	Dingqing, Tibet
T2	4	Yang Y.H. 2012XZ014-A	Dingqing, Tibet
5	Yang Y.H. 2012XZ014-B	Dingqing, Tibet
6	Yang Y.H. 2012XZ014-C	Dingqing, Tibet
K1	7	Ni L.H.et al GS201301-1	Maqu, Gansu
8	Ni L.H.et al GS201301-2	Maqu, Gansu
9	Ni L.H.et al GS201301-3	Maqu, Gansu
10	Ni L.H.et al GS201301-4	Maqu, Gansu
11	Ni L.H.et al GS201301-5	Maqu, Gansu
K2	12	Ni L.H. GS201601-1	Maqu, Gansu
13	Ni L.H. GS201601-2	Maqu, Gansu
14	Ni L.H. GS201601-3	Maqu, Gansu
15	Ni L.H. GS201601-4	Maqu, Gansu
16	Ni L.H. GS201601-5	Maqu, Gansu
17	Ni L.H. GS201601-6	Maqu, Gansu
18	Ni L.H. GS201601-7	Maqu, Gansu
19	Ni L.H. GS201601-8	Maqu, Gansu
20	Ni L.H. GS201601-9	Maqu, Gansu
21	Ni L.H. GS201601-10	Maqu, Gansu
22	Ni L.H. GS201601-11	Maqu, Gansu
23	Ni L.H. GS201601-12	Maqu, Gansu
24	Ni L.H. GS201601-13	Maqu, Gansu
25	Ni L.H. GS201601-14	Maqu, Gansu
26	Ni L.H. GS201601-15	Maqu, Gansu
G1	27	Ni L.H.et al GZ201601-1	Weining, Guizhou
28	Ni L.H.et al GZ201601-2	Weining, Guizhou
29	Ni L.H.et al GZ201601-3	Weining, Guizhou
30	Ni L.H.et al GZ201601-4	Weining, Guizhou
31	Ni L.H.et al GZ201601-5	Weining, Guizhou
32	Ni L.H.et al GZ201601-6	Weining, Guizhou
33	Ni L.H.et al GZ201601-7	Weining, Guizhou
34	Ni L.H.et al GZ201601-8	Weining, Guizhou
35	Ni L.H.et al GZ201601-9	Weining, Guizhou
36	Ni L.H.et al GZ201601-10	Weining, Guizhou
37	Ni L.H.et al GZ201601-11	Weining, Guizhou
38	Ni L.H.et al GZ201601-12	Weining, Guizhou
39	Ni L.H.et al GZ201601-13	Weining, Guizhou
40	Ni L.H.et al GZ201601-14	Weining, Guizhou
41	Ni L.H.et al GZ201601-15	Weining, Guizhou
G2	42	Ni L.H.et al GZ201602-1	Weining, Guizhou
43	Ni L.H.et al GZ201602-2	Weining, Guizhou
44	Ni L.H.et al GZ201602-3	Weining, Guizhou
45	Ni L.H.et al GZ201602-4	Weining, Guizhou
46	Ni L.H.et al GZ201602-5	Weining, Guizhou
47	Ni L.H.et al GZ201602-6	Weining, Guizhou
48	Ni L.H.et al GZ201602-7	Weining, Guizhou
49	Ni L.H.et al GZ201602-8	Weining, Guizhou
50	Ni L.H.et al GZ201602-9	Weining, Guizhou
51	Ni L.H.et al GZ201602-10	Weining, Guizhou
52	Ni L.H.et al GZ201601-11	Weining, Guizhou
53	Ni L.H.et al GZ201602-12	Weining, Guizhou
54	Ni L.H.et al GZ201602-13	Weining, Guizhou
55	Ni L.H.et al GZ201602-14	Weining, Guizhou
56	Ni L.H.et al GZ201602-15	Weining, Guizhou
T3	57	Ni L.H.et al 2016XZ008-1	Basu, Tibet
58	Ni L.H.et al 2016XZ008-2	Basu, Tibet
59	Ni L.H.et al 2016XZ008-3	Basu, Tibet
60	Ni L.H.et al 2016XZ008-4	Basu, Tibet
61	Ni L.H.et al 2016XZ008-5	Basu, Tibet
T4	62	Ni L.H.et al 2016XZ009-1	Basu, Tibet
63	Ni L.H.et al 2016XZ009-2	Basu, Tibet
64	Ni L.H.et al 2016XZ009-3	Basu, Tibet
T5	65	Ni L.H.et al 2016XZ011-1	Changdu, Tibet
66	Ni L.H.et al 2016XZ011-2	Changdu, Tibet
67	Ni L.H.et al 2016XZ011-3	Changdu, Tibet
68	Ni L.H.et al 2016XZ011-4	Changdu, Tibet
69	Ni L.H.et al 2016XZ011-5	Changdu, Tibet
70	Ni L.H.et al 2016XZ011-6	Changdu, Tibet
71	Ni L.H.et al 2016XZ011-7	Changdu, Tibet
72	Ni L.H.et al 2016XZ011-8	Changdu, Tibet
73	Ni L.H.et al 2016XZ011-9	Changdu, Tibet
74	Ni L.H.et al 2016XZ011-10	Changdu, Tibet
75	Ni L.H.et al 2016XZ011-11	Changdu, Tibet
76	Ni L.H.et al 2016XZ011-12	Changdu, Tibet
77	Ni L.H.et al 2016XZ011-13	Changdu, Tibet
78	Ni L.H.et al 2016XZ011-14	Changdu, Tibet
79	Ni L.H.et al 2016XZ011-15	Changdu, Tibet
S1	80	Ni L.H. SC201601-1	Kangding, Sichuan
81	Ni L.H. SC201601-2	Kangding, Sichuan
82	Ni L.H. SC201601-3	Kangding, Sichuan
83	Ni L.H. SC201601-4	Kangding, Sichuan
84	Ni L.H. SC201601-5	Kangding, Sichuan
85	Ni L.H. SC201601-6	Kangding, Sichuan
86	Ni L.H. SC201601-7	Kangding, Sichuan
87	Ni L.H. SC201601-8	Kangding, Sichuan
88	Ni L.H. SC201601-9	Kangding, Sichuan
89	Ni L.H. SC201601-10	Kangding, Sichuan
S2	90	Ni L.H. SC201602-1	Daofu, Sichuan
91	Ni L.H. SC201602-2	Daofu, Sichuan
92	Ni L.H. SC201602-3	Daofu, Sichuan
93	Ni L.H. SC201602-4	Daofu, Sichuan
94	Ni L.H. SC201602-5	Daofu, Sichuan
95	Ni L.H. SC201602-6	Daofu, Sichuan
96	Ni L.H. SC201602-7	Daofu, Sichuan
97	Ni L.H. SC201602-8	Daofu, Sichuan
98	Ni L.H. SC201602-9	Daofu, Sichuan
99	Ni L.H. SC201602-10	Daofu, Sichuan
100	Ni L.H. SC201602-11	Daofu, Sichuan
101	Ni L.H. SC201602-12	Daofu, Sichuan
102	Ni L.H. SC201602-13	Daofu, Sichuan
103	Ni L.H. SC201602-14	Daofu, Sichuan
104	Ni L.H. SC201602-15	Daofu, Sichuan
Y1	105	Ni L.H.et al YN201601-1	Lijiang, Yunnan
106	Ni L.H.et al YN201601-2	Lijiang, Yunnan
107	Ni L.H.et al YN201601-3	Lijiang, Yunnan
108	Ni L.H.et al YN201601-4	Lijiang, Yunnan
109	Ni L.H.et al YN201601-5	Lijiang, Yunnan
110	Ni L.H.et al YN201601-6	Lijiang, Yunnan
111	Ni L.H.et al YN201601-7	Lijiang, Yunnan
112	Ni L.H.et al YN201601-8	Lijiang, Yunnan
113	Ni L.H.et al YN201601-9	Lijiang, Yunnan
114	Ni L.H.et al YN201601-10	Lijiang, Yunnan
115	Ni L.H.et al YN201601-11	Lijiang, Yunnan
116	Ni L.H.et al YN201601-12	Lijiang, Yunnan
117	Ni L.H.et al YN201601-13	Lijiang, Yunnan
118	Ni L.H.et al YN201601-14	Lijiang, Yunnan
119	Ni L.H.et al YN201601-15	Lijiang, Yunnan
Y2	120	Ni L.H. YN201603-1	Lijiang, Yunnan
121	Ni L.H. YN201603-2	Lijiang, Yunnan
122	Ni L.H. YN201603-3	Lijiang, Yunnan
123	Ni L.H. YN201603-4	Lijiang, Yunnan
124	Ni L.H. YN201603-5	Lijiang, Yunnan
Y3^*	125	Ni L.H. YN201301-1	Lijiang, Yunnan
126	Ni L.H. YN201301-2	Lijiang, Yunnan
127	Ni L.H. YN201301-3	Lijiang, Yunnan
128	Ni L.H. YN201301-4	Lijiang, Yunnan
Q	129	Ni L.H.et al QH201405-1	Banma, Qinghai
130	Ni L.H.et al QH201405-2	Banma, Qinghai
131	Ni L.H.et al QH201405-3	Banma, Qinghai
T6	132	Zhao Z.L.et al 2017XZ006-1	Bomi, Tibet
133	Zhao Z.L.et al 2017XZ006-2	Bomi, Tibet
134	Zhao Z.L.et al 2017XZ006-3	Bomi, Tibet
135	Zhao Z.L.et al 2017XZ006-4	Bomi, Tibet
136	Zhao Z.L.et al 2017XZ006-5	Bomi, Tibet
137	Zhao Z.L.et al 2017XZ006-6	Bomi, Tibet
138	Zhao Z.L.et al 2017XZ006-7	Bomi, Tibet
139	Zhao Z.L.et al 2017XZ006-8	Bomi, Tibet
140	Zhao Z.L.et al 2017XZ006-9	Bomi, Tibet
141	Zhao Z.L.et al 2017XZ006-10	Bomi, Tibet
T7	142	Zhao Z.L.et al 2017XZ007-1	Basu, Tibet
143	Zhao Z.L.et al 2017XZ007-2	Basu, Tibet
144	Zhao Z.L.et al 2017XZ007-3	Basu, Tibet
145	Zhao Z.L.et al 2017XZ007-4	Basu, Tibet
146	Zhao Z.L.et al 2017XZ007-5	Basu, Tibet
147	Zhao Z.L.et al 2017XZ007-6	Basu, Tibet
148	Zhao Z.L.et al 2017XZ007-7	Basu, Tibet
149	Zhao Z.L.et al 2017XZ007-8	Basu, Tibet
T8	150	Zhao Z.L.et al 2017XZ008-1	Zuogong, Tibet
151	Zhao Z.L.et al 2017XZ008-2	Zuogong, Tibet
152	Zhao Z.L.et al 2017XZ008-3	Zuogong, Tibet
153	Zhao Z.L.et al 2017XZ008-4	Zuogong, Tibet
154	Zhao Z.L.et al 2017XZ008-5	Zuogong, Tibet
155	Zhao Z.L.et al 2017XZ008-6	Zuogong, Tibet
T9	156	Zhao Z.L.et al 2017XZ009-1	Zuogong, Tibet
157	Zhao Z.L.et al 2017XZ009-2	Zuogong, Tibet
158	Zhao Z.L.et al 2017XZ009-3	Zuogong, Tibet
159	Zhao Z.L.et al 2017XZ009-4	Zuogong, Tibet
160	Zhao Z.L.et al 2017XZ009-5	Zuogong, Tibet
Y4^*	161	Yang S.H. YN201701-1	Lijiang, Yunnan
162	Yang S.H. YN201701-2	Lijiang, Yunnan
163	Yang S.H. YN201701-3	Lijiang, Yunnan
M^**	164	Zhao Z.L.et al 2017XZ005-1	Bomi, Tibet
165	Zhao Z.L.et al 2017XZ005-2	Bomi, Tibet
H^**	166	Ni L.H.et al GZ201603-1	Weining, Guizhou
167	Ni L.H.et al GZ201603-2	Weining, Guizhou

Primer combination	Amplified loci	PL	PPL/%		Primer combination	Amplified loci	PL	PPL/%
MseⅠ+CAG/EcoRⅠ+AAC	31	29	93.55		MseI+CTG/EcoRⅠ+ACC	41	40	97.56
MseⅠ+CAG/EcoRⅠ+ACG	26	23	88.46	MseI+CTG/EcoRⅠ+ACG	12	10	83.33
MseⅠ+CTC/EcoRⅠ+AAC	42	36	85.71	MseI+CTT/EcoRⅠ+AAG	9	6	66.67
MseⅠ+CTG/EcoRⅠ+AAC	36	28	77.78	MseI+CTT/EcoRⅠ+ACA	18	12	66.67
MseⅠ+CTG/EcoRⅠ+ACA	19	12	63.16	MseI+CTT/EcoRⅠ+AAG	41	25	60.98
MseⅠ+CTG/EcoRⅠ+ACT	21	19	90.48	MseI+CTT/EcoRⅠ+ACG	19	14	73.68
Total					315	254	80.63

Primer combination	Amplified loci	PL	PPL/%		Primer combination	Amplified loci	PL	PPL/%
MseⅠ+CAG/EcoRⅠ+AAC	31	29	93.55		MseI+CTG/EcoRⅠ+ACC	41	40	97.56
MseⅠ+CAG/EcoRⅠ+ACG	26	23	88.46	MseI+CTG/EcoRⅠ+ACG	12	10	83.33
MseⅠ+CTC/EcoRⅠ+AAC	42	36	85.71	MseI+CTT/EcoRⅠ+AAG	9	6	66.67
MseⅠ+CTG/EcoRⅠ+AAC	36	28	77.78	MseI+CTT/EcoRⅠ+ACA	18	12	66.67
MseⅠ+CTG/EcoRⅠ+ACA	19	12	63.16	MseI+CTT/EcoRⅠ+AAG	41	25	60.98
MseⅠ+CTG/EcoRⅠ+ACT	21	19	90.48	MseI+CTT/EcoRⅠ+ACG	19	14	73.68
Total					315	254	80.63

Pop. ID	Indiv. No.	PL	PPL /%	N_a	N_e	H	I_m
T1	3	4	1.27	1.012 7	1.005 4	0.003 8	0.006 1
T2	3	3	0.95	1.009 5	1.005 7	0.003 5	0.005 2
K1	5	14	4.44	1.044 4	1.028 5	0.016 1	0.023 9
K2	15	12	3.81	1.038 1	1.019 6	0.012 3	0.018 8
G1	15	29	9.21	1.092 1	1.049 1	0.028 1	0.042 2
G2	15	37	11.75	1.117 5	1.068 8	0.038 8	0.057 9
T3	5	18	5.71	1.057 1	1.034 8	0.020 0	0.029 9
T4	3	3	0.95	1.009 5	1.005 7	0.003 5	0.005 2
T5	15	20	6.35	1.063 5	1.031 2	0.018 8	0.028 7
S1	10	50	15.87	1.158 7	1.099 8	0.056 7	0.084 2
S2	15	19	6.03	1.060 3	1.030 6	0.018 2	0.027 7
Y1	15	46	14.6	1.146 0	1.082 2	0.048 7	0.073 3
Y2	5	13	4.13	1.041 3	1.024 3	0.014 4	0.021 7
Y3	4	38	12.06	1.120 6	1.072 0	0.042 2	0.063 6
Q	3	5	1.09	1.015 9	1.006 8	0.004 8	0.007 6
T6	10	23	7.30	1.073 0	1.037 7	0.022 0	0.033 4
T7	8	7	2.22	1.022 2	1.014 9	0.008 2	0.012 0
T8	6	11	3.49	1.034 9	1.013 7	0.009 0	0.014 5
T9	5	20	6.35	1.063 5	1.041 0	0.023 2	0.034 4
Y4	3	61	19.37	1.193 7	1.126 2	0.073 6	0.109 2

Pop. ID	Indiv. No.	PL	PPL /%	N_a	N_e	H	I_m
T1	3	4	1.27	1.012 7	1.005 4	0.003 8	0.006 1
T2	3	3	0.95	1.009 5	1.005 7	0.003 5	0.005 2
K1	5	14	4.44	1.044 4	1.028 5	0.016 1	0.023 9
K2	15	12	3.81	1.038 1	1.019 6	0.012 3	0.018 8
G1	15	29	9.21	1.092 1	1.049 1	0.028 1	0.042 2
G2	15	37	11.75	1.117 5	1.068 8	0.038 8	0.057 9
T3	5	18	5.71	1.057 1	1.034 8	0.020 0	0.029 9
T4	3	3	0.95	1.009 5	1.005 7	0.003 5	0.005 2
T5	15	20	6.35	1.063 5	1.031 2	0.018 8	0.028 7
S1	10	50	15.87	1.158 7	1.099 8	0.056 7	0.084 2
S2	15	19	6.03	1.060 3	1.030 6	0.018 2	0.027 7
Y1	15	46	14.6	1.146 0	1.082 2	0.048 7	0.073 3
Y2	5	13	4.13	1.041 3	1.024 3	0.014 4	0.021 7
Y3	4	38	12.06	1.120 6	1.072 0	0.042 2	0.063 6
Q	3	5	1.09	1.015 9	1.006 8	0.004 8	0.007 6
T6	10	23	7.30	1.073 0	1.037 7	0.022 0	0.033 4
T7	8	7	2.22	1.022 2	1.014 9	0.008 2	0.012 0
T8	6	11	3.49	1.034 9	1.013 7	0.009 0	0.014 5
T9	5	20	6.35	1.063 5	1.041 0	0.023 2	0.034 4
Y4	3	61	19.37	1.193 7	1.126 2	0.073 6	0.109 2

Variation source	df	Est.Var.	Percentage /%	PhiPT	P value
Among population	19	27.305	87	0.866	< 0.001
Within population	143	4.232	13
Total variation	162	31.536	100

Variation source	df	Est.Var.	Percentage /%	PhiPT	P value
Among population	19	27.305	87	0.866	< 0.001
Within population	143	4.232	13
Total variation	162	31.536	100

Pop.ID	psbI-trnS (GCU) 76/104^*	trnS(GCU)- trnG (UCC) 149/539	rpoB-trnC (GCA) 1157/1264	ycf3 intron1 1825/1984
S1a (n=3)	10A	11A	9A	10A
S1b (n=7)	9A	12A	9A	11A
S2(n=3)	9A	12A	10A	11A

Pop.ID	psbI-trnS (GCU) 76/104^*	trnS(GCU)- trnG (UCC) 149/539	rpoB-trnC (GCA) 1157/1264	ycf3 intron1 1825/1984
S1a (n=3)	10A	11A	9A	10A
S1b (n=7)	9A	12A	9A	11A
S2(n=3)	9A	12A	10A	11A

Pop.ID	ccsA 978/984^*	petA-psbJ 90/840	rpl16 intron 167/1170	rpl33 176/201
S1a (n= 3)	T	C	A	C
S1b (n= 7)	G	A	G	A
S2(n= 3)	G	A	G	A

Pop.ID	ccsA 978/984^*	petA-psbJ 90/840	rpl16 intron 167/1170	rpl33 176/201
S1a (n= 3)	T	C	A	C
S1b (n= 7)	G	A	G	A
S2(n= 3)	G	A	G	A

藏药解吉那保基原植物粗茎秦艽品种鉴定及种内遗传多样性分析

PDF下载

宗粉粉 ¹ , 赵志礼 ¹^,^* , 倪梁红 ¹ , 嘎务 ² , 刘铜华 ²^,³

药学学报 | 研究论文 2020,55(8): 1941-1950

收起

药学学报 | 研究论文 2020, 55(8): 1941-1950

藏药解吉那保基原植物粗茎秦艽品种鉴定及种内遗传多样性分析

全屏

宗粉粉¹, 赵志礼¹^,^*, 倪梁红¹, 嘎务², 刘铜华²^,³

作者信息

1.上海中医药大学, 上海 201203

2.西藏藏医药大学, 西藏拉萨 850000

3.北京中医药大学, 北京 100029

通讯作者:

*赵志礼, Tel:86-21-51322202, E-mail:zhilzhao@sohu.com

DNA fingerprinting and genetic diversity in a threatened alpine medicinal plant, Gentiana crassicaulis (Gentianaceae) using AFLP

Fen-fen ZONG¹, Zhi-li ZHAO¹^,^*, Liang-hong NI¹, Dorje GAAWE², Tong-hua LIU²^,³

Affiliations

1. Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

2. Tibetan Traditional Medical College, Lhasa 850000, China

3. Beijing University of Chinese Medicine, Beijing 100029, China

出版时间: 2020-08-12 doi: 10.16438/j.0513-4870.2020-0246

文章导航

摘要

收起

藏药“解吉那保”（རྒྱ་རྒྱུད་ནག་པོ་），能消肿，治白喉等。基于民族药物学考察、自然分布区广泛取样（6省区20个居群163株个体），结合模式标本查阅与形态学比较，将其主流品种之一鉴定为龙胆科龙胆属Gentiana粗茎秦艽Gentiana crassicaulis。粗茎秦艽，我国特有物种，仅在西藏、云南、四川、贵州、青海及甘肃等省区有分布，多见于海拔2 100~4 500 m的高山草甸、林下及林缘。应用AFLP技术，以龙胆科花锚属椭圆叶花锚Halenia elliptica及龙胆属近缘种麻花艽G.straminea为外类群，构建DNA指纹谱。从64对引物组合中筛选出12对，共扩增出315个条带，其中多态性条带254条，占比为80.63%。粗茎秦艽种内遗传多样性丰富，遗传变异主要存在于居群间（87%），居群内部遗传变异较小（13%）。非加权组平均法（unweighted pair-group method with arithmetic means，UPGMA）聚类树物种间拓扑结构与经典分类学观点相一致；物种内分为两大支：支I由西藏、甘肃、青海及四川各居群组成，支Ⅱ由贵州及云南各居群组成。PCA分析及Mantel检验等均表明粗茎秦艽居群间遗传距离与空间地理距离具有显著的正相关关系。同时，结合SSR与SNP标记，探讨了四川康定县居群S1的分化问题。本工作可为藏药“解吉那保”种质资源保护、药材生药学鉴定及道地性探究等提供基础资料。

关键词

藏药 / 解吉那保 / 粗茎秦艽 / 扩增片段长度多态性 / DNA指纹谱

Abstract

收起

Jieji Nabao is a common Tibetan herb. According to our ethnobotanical studies, one of its original plants is identified as Gentiana crassicaulis Duthie ex Burk. (Gentianaceae). Endemic to the Qinghai-Tibet Plateau, this medicinal alpine plant is a threatened species. In this study, 163 individuals from 20 populations of G. crassicaulis were collected throughout its geographical range and amplified fragment length polymorphism (AFLP) was used to investigate genetic variation in this species. A cluster analysis was performed on the AFLP data with Halenia elliptica and Gentiana straminea as the outgroups. From 64 pairs of AFLP primer combinations, 12 pairs were selected for amplification and a total of 315 bands were amplified, of which 254 bands were polymorphic, accounting for 80.63%. High genetic differentiation was detected between populations (87%), and low within populations (13%). The UPGMA (unweighted pair-group method with arithmetic means) tree was topologically consistent with the traditional taxonomic treatments at the species level, and the populations of G. crassicaulis were divided into two branches:one from Yunnan and Guizhou, the other from Tibet, Qinghai, Sichuan and Gansu. PCA analysis and the Mantel test showed that there was a positive correlation between genetic distance and geographical distance. In addition, combined with SSR and SNP markers within cpDNA, the genetic differentiation within the Sichuan population S1 was validated.

Key words

Tibetan herb / Jieji Nabao / Gentiana crassicaulis / amplified fragment length polymorphism / DNA fingerprinting

引用本文

宗粉粉, 赵志礼, 倪梁红, 嘎务, 刘铜华. 藏药解吉那保基原植物粗茎秦艽品种鉴定及种内遗传多样性分析. 药学学报, 2020 , 55 (8) : 1941 -1950 . DOI: 10.16438/j.0513-4870.2020-0246

Fen-fen ZONG, Zhi-li ZHAO, Liang-hong NI, Dorje GAAWE, Tong-hua LIU. DNA fingerprinting and genetic diversity in a threatened alpine medicinal plant, Gentiana crassicaulis (Gentianaceae) using AFLP[J]. Acta Pharmaceutica Sinica, 2020 , 55 (8) : 1941 -1950 . DOI: 10.16438/j.0513-4870.2020-0246

正文

收起

青藏高原复杂的地质历史、多样化的生境及气候条件, 蕴育出丰富的动植物资源, 是研究物种分化和物种形成机制的理想地区; 同时, 高山植物常具有独特的次生产物结构类型及生物活性特质, 为寻找安全、高效的临床药物提供了更为广阔的研究空间。

龙胆科龙胆属Gentiana秦艽组Sect. Cruciata植物多分布于我国青藏高原^[1], 物种及遗传背景丰富多样。该组多个物种常作为中药秦艽及“解吉那保” (རྒྱ་རྒྱུད་ནག་པོ་)等藏药材的基原植物, 具有重要的药用价值^{[2, 3]}。

藏医药学是祖国民族医药学的重要组成部分, 具有悠久的发展历史及丰富的科学与藏族文化内涵。深入开展藏药品种整理, 探讨高山植物物种遗传多样性及藏药材品质与具体产地的相关性等问题, 可为藏药材客观、准确的品种鉴定、活性物质基础研究、质量标准的完善、濒危藏药植物的就地保护及栽培学研究等工作打下坚实基础, 具有重要的科学意义。但由于种种原因, 藏药品种整理等工作尚不能适应藏医药学发展的需要, 当务之急是开展相关深入的基础性研究工作。藏药“解吉那保”, 花、根入药, 能消肿, 治白喉等^{[3, 4]}。课题组前期与西藏藏医药大学合作开展了相关民族药物学考察、基原植物标本采集、物种分类学鉴定及遗传背景初步分析等工作^[5-10]。

粗茎秦艽Gentiana crassicaulis Duthie ex Burk., “解吉那保”主流品种之一, 也是中药秦艽基原植物之一。我国特有物种, 自然分布区涉及西藏、云南、四川、贵州、青海及甘肃等省区, 多见于海拔2 100~4 500 m的高山草甸、林下及林缘^[1]。

近年来, 野生资源状况不容乐观, 粗茎秦艽G. crassicaulis Duthie ex Burk.及其秦艽组近缘物种麻花艽G. straminea Maxim.、小秦艽G. dahurica Fisch.及大叶秦艽G. macrophylla Pall.均被列入《国家重点保护野生药材物种名录》^[11]。药用植物种质资源是中药生产的源头, 也是中药可持续利用的物质基础。对粗茎秦艽及其近缘药用种类开展深入的遗传背景分析, 进而开展种质资源评价与就地保护工作, 紧迫性不言而喻。

扩增片段长度多态性(amplified fragment length polymorphism, AFLP)是一种新的分子标记技术, 通过对基因组DNA的限制性酶切片段进行选择性扩增而揭示其多态性, 具有灵敏、稳定、多态性丰富等优点^[12]。近年来, 该技术在中药种质资源的遗传多样性研究等领域得到广泛应用, 如对枸杞属Lycium^[13]、滇重楼Paris polyphylla var. yunnanensis^[14]、短柄乌头Aconitum brachypodum^[15]及三叶木通Akebia trifoliata^[16]等药用植物的遗传多样性分析等。为此, 在品种整理、自然分布区广泛取样及分类学鉴定基础上, 采用AFLP技术, 对163份粗茎秦艽种质样本遗传背景进行深入分析, 构建DNA指纹谱; 以期为高山药用植物粗茎秦艽核心种质构建、深入开展藏药“解吉那保”及秦艽类品种的种质资源保护、药材生药学鉴定及道地性研究等提供基础资料。

材料与方法

收起

民族植物学考察及分类学鉴定 自2010年起与西藏藏医药大学合作开展藏药“解吉那保”主流品种整理工作, 在西藏及云南等6省区粗茎秦艽G. crassicaulis自然分布区广泛采样, 共计20个居群, 163份种质样本(图 1和表 1); 同时, 取新鲜叶片硅胶快速干燥, 备用。采集外类群标本, 麻花艽G. straminea 2份、椭圆叶花锚Halenia elliptica 2份。去英国皇家植物园(Kew)植物标本馆查阅粗茎秦艽G. crassicaulis及其近缘物种的模式标本, 以确保物种各居群样本分类学鉴定的准确性。凭证标本存放于上海中医药大学中药学院药用植物标本室。

总DNA提取 取适量干燥叶片, 加入液氮研磨成细粉状, 参考Doyle的CTAB法^[17]并适当改良提取粗茎秦艽基因组DNA。

接头和引物 参考Vos等^[18]方法合成AFLP接头和引物。EcoRⅠ接头1: 5′-CTCGTAGACTGCGCGTA CC-3′, 接头2: 5′-AATTGGTACGCAGTCTAC-3′; MseⅠ接头1: 5′-GACGATGAGTCCTGAG-3′, 接头2: 5′-TAC TCAGGACTCAT-3′。预扩增引物: EcoRI+A: 5′-GAC TGCGTACCAATTCA-3′; MseI+C: 5′-GATGAGTCCT GAGTAAC-3′。从64对引物中筛选出12对带型分布均匀、多态性高且分辨力强的引物进行AFLP分析。

酶切和连接 酶切、连接一步进行, 反应体系:模板DNA 500 ng, 10×Cut Smart Buffer 3 μL, EcoRⅠ-HF 0.5 μL, MseⅠ 0.5 μL, 10×T4 DNA Ligase Buffer 3 μL, T4 DNA Ligase (350 U·μL^-1) 0.1 μL, 5 μmol·L^-1EcoRI双链接头2 μL, 50 μmol·L^-1 MseⅠ双链接头1 μL, ddH₂O补充至30 μL, 混匀。反应程序: 37 ℃, 4 h; 65 ℃, 20 min。酶切-连接产物稀释10倍进行预扩增。

预扩增 体系:10×PCR Buffer 2 μL, MgCl₂(25 mmol·L^-1)1.2 μL, dNTP mixture (10 mmol·L^-1)0.4 μL, 引物EA (10 μmol·L^-1)1 μL, 引物MC (10 μmol·L^-1)1 μL, Taq DNA聚合酶0.4 μL, 酶连产物10倍稀释液2 μL, ddH₂O补充至20 μL, 离心混匀。循环参数:95℃, 3 min; 94℃, 30 s, 56℃, 45 s, 72℃, 80 s, 30 cycles; 72℃, 8 min。预扩增产物稀释20倍作为选择性扩增模板。

选择性扩增 体系:10×PCR Buffer 3 μL, MgCl₂(25 mmol·L^-1)1.8 μL, dNTP mixture (10 mmol·L^-1)0.6 μL, 引物1(10 μmol·L^-1)1 μL, 引物2(10 μmol·L^-1)1 μL, Taq DNA聚合酶0.4 μL, 预扩产物20倍稀释液2 μL, ddH₂O补充至30 μL, 离心混匀。循环参数:95℃, 5 min; 94℃, 30 s, 65℃(每循环降低0.7℃), 30 s, 72℃, 60 s, 12 cycles; 94℃, 30 s, 56℃, 45 s, 72℃, 80 s, 23 cycles; 72℃, 5 min。

变性聚丙烯酰胺凝胶电泳 上样, 10%变性聚丙烯酰胺凝胶电泳。银染法染色, 拍照。样品1~167按顺序点样(三块胶); 同时为尽量减少由于样品处于不同胶板造成的读带误差, 粗茎秦艽每个居群均再取前3份样品平行点于另一块胶板中, 以做读带参照(图 2)。

数据分析 在相同迁移率位置, 每个样品的扩增带按有(记为“1”)或无(记为“0”)记录, 只记录清晰易辨条带, 由此得到“0, 1”矩阵。使用POPGENE 32软件计算相关遗传参数; 同时使用NTSYS pc-V.2.10e进行UPGMA聚类分析, 以检验居群间和个体间的亲缘关系。利用GenAlEx 6.5软件进行PCA分析、分子方差分析(AMOVA)、Mantel检验以及空间自相关分析。

cpDNA SSR及SNP标记分析 为探讨四川康定县居群S1的分化问题, 测定cpDNA psbI-trnS (GCU)、trnS (GCU)-trnG (UCC)、rpoB-trnC (GCA)及ycf3 intron1序列, 以筛选SSR标记; 测定cpDNA ccsA、petA-psbJ、rpl16 intron及rpl33序列, 以筛选SNP标记。

结果

收起

1 “解吉那保”基原植物形态特征及分类学位置

多年生草本。茎基部被枯存的纤维状叶鞘包裹。基生叶莲座丛状; 茎生叶对生, 最上部叶密集呈苞叶状包被花序。花多数, 茎顶簇生呈头状; 花冠冠檐蓝紫色, 长2~2.2 cm。我国特有物种。分布于西藏、云南、四川、贵州、青海及甘肃等省区。生于山坡草地、高山草甸、灌丛等, 海拔2 100~4 500 m。形态特征及后选模式标本(Lectotype, Pratt 463; K!)见图 3。

经查阅模式标本、参考相关分类学文献并进行分类学鉴定, 其基原为龙胆科龙胆属Gentiana秦艽组Sect. Cruciata植物粗茎秦艽Gentiana crassicaulis Duthie ex Burk.。据此, 对实验中所涉及的各居群标本逐一鉴定, 以确保物种遗传背景分析结果的科学性。

2 AFLP扩增结果

从64对“3+3”引物组合中筛选出扩增带纹清晰, 多态性丰富且多态性位点百分率较高的引物组合12对, 对样品进行扩增。扩增片段多在50~500 bp之间, 共获得315个位点, 其中多态性位点(PL) 254个, 多态位点百分率(PPL)平均值为80.63%, 表明粗茎秦艽种质资源的遗传背景存在一定多样性。每对引物扩增情况见表 2, 其中MseⅠ+CAG/EcoRⅠ+ACG扩增图谱见图 2。

3 粗茎秦艽遗传多样性以及居群的遗传分化

对检测群体遗传多样性指标多态位点百分率(PPL)、观察等位基因数(N_a)、有效等位基因数(N_e)、Nei's基因多样性(H)及Shannon信息指数(I_m)进行统计, 结果见表 3。粗茎秦艽居群PPL变化区间为0.95%~19.37%, 平均百分比为6.85%; N_a变化区间为1.009 5~1.193 7, 平均为1.068 7; N_e变化区间为1.005 4~1.126 2, 平均为1.039 9; H变化区间为0.0038~0.073 6, 平均为0.023 3; I_m变化区间为0.005 2~0.109 2, 平均为0.035 0。物种水平的N_a则为1.727 0, N_e为1.315 0, H为0.189 9, I_m为0.292 7。显示粗茎秦艽物种不同居群间遗传多样性有差异, 其中Y4居群遗传多样性水平最高, Nei's基因多样性和Shannon信息指数最大, 分别为0.073 6和0.109 2。

POPGENE分析软件显示:粗茎秦艽居群总变异系数(H_t: total genetic diversity for species)为0.185 3, 群体内的遗传多样性(H_s: genetic diversity within populations)为0.023 3, 群体间的遗传多样性(D_st: inter-subpopulation gene diversity)为0.162 0, 遗传分化系数(G_st: coefficient of gene differentiation)为0.874 3, 基因流(N_m: gene flow)为0.071 9。居群间遗传分化远高于居群内, 遗传变异主要存在于居群间(87%), 居群内部的遗传变异较小(13%)。

GenAlEx 6.5软件AMOVA分析时, 居群间的遗传分化系数一般用分化指数F_st表示, 它通过等位基因频率计算得到, 但其需要居群处于Hardy-Weinberg平衡时才有意义^{[19, 20]}。由于AFLP为显性标记, 不能满足此条件, 通常以AMOVA分析计算的F_st相似数Φ_st (即表 4中的PhiPT值)来代替, 而G_st是POPGENE1.32软件在假定Hardy-Weinberg平衡情形下估算的居群间的遗传分化值。因此, G_st与Φ_st都是F_st的近似值^[21]。由此, G_st和Φ_st分别为0.874 3和0.866, 二者非常接近, AMOVA分析和POPGENE分析软件的相关推断相吻合, 见表 4。遗传分化指数F_st在0~0.05之间表示群体间无分化; 0.05~0.15间为中度分化; 0.15~0.25间为高度分化; 大于0.25表示极高的分化^[22]。显然, 粗茎秦艽不同居群间遗传分化高, 遗传差异显著性极大。

4 聚类分析

4.1 居群聚类分析

采用非加权组平均法(unweighted pair-group method with arithmetic means, UPGMA)进行聚类分析, 结果显示:首先外类群龙胆科花锚属椭圆叶花锚Halenia elliptica及龙胆属近缘种麻花艽G. straminea被依次分出; 而粗茎秦艽G. crassicaulis所有20个居群共聚为另一大支。粗茎秦艽物种内, 则分为I、II两支:支I由西藏、甘肃、青海及四川各居群组成, 支II由贵州及云南各居群组成; 同时, 除云南Y4居群(栽培)外, 相同省区内各居群均分别聚为一支, 显现出高度的地域相关性, 即相同居群个体或空间距离较近的居群多显示出更为接近的亲缘关系(图 4)。

4.2 个体聚类分析

基于粗茎秦艽物种163份种质构建UPGMA聚类树。拓扑结构基本一致:首先, 外类群分出; 物种内分为两大支, 西藏、甘肃、青海及四川大部分个体(S2居群全部个体及由S1居群7株个体组成的S1b)构成支I; 值得注意的是, 支II除了包括贵州及云南各居群个体, 四川少数个体(S1居群3个组成的S1a)也在此聚类(图 5)。

5 PCA分析

主成分分析(PCA)可直观反映种质之间的遗传及分类学特性。GenAlEx 6.5软件^[23-25]分析显示:前两个坐标分别占总遗传变异的40.46%和11.57%;总体结果与UPGMA聚类树拓扑结构基本一致, 均表现出明显的地理相关性。外类群椭圆叶花锚H. elliptica及麻花艽G. straminea (M, H)在第Ⅲ象限。四川居群S1共有10株个体, 除了其中3个外, 甘肃、青海及四川其余个体均位于第Ⅳ象限, 西藏样品均位于第Ⅰ象限。贵州居群集中在第Ⅱ象限。云南居群均位于第Ⅲ象限, 其中, 2个野生居群(Y1、Y2)个体位置较为集中, 而栽培个体(Y3、Y4)较为分散; 四川居群S1另3株个体亦在此象限(图 6)。

6 Mantel检验及空间自相关分析

Mantel检验显示:粗茎秦艽居群间遗传距离与地理距离具有显著正相关关系, 相关系数R_xy=0.541, 且极其显著(P < 0.01)。随着地理距离增加, 物种内居群间遗传距离显著增大, 地理分布特性是影响粗茎秦艽种群遗传结构的重要因素之一。

空间自相关分析(spatial autocorrelation analysis), 将采样点地理距离划分为不同等级, 用空间自相关系数检验特定距离等级内各群体的遗传结构是否符合随机分布假设。若群体存在显著遗传差异, 自相关系数可以量化差异的程度^[26]。根据粗茎秦艽各居群分布, 划分100、200、300、…、1 000 km共10个距离等级, 计算不同距离等级下的多位点相关系数r^[27]。该系数值介于-1到1之间, 为0时表示不存在空间自相关^[28]。结果显示:居群间距离在小于350 km时, r > 0, 呈现显著性正相关; 在350~1 000 km时, r < 0, 呈现显著性负相关。在一定范围内, 距离越小, r值越大。提示:居群间距离对于其遗传相似度有显著影响, 邻近群体具有较高的遗传相似度(图 7)。

7 四川康定县居群S1的cpDNA SSR及SNP标记

对cpDNA psbI-trnS (GCU)、trnS (GCU)-trnG (UCC)、rpoB-trnC (GCA)及ycf3 intron1序列中SSR标记分析显示: S1a 3份种质内与S1b 7份种质内位点分别保持一致, 两者只有1个位点相同, 差异较为明显; 而S1b和居群S2有3个位点相同, 显示了更为接近的亲缘关系(表 5)。cpDNA ccsA、petA-psbJ、rpl16 intron及rpl33序列中SNP标记分析显示: S1a 3份种质内与S1b 7份种质内位点分别保持一致, 两者存在4个位点变异, 而S1b和居群S2则无差异; SSR与SNP标记均支持AFLP指纹谱分析结果(表 6)。

讨论

收起

国产特有物种粗茎秦艽G. crassicaulis, 自然分布区涉及范围广, 各居群个体生长小环境复杂多样; 植株高矮, 基生叶的较宽或较窄, 花色(蓝色、灰色及乳白色等)等亦有变化。如何把握种内个体形态特征的变异幅度, 准确鉴定不同产地实验样本, 是构建物种AFLP指纹谱关键的第一步。课题组在多年野外考察、分布区广泛取样(6省区20个居群163株个体)基础上, 结合模式标本查阅与形态学观察与比较, 进而开展物种鉴定; 同时, 以龙胆科花锚属椭圆叶花锚H. elliptica及龙胆属近缘种麻花艽G. straminea为外类群, 用于系统学分析等, 结果显示, 首先外类群被依次分出; 而粗茎秦艽物种所有20个居群共聚为另一大支, 鉴定工作达到预期目标。

道地药材是传统公认且来源于特定产地的名优正品药材, 为进一步揭示中药材道地性的科学内涵, 有许多工作要做^[29]。内生菌与植物的互作关系及对宿主代谢的影响是近年来热点领域之一, 其在探讨药用植物道地性成因方面, 拓展了新的思路^[30]。本工作基于筛选的12对引物组合, 构建的AFLP指纹谱, 在一定程度上反映出粗茎秦艽物种的遗传特性; 亦显示了AFLP分子标记技术在解析物种内不同个体遗传背景与产地空间地理相关性等方面的优势。因此, AFLP分子标记结合多学科新方法开展相关工作, 将深化对中药材道地性的认知。

藏药材传统生产方式多为就地采集野生资源加以利用。“解吉那保”主流品种之一粗茎秦艽分布于西藏、云南、四川、贵州、青海及甘肃等省区, 自然分布区内不同产地(居群) AFLP指纹谱各有特性, 其次生产物是否亦有一定差异, 对药材质量是否会产生影响, 值得进一步探讨。

有研究表明^[31], 青藏高原特有种菊叶红景天Rhodiola chrysanthemifolia (Crassulaceae)遗传变异主要存在于居群间(77.3%); 造成居群间高度分化的主要因素可能是在恶劣的山地环境中种群的历史、地理和遗传隔离。本研究工作显示, 粗茎秦艽种内存在丰富的遗传多样性, 且遗传变异亦主要存在于居群间。遗传多样性丰富可能与物种生长环境复杂, 基因流受阻以及长期的自然选择有关。同时, Mantel检验表明粗茎秦艽居群间遗传距离与地理距离具有显著的正相关关系; 有关高山植物紫花针茅Stipa purpurea的研究^[32]也发现了同样的规律。

在个体聚类与PCA等分析中注意到:来自四川康定县的居群S1, 其10个种质被分为S1a、S1b两类, 比例为3:7。S1a显示了与S1b及四川道孚县居群S2较远的亲缘关系。此状况, 在指纹谱中亦有反映(图 2, 泳道80~89)。为此, 尝试结合序列分子标记探究其原因。获得的叶绿体基因组SSR及SNP标记均支持AFLP指纹谱分析结果(表 5、6)。

聚类分析显示, 粗茎秦艽在自然分布区内种群分化为两大支, 一支由西藏、甘肃、青海及四川各居群组成, 另一支由贵州及云南各居群组成。这是否暗含物种起源、迁移路线与分化的规律性？值得应用植物群体遗传学、谱系地理学以及传粉生物学等多学科的证据来深入探究。同时, AFLP指纹技术显现了在种下居群甚至个体遗传特性间的分辨率, 相信结合居群次生产物指纹分析等, 将在药材道地性探究及系统的种质资源保护等方面有更为广阔的应用前景。

作者贡献:宗粉粉是本文的第一作者, 负责分子生物学实验及稿件撰写; 倪梁红负责样品采集工作; 嘎务负责民族植物学考察; 刘铜华参与研究工作总体设计; 赵志礼为本文的通讯作者, 负责样品分类学鉴定、研究工作总体设计及稿件修改等工作。

利益冲突:本文的研究内容无任何利益冲突。

基金

收起

国家自然科学基金面上项目(81173654)
国家自然科学基金青年科学基金项目(81503354)
藏医药区域协同创新中心项目(2018XTCX005)

参考文献

收起

文献

收起

参考文献引证文献

排序方式：

[1]

. Gentianaceae in Flora Reipublicae Popularis Sinicae:Vol 62(中国植物志:62卷)[M]. Beijing: Science Press, 1988: 59-75.

[2]

Chinese Pharmacopoeia Commission.Pharmacopoeia of the People's Republic of China: Vol I (中华人民共和国药典: 一部)[S]. Beijing: China Medical Science Press, 2015: 270-271.

[3]

Yang

. Tibetan Medicine (藏药志)[M]. Xining: Qinghai People Press, 1991: 11-12.

[4]

Dimaer

Danzeng Pengcuo

. Jing Zhu Ben Cao (晶珠本草)[M]. Shanghai: Shanghai Science & Technology Press, 1986: 119.

[5]

Zhao

, Dorje

Gaawe

, Wang

. Identification of medicinal plants used as Tibetan Traditional Medicine Jie-Ji[J]. J Ethnopharmacol, 2010, 132: 122-126.

https://doi.org/10.1016/j.jep.2010.07.051

[6]

, Zhao

. A morphometric comparison of three closely related species of Gentiana (Gentianaceae), endemic to the region of the Qinghai-Tibet Plateau[J]. Botany, 2018, 96: 209-215.

https://doi.org/10.1139/cjb-2017-0166

[7]

, Zhao

, Xu

et al . Chloroplast genome structures in Gentiana (Gentianaceae), based on three medicinal alpine plants used in Tibetan herbal medicine[J]. Curr Genet, 2017, 63: 241-252.

https://doi.org/10.1007/s00294-016-0631-1

[8]

, Zhao

, Xiong

et al . A strategy for identifying six species of Sect. Cruciata (Gentiana) in Gansu using DNA barcode sequences[J]. Acta Pharm Sin (药学学报), 2016, 51: 821-827. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=yxxb201605022

[9]

Wei

, Zhao

, Ni

et al . Taxonomic identification of Tibetan herb Bawo Sebo and its chloroplast genome structure[J]. Acta Pharm Sin (药学学报), 2018, 53: 1009-1015. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=yxxb201806023

[10]

, Zhao

, Ni

et al . The identification of Sect. Cruciata (Gentiana) species using mtDNA nad1/b-c and nad5/d-e fragments[J]. Acta Pharm Sin (药学学报), 2019, 54: 166-172. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=yxxb201901025

[11]

Zhou

, Xu

, Shun

. Resource Science of Chinese Medicinal Materials (中药资源学)[M]. Shanghai: Shanghai Science and Technology Literature Press, 2007: 370.

[12]

Arif

, Bakir

, Khan

et al . A brief review of molecular techniques to assess plant diversity[J]. Int J Mol Sci, 2010, 11: 2079-2096.

https://doi.org/10.3390/ijms11052079

[13]

, Fan

, Dai

et al . Analysis of genetic diversity for wolfberry germplasms by AFLP technology[J]. Chin Tradit Herb Drugs (中草药), 2011, 42: 770-773. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zcy201104037

[14]

, Wang

, He

et al . AFLP analysis on genetic diversity of P. polyphylla var. yunnanensis[J]. Bot Res (植物学研究), 2018, 7: 45-53. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=HANS201803200220

[15]

, Pu

, Liu

et al . AFLP analysis of genetic diversity of Aconitum brachypodum[J]. Chin Tradit Herb Drugs (中草药), 2018, 49: 439-443. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=zcy201802026

[16]

Zhang

, Jian

, Wang

. AFLP analysis on genetic diversity of Akebia trifoliata in Qinling Mountains of Shaanxi[J]. Chin Tradit Herb Drugs (中草药), 2016, 47: 3890-3895.

[17]

Doyle

, Doyle

. A rapid DNA isolation procedure for small quantities of fresh leaf material[J]. Phyochem Bull, 1987, 19: 11-15. https://www.researchgate.net/publication/245035737_A_Rapid_DNA_Isolation_Procedure_from_Small_Quantities_of_Fresh_Leaf_Tissues

[18]

Vos

, Hogers

, Bleeker

et al . AFLP:a new technique for DNA fingerprinting[J]. Nucleic Acids Res, 1995, 23: 4407-4414.

https://doi.org/10.1093/nar/23.21.4407

[19]

Lynch

, Milligan

. Analysis of population genetic structure with RAPD markers[J]. Mol Ecol, 1994, 3: 91-99.

https://doi.org/10.1111/j.1365-294X.1994.tb00109.x

[20]

Palacios

, González-Candelas

. Analysis of population genetic structure and variability using RAPD markers in the endemic and endangered Limonium duforii (Plumbaginaceae)[J]. Mol Ecol, 1997, 6: 1107-1121.

https://doi.org/10.1046/j.1365-294X.1997.00283.x

[21]

Zhang

, Yang

. A statistical and comparative analysis of genetic diversity detected by different molecular markers[J]. Acta Bot Yunnan (云南植物研究), 2008, 30: 159-167. http://en.cnki.com.cn/Article_en/CJFDTOTAL-YOKE200802005.htm

[22]

Wright

. Evolution in mendelian populations[J]. Genetics, 1931, 16: 97-159. https://link.springer.com/article/10.1007/BF02459575

[23]

Peakall

, Smouse

. GenAlEx 6.5:genetic analysis in Excel. Population genetic software for teaching and research-an update[J]. Bioinformatics, 2012, 28: 2537-2539.

https://doi.org/10.1093/bioinformatics/bts460

[24]

Peakall

, Smouse

. GENALEX 6:genetic analysis in Excel. Population genetic software for teaching and research[J]. Mol Ecol Notes, 2010, 6: 288-295. https://academic.oup.com/bioinformatics/article/28/19/2537/288671

[25]

. Introduction to a population genetic software-GENALEX 6[J]. Chin Wind Plant Resour (中国野生植物资源), 2008, 27: 59-62. http://en.cnki.com.cn/Article_en/CJFDTOTAL-ZYSZ200804018.htm

[26]

Wen B. Y Chromosome, mtDNA Polymorphisms and Genetic Structure of East Asian Population (Y染色体、mtDNA多态性与东亚人群的遗传结构)[D]. Shanghai: Fudan University, 2003.

[27]

Smouse

, Peakall

. Spatial autocorrelation analysis of individual multiallele and multilocus genetic structure[J]. Heredity, 1999, 82: 561-573.

https://doi.org/10.1038/sj.hdy.6885180

[28]

Yang

, Zhang

, Tian

et al . Microsatellite genetic diversity and fine-scale spatial genetic structure within a natural stand of Liriodendron chinense (Magnoliaceae) in Lanmushan, Duyun City, Guizhou Province[J]. Biodiv Sci (生物多样性), 2014, 22: 375-384.

https://doi.org/10.3724/SP.J.1003.2014.14013

[29]

Xiao

, Chen

, Huang

et al . Survey of investigation on Daodi Chinese medicinal materials in China since 1980s[J]. China J Chin Mater Med (中国中药杂志), 2009, 34: 519-523.

[30]

Cui

, Guo

, Xiao

. Interaction between endophytes and host plant and the role of endophytes in genuineness analysis of medicinal plant[J]. Acta Pharm Sin (药学学报), 2017, 52: 214-221. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=yxxb201702005

[31]

Xia

, Chen

et al . ISSR analysis of genetic diversity of the Qinghai-Tibet Plateau endemic Rhodiola chrysanthemifolia (Crassulaceae)[J]. Biochem System Ecol, 2007, 35: 209-214.

https://doi.org/10.1016/j.bse.2006.09.016

[32]

Liu

, Dong

, Song

et al . Genetic diversity pattern of Stipa purpurea populations in the hinterland of Qinghai-Tibet Plateau[J]. Ann Appl Biol, 2009, 154: 57-65.

https://doi.org/10.1111/j.1744-7348.2008.00274.x

2020年第55卷第8期

PDF下载

145

引用本文

BibTeX

文章信息

doi: 10.16438/j.0513-4870.2020-0246

接收时间：2020-03-06
首发时间：2026-01-21
出版时间：2020-08-12

补充材料

相关文章

文章信息

作者

出版历史

收稿日期：2020-03-06
修回日期：2020-03-30

基金

国家自然科学基金面上项目(81173654)

国家自然科学基金青年科学基金项目(81503354)

藏医药区域协同创新中心项目(2018XTCX005)

作者信息

1.上海中医药大学, 上海 201203

2.西藏藏医药大学, 西藏拉萨 850000

3.北京中医药大学, 北京 100029

通讯作者:

*赵志礼, Tel:86-21-51322202, E-mail:zhilzhao@sohu.com

参考文献

分享链接

https://castjournals.cast.org.cn/joweb/yxxb/CN/10.16438/j.0513-4870.2020-0246

分享至

全文二维码

扫描看全文

引用本文

BibTeX

本文的引用情况

2种不同金属材料的力学参数

科 Family	属数 Number of genus	种数 Number of species	占总种数比例 Percentage of total species (%)	属 Genus	种数 Number of species	占总种数比例 Percentage of total species (%)
鹅膏菌科Amanitaceae	2	11	5.26	鹅膏菌属 Amanita	10	4.78
小菇科 Mycenaceae	2	12	5.74	丝盖伞属 Inocybe	5	2.39
多孔菌科 Polyporaceae	8	14	6.70	蜡蘑属 Laccaria	5	2.39
红菇科 Russulaceae	3	23	11.00	小皮伞属 Marasmius	6	2.87
				小菇属 Mycena	11	5.26
				光柄菇属 Pluteus	5	2.39
				红菇属 Russula	17	8.13
				栓菌属 Trametes	5	2.39

关闭全屏

BibTeX
EndNote
RefWorks
TxT

Pop. ID	Sample No.	Voucher specimens	Locality
T1	1	Ni L.H.et al XZ201214-4	Dingqing, Tibet
2	Ni L.H.et al XZ201214-5	Dingqing, Tibet
3	Ni L.H.et al XZ201214-6	Dingqing, Tibet
T2	4	Yang Y.H. 2012XZ014-A	Dingqing, Tibet
5	Yang Y.H. 2012XZ014-B	Dingqing, Tibet
6	Yang Y.H. 2012XZ014-C	Dingqing, Tibet
K1	7	Ni L.H.et al GS201301-1	Maqu, Gansu
8	Ni L.H.et al GS201301-2	Maqu, Gansu
9	Ni L.H.et al GS201301-3	Maqu, Gansu
10	Ni L.H.et al GS201301-4	Maqu, Gansu
11	Ni L.H.et al GS201301-5	Maqu, Gansu
K2	12	Ni L.H. GS201601-1	Maqu, Gansu
13	Ni L.H. GS201601-2	Maqu, Gansu
14	Ni L.H. GS201601-3	Maqu, Gansu
15	Ni L.H. GS201601-4	Maqu, Gansu
16	Ni L.H. GS201601-5	Maqu, Gansu
17	Ni L.H. GS201601-6	Maqu, Gansu
18	Ni L.H. GS201601-7	Maqu, Gansu
19	Ni L.H. GS201601-8	Maqu, Gansu
20	Ni L.H. GS201601-9	Maqu, Gansu
21	Ni L.H. GS201601-10	Maqu, Gansu
22	Ni L.H. GS201601-11	Maqu, Gansu
23	Ni L.H. GS201601-12	Maqu, Gansu
24	Ni L.H. GS201601-13	Maqu, Gansu
25	Ni L.H. GS201601-14	Maqu, Gansu
26	Ni L.H. GS201601-15	Maqu, Gansu
G1	27	Ni L.H.et al GZ201601-1	Weining, Guizhou
28	Ni L.H.et al GZ201601-2	Weining, Guizhou
29	Ni L.H.et al GZ201601-3	Weining, Guizhou
30	Ni L.H.et al GZ201601-4	Weining, Guizhou
31	Ni L.H.et al GZ201601-5	Weining, Guizhou
32	Ni L.H.et al GZ201601-6	Weining, Guizhou
33	Ni L.H.et al GZ201601-7	Weining, Guizhou
34	Ni L.H.et al GZ201601-8	Weining, Guizhou
35	Ni L.H.et al GZ201601-9	Weining, Guizhou
36	Ni L.H.et al GZ201601-10	Weining, Guizhou
37	Ni L.H.et al GZ201601-11	Weining, Guizhou
38	Ni L.H.et al GZ201601-12	Weining, Guizhou
39	Ni L.H.et al GZ201601-13	Weining, Guizhou
40	Ni L.H.et al GZ201601-14	Weining, Guizhou
41	Ni L.H.et al GZ201601-15	Weining, Guizhou
G2	42	Ni L.H.et al GZ201602-1	Weining, Guizhou
43	Ni L.H.et al GZ201602-2	Weining, Guizhou
44	Ni L.H.et al GZ201602-3	Weining, Guizhou
45	Ni L.H.et al GZ201602-4	Weining, Guizhou
46	Ni L.H.et al GZ201602-5	Weining, Guizhou
47	Ni L.H.et al GZ201602-6	Weining, Guizhou
48	Ni L.H.et al GZ201602-7	Weining, Guizhou
49	Ni L.H.et al GZ201602-8	Weining, Guizhou
50	Ni L.H.et al GZ201602-9	Weining, Guizhou
51	Ni L.H.et al GZ201602-10	Weining, Guizhou
52	Ni L.H.et al GZ201601-11	Weining, Guizhou
53	Ni L.H.et al GZ201602-12	Weining, Guizhou
54	Ni L.H.et al GZ201602-13	Weining, Guizhou
55	Ni L.H.et al GZ201602-14	Weining, Guizhou
56	Ni L.H.et al GZ201602-15	Weining, Guizhou
T3	57	Ni L.H.et al 2016XZ008-1	Basu, Tibet
58	Ni L.H.et al 2016XZ008-2	Basu, Tibet
59	Ni L.H.et al 2016XZ008-3	Basu, Tibet
60	Ni L.H.et al 2016XZ008-4	Basu, Tibet
61	Ni L.H.et al 2016XZ008-5	Basu, Tibet
T4	62	Ni L.H.et al 2016XZ009-1	Basu, Tibet
63	Ni L.H.et al 2016XZ009-2	Basu, Tibet
64	Ni L.H.et al 2016XZ009-3	Basu, Tibet
T5	65	Ni L.H.et al 2016XZ011-1	Changdu, Tibet
66	Ni L.H.et al 2016XZ011-2	Changdu, Tibet
67	Ni L.H.et al 2016XZ011-3	Changdu, Tibet
68	Ni L.H.et al 2016XZ011-4	Changdu, Tibet
69	Ni L.H.et al 2016XZ011-5	Changdu, Tibet
70	Ni L.H.et al 2016XZ011-6	Changdu, Tibet
71	Ni L.H.et al 2016XZ011-7	Changdu, Tibet
72	Ni L.H.et al 2016XZ011-8	Changdu, Tibet
73	Ni L.H.et al 2016XZ011-9	Changdu, Tibet
74	Ni L.H.et al 2016XZ011-10	Changdu, Tibet
75	Ni L.H.et al 2016XZ011-11	Changdu, Tibet
76	Ni L.H.et al 2016XZ011-12	Changdu, Tibet
77	Ni L.H.et al 2016XZ011-13	Changdu, Tibet
78	Ni L.H.et al 2016XZ011-14	Changdu, Tibet
79	Ni L.H.et al 2016XZ011-15	Changdu, Tibet
S1	80	Ni L.H. SC201601-1	Kangding, Sichuan
81	Ni L.H. SC201601-2	Kangding, Sichuan
82	Ni L.H. SC201601-3	Kangding, Sichuan
83	Ni L.H. SC201601-4	Kangding, Sichuan
84	Ni L.H. SC201601-5	Kangding, Sichuan
85	Ni L.H. SC201601-6	Kangding, Sichuan
86	Ni L.H. SC201601-7	Kangding, Sichuan
87	Ni L.H. SC201601-8	Kangding, Sichuan
88	Ni L.H. SC201601-9	Kangding, Sichuan
89	Ni L.H. SC201601-10	Kangding, Sichuan
S2	90	Ni L.H. SC201602-1	Daofu, Sichuan
91	Ni L.H. SC201602-2	Daofu, Sichuan
92	Ni L.H. SC201602-3	Daofu, Sichuan
93	Ni L.H. SC201602-4	Daofu, Sichuan
94	Ni L.H. SC201602-5	Daofu, Sichuan
95	Ni L.H. SC201602-6	Daofu, Sichuan
96	Ni L.H. SC201602-7	Daofu, Sichuan
97	Ni L.H. SC201602-8	Daofu, Sichuan
98	Ni L.H. SC201602-9	Daofu, Sichuan
99	Ni L.H. SC201602-10	Daofu, Sichuan
100	Ni L.H. SC201602-11	Daofu, Sichuan
101	Ni L.H. SC201602-12	Daofu, Sichuan
102	Ni L.H. SC201602-13	Daofu, Sichuan
103	Ni L.H. SC201602-14	Daofu, Sichuan
104	Ni L.H. SC201602-15	Daofu, Sichuan
Y1	105	Ni L.H.et al YN201601-1	Lijiang, Yunnan
106	Ni L.H.et al YN201601-2	Lijiang, Yunnan
107	Ni L.H.et al YN201601-3	Lijiang, Yunnan
108	Ni L.H.et al YN201601-4	Lijiang, Yunnan
109	Ni L.H.et al YN201601-5	Lijiang, Yunnan
110	Ni L.H.et al YN201601-6	Lijiang, Yunnan
111	Ni L.H.et al YN201601-7	Lijiang, Yunnan
112	Ni L.H.et al YN201601-8	Lijiang, Yunnan
113	Ni L.H.et al YN201601-9	Lijiang, Yunnan
114	Ni L.H.et al YN201601-10	Lijiang, Yunnan
115	Ni L.H.et al YN201601-11	Lijiang, Yunnan
116	Ni L.H.et al YN201601-12	Lijiang, Yunnan
117	Ni L.H.et al YN201601-13	Lijiang, Yunnan
118	Ni L.H.et al YN201601-14	Lijiang, Yunnan
119	Ni L.H.et al YN201601-15	Lijiang, Yunnan
Y2	120	Ni L.H. YN201603-1	Lijiang, Yunnan
121	Ni L.H. YN201603-2	Lijiang, Yunnan
122	Ni L.H. YN201603-3	Lijiang, Yunnan
123	Ni L.H. YN201603-4	Lijiang, Yunnan
124	Ni L.H. YN201603-5	Lijiang, Yunnan
Y3^*	125	Ni L.H. YN201301-1	Lijiang, Yunnan
126	Ni L.H. YN201301-2	Lijiang, Yunnan
127	Ni L.H. YN201301-3	Lijiang, Yunnan
128	Ni L.H. YN201301-4	Lijiang, Yunnan
Q	129	Ni L.H.et al QH201405-1	Banma, Qinghai
130	Ni L.H.et al QH201405-2	Banma, Qinghai
131	Ni L.H.et al QH201405-3	Banma, Qinghai
T6	132	Zhao Z.L.et al 2017XZ006-1	Bomi, Tibet
133	Zhao Z.L.et al 2017XZ006-2	Bomi, Tibet
134	Zhao Z.L.et al 2017XZ006-3	Bomi, Tibet
135	Zhao Z.L.et al 2017XZ006-4	Bomi, Tibet
136	Zhao Z.L.et al 2017XZ006-5	Bomi, Tibet
137	Zhao Z.L.et al 2017XZ006-6	Bomi, Tibet
138	Zhao Z.L.et al 2017XZ006-7	Bomi, Tibet
139	Zhao Z.L.et al 2017XZ006-8	Bomi, Tibet
140	Zhao Z.L.et al 2017XZ006-9	Bomi, Tibet
141	Zhao Z.L.et al 2017XZ006-10	Bomi, Tibet
T7	142	Zhao Z.L.et al 2017XZ007-1	Basu, Tibet
143	Zhao Z.L.et al 2017XZ007-2	Basu, Tibet
144	Zhao Z.L.et al 2017XZ007-3	Basu, Tibet
145	Zhao Z.L.et al 2017XZ007-4	Basu, Tibet
146	Zhao Z.L.et al 2017XZ007-5	Basu, Tibet
147	Zhao Z.L.et al 2017XZ007-6	Basu, Tibet
148	Zhao Z.L.et al 2017XZ007-7	Basu, Tibet
149	Zhao Z.L.et al 2017XZ007-8	Basu, Tibet
T8	150	Zhao Z.L.et al 2017XZ008-1	Zuogong, Tibet
151	Zhao Z.L.et al 2017XZ008-2	Zuogong, Tibet
152	Zhao Z.L.et al 2017XZ008-3	Zuogong, Tibet
153	Zhao Z.L.et al 2017XZ008-4	Zuogong, Tibet
154	Zhao Z.L.et al 2017XZ008-5	Zuogong, Tibet
155	Zhao Z.L.et al 2017XZ008-6	Zuogong, Tibet
T9	156	Zhao Z.L.et al 2017XZ009-1	Zuogong, Tibet
157	Zhao Z.L.et al 2017XZ009-2	Zuogong, Tibet
158	Zhao Z.L.et al 2017XZ009-3	Zuogong, Tibet
159	Zhao Z.L.et al 2017XZ009-4	Zuogong, Tibet
160	Zhao Z.L.et al 2017XZ009-5	Zuogong, Tibet
Y4^*	161	Yang S.H. YN201701-1	Lijiang, Yunnan
162	Yang S.H. YN201701-2	Lijiang, Yunnan
163	Yang S.H. YN201701-3	Lijiang, Yunnan
M^**	164	Zhao Z.L.et al 2017XZ005-1	Bomi, Tibet
165	Zhao Z.L.et al 2017XZ005-2	Bomi, Tibet
H^**	166	Ni L.H.et al GZ201603-1	Weining, Guizhou
167	Ni L.H.et al GZ201603-2	Weining, Guizhou

Pop.
ID

Sample
No.

Voucher specimens

Locality

Ni L.H.et al XZ201214-4

Dingqing, Tibet

Ni L.H.et al XZ201214-5

Dingqing, Tibet

Ni L.H.et al XZ201214-6

Dingqing, Tibet

Yang Y.H. 2012XZ014-A

Dingqing, Tibet

Yang Y.H. 2012XZ014-B

Dingqing, Tibet

Yang Y.H. 2012XZ014-C

Dingqing, Tibet

Ni L.H.et al GS201301-1

Maqu, Gansu

Ni L.H.et al GS201301-2

Maqu, Gansu

Ni L.H.et al GS201301-3

Maqu, Gansu

Ni L.H.et al GS201301-4

Maqu, Gansu

Ni L.H.et al GS201301-5

Maqu, Gansu

Ni L.H. GS201601-1

Maqu, Gansu

Ni L.H. GS201601-2

Maqu, Gansu

Ni L.H. GS201601-3

Maqu, Gansu

Ni L.H. GS201601-4

Maqu, Gansu

Ni L.H. GS201601-5

Maqu, Gansu

Ni L.H. GS201601-6

Maqu, Gansu

Ni L.H. GS201601-7

Maqu, Gansu

Ni L.H. GS201601-8

Maqu, Gansu

Ni L.H. GS201601-9

Maqu, Gansu

Ni L.H. GS201601-10

Maqu, Gansu

Ni L.H. GS201601-11

Maqu, Gansu

Ni L.H. GS201601-12

Maqu, Gansu

Ni L.H. GS201601-13

Maqu, Gansu

Ni L.H. GS201601-14

Maqu, Gansu

Ni L.H. GS201601-15

Maqu, Gansu

Ni L.H.et al GZ201601-1

Weining, Guizhou

Ni L.H.et al GZ201601-2

Weining, Guizhou

Ni L.H.et al GZ201601-3

Weining, Guizhou

Ni L.H.et al GZ201601-4

Weining, Guizhou

Ni L.H.et al GZ201601-5

Weining, Guizhou

Ni L.H.et al GZ201601-6

Weining, Guizhou

Ni L.H.et al GZ201601-7

Weining, Guizhou

Ni L.H.et al GZ201601-8

Weining, Guizhou

Ni L.H.et al GZ201601-9

Weining, Guizhou

Ni L.H.et al GZ201601-10

Weining, Guizhou

Ni L.H.et al GZ201601-11

Weining, Guizhou

Ni L.H.et al GZ201601-12

Weining, Guizhou

Ni L.H.et al GZ201601-13

Weining, Guizhou

Ni L.H.et al GZ201601-14

Weining, Guizhou

Ni L.H.et al GZ201601-15

Weining, Guizhou

Ni L.H.et al GZ201602-1

Weining, Guizhou

Ni L.H.et al GZ201602-2

Weining, Guizhou

Ni L.H.et al GZ201602-3

Weining, Guizhou

Ni L.H.et al GZ201602-4

Weining, Guizhou

Ni L.H.et al GZ201602-5

Weining, Guizhou

Ni L.H.et al GZ201602-6

Weining, Guizhou

Ni L.H.et al GZ201602-7

Weining, Guizhou

Ni L.H.et al GZ201602-8

Weining, Guizhou

Ni L.H.et al GZ201602-9

Weining, Guizhou

Ni L.H.et al GZ201602-10

Weining, Guizhou

Ni L.H.et al GZ201601-11

Weining, Guizhou

Ni L.H.et al GZ201602-12

Weining, Guizhou

Ni L.H.et al GZ201602-13

Weining, Guizhou

Ni L.H.et al GZ201602-14

Weining, Guizhou

Ni L.H.et al GZ201602-15

Weining, Guizhou

Ni L.H.et al 2016XZ008-1

Basu, Tibet

Ni L.H.et al 2016XZ008-2

Basu, Tibet

Ni L.H.et al 2016XZ008-3

Basu, Tibet

Ni L.H.et al 2016XZ008-4

Basu, Tibet

Ni L.H.et al 2016XZ008-5

Basu, Tibet

Ni L.H.et al 2016XZ009-1

Basu, Tibet

Ni L.H.et al 2016XZ009-2

Basu, Tibet

Ni L.H.et al 2016XZ009-3

Basu, Tibet

Ni L.H.et al 2016XZ011-1

Changdu, Tibet

Ni L.H.et al 2016XZ011-2

Changdu, Tibet

Ni L.H.et al 2016XZ011-3

Changdu, Tibet

Ni L.H.et al 2016XZ011-4

Changdu, Tibet

Ni L.H.et al 2016XZ011-5

Changdu, Tibet

Ni L.H.et al 2016XZ011-6

Changdu, Tibet

Ni L.H.et al 2016XZ011-7

Changdu, Tibet

Ni L.H.et al 2016XZ011-8

Changdu, Tibet

Ni L.H.et al 2016XZ011-9

Changdu, Tibet

Ni L.H.et al 2016XZ011-10

Changdu, Tibet

Ni L.H.et al 2016XZ011-11

Changdu, Tibet

Ni L.H.et al 2016XZ011-12

Changdu, Tibet

Ni L.H.et al 2016XZ011-13

Changdu, Tibet

Ni L.H.et al 2016XZ011-14

Changdu, Tibet

Ni L.H.et al 2016XZ011-15

Changdu, Tibet

Ni L.H. SC201601-1

Kangding, Sichuan

Ni L.H. SC201601-2

Kangding, Sichuan

Ni L.H. SC201601-3

Kangding, Sichuan

Ni L.H. SC201601-4

Kangding, Sichuan

Ni L.H. SC201601-5

Kangding, Sichuan

Ni L.H. SC201601-6

Kangding, Sichuan

Ni L.H. SC201601-7

Kangding, Sichuan

Ni L.H. SC201601-8

Kangding, Sichuan

Ni L.H. SC201601-9

Kangding, Sichuan

Ni L.H. SC201601-10

Kangding, Sichuan

Ni L.H. SC201602-1

Daofu, Sichuan

Ni L.H. SC201602-2

Daofu, Sichuan

Ni L.H. SC201602-3

Daofu, Sichuan

Ni L.H. SC201602-4

Daofu, Sichuan

Ni L.H. SC201602-5

Daofu, Sichuan

Ni L.H. SC201602-6

Daofu, Sichuan

Ni L.H. SC201602-7

Daofu, Sichuan

Ni L.H. SC201602-8

Daofu, Sichuan

Ni L.H. SC201602-9

Daofu, Sichuan

Ni L.H. SC201602-10

Daofu, Sichuan

100

Ni L.H. SC201602-11

Daofu, Sichuan

101

Ni L.H. SC201602-12

Daofu, Sichuan

102

Ni L.H. SC201602-13

Daofu, Sichuan

103

Ni L.H. SC201602-14

Daofu, Sichuan

104

Ni L.H. SC201602-15

Daofu, Sichuan

105

Ni L.H.et al YN201601-1

Lijiang, Yunnan

106

Ni L.H.et al YN201601-2

Lijiang, Yunnan

107

Ni L.H.et al YN201601-3

Lijiang, Yunnan

108

Ni L.H.et al YN201601-4

Lijiang, Yunnan

109

Ni L.H.et al YN201601-5

Lijiang, Yunnan

110

Ni L.H.et al YN201601-6

Lijiang, Yunnan

111

Ni L.H.et al YN201601-7

Lijiang, Yunnan

112

Ni L.H.et al YN201601-8

Lijiang, Yunnan

113

Ni L.H.et al YN201601-9

Lijiang, Yunnan

114

Ni L.H.et al YN201601-10

Lijiang, Yunnan

115

Ni L.H.et al YN201601-11

Lijiang, Yunnan

116

Ni L.H.et al YN201601-12

Lijiang, Yunnan

117

Ni L.H.et al YN201601-13

Lijiang, Yunnan

118

Ni L.H.et al YN201601-14

Lijiang, Yunnan

119

Ni L.H.et al YN201601-15

Lijiang, Yunnan

120

Ni L.H. YN201603-1

Lijiang, Yunnan

121

Ni L.H. YN201603-2

Lijiang, Yunnan

122

Ni L.H. YN201603-3

Lijiang, Yunnan

123

Ni L.H. YN201603-4

Lijiang, Yunnan

124

Ni L.H. YN201603-5

Lijiang, Yunnan

Y3^*

125

Ni L.H. YN201301-1

Lijiang, Yunnan

126

Ni L.H. YN201301-2

Lijiang, Yunnan

127

Ni L.H. YN201301-3

Lijiang, Yunnan

128

Ni L.H. YN201301-4

Lijiang, Yunnan

129

Ni L.H.et al QH201405-1

Banma, Qinghai

130

Ni L.H.et al QH201405-2

Banma, Qinghai

131

Ni L.H.et al QH201405-3

Banma, Qinghai

132

Zhao Z.L.et al 2017XZ006-1

Bomi, Tibet

133

Zhao Z.L.et al 2017XZ006-2

Bomi, Tibet

134

Zhao Z.L.et al 2017XZ006-3

Bomi, Tibet

135

Zhao Z.L.et al 2017XZ006-4

Bomi, Tibet

136

Zhao Z.L.et al 2017XZ006-5

Bomi, Tibet

137

Zhao Z.L.et al 2017XZ006-6

Bomi, Tibet

138

Zhao Z.L.et al 2017XZ006-7

Bomi, Tibet

139

Zhao Z.L.et al 2017XZ006-8

Bomi, Tibet

140

Zhao Z.L.et al 2017XZ006-9

Bomi, Tibet

141

Zhao Z.L.et al 2017XZ006-10

Bomi, Tibet

142

Zhao Z.L.et al 2017XZ007-1

Basu, Tibet

143

Zhao Z.L.et al 2017XZ007-2

Basu, Tibet

144

Zhao Z.L.et al 2017XZ007-3

Basu, Tibet

145

Zhao Z.L.et al 2017XZ007-4

Basu, Tibet

146

Zhao Z.L.et al 2017XZ007-5

Basu, Tibet

147

Zhao Z.L.et al 2017XZ007-6

Basu, Tibet

148

Zhao Z.L.et al 2017XZ007-7

Basu, Tibet

149

Zhao Z.L.et al 2017XZ007-8

Basu, Tibet

150

Zhao Z.L.et al 2017XZ008-1

Zuogong, Tibet

151

Zhao Z.L.et al 2017XZ008-2

Zuogong, Tibet

152

Zhao Z.L.et al 2017XZ008-3

Zuogong, Tibet

153

Zhao Z.L.et al 2017XZ008-4

Zuogong, Tibet

154

Zhao Z.L.et al 2017XZ008-5

Zuogong, Tibet

155

Zhao Z.L.et al 2017XZ008-6

Zuogong, Tibet

156

Zhao Z.L.et al 2017XZ009-1

Zuogong, Tibet

157

Zhao Z.L.et al 2017XZ009-2

Zuogong, Tibet

158

Zhao Z.L.et al 2017XZ009-3

Zuogong, Tibet

159

Zhao Z.L.et al 2017XZ009-4

Zuogong, Tibet

160

Zhao Z.L.et al 2017XZ009-5

Zuogong, Tibet

Y4^*

161

Yang S.H. YN201701-1

Lijiang, Yunnan

162

Yang S.H. YN201701-2

Lijiang, Yunnan

163

Yang S.H. YN201701-3

Lijiang, Yunnan

M^**

164

Zhao Z.L.et al 2017XZ005-1

Bomi, Tibet

165

Zhao Z.L.et al 2017XZ005-2

Bomi, Tibet

H^**

166

Ni L.H.et al GZ201603-1

Weining, Guizhou

167

Ni L.H.et al GZ201603-2

Weining, Guizhou

Primer combination	Amplified loci	PL	PPL/%		Primer combination	Amplified loci	PL	PPL/%
MseⅠ+CAG/EcoRⅠ+AAC	31	29	93.55		MseI+CTG/EcoRⅠ+ACC	41	40	97.56
MseⅠ+CAG/EcoRⅠ+ACG	26	23	88.46	MseI+CTG/EcoRⅠ+ACG	12	10	83.33
MseⅠ+CTC/EcoRⅠ+AAC	42	36	85.71	MseI+CTT/EcoRⅠ+AAG	9	6	66.67
MseⅠ+CTG/EcoRⅠ+AAC	36	28	77.78	MseI+CTT/EcoRⅠ+ACA	18	12	66.67
MseⅠ+CTG/EcoRⅠ+ACA	19	12	63.16	MseI+CTT/EcoRⅠ+AAG	41	25	60.98
MseⅠ+CTG/EcoRⅠ+ACT	21	19	90.48	MseI+CTT/EcoRⅠ+ACG	19	14	73.68
Total					315	254	80.63

Primer combination

Amplified loci

PPL/%

Primer combination

Amplified loci

PPL/%

MseⅠ+CAG/EcoRⅠ+AAC

93.55

MseI+CTG/EcoRⅠ+ACC

97.56

MseⅠ+CAG/EcoRⅠ+ACG

88.46

MseI+CTG/EcoRⅠ+ACG

83.33

MseⅠ+CTC/EcoRⅠ+AAC

85.71

MseI+CTT/EcoRⅠ+AAG

66.67

MseⅠ+CTG/EcoRⅠ+AAC

77.78

MseI+CTT/EcoRⅠ+ACA

66.67

MseⅠ+CTG/EcoRⅠ+ACA

63.16

MseI+CTT/EcoRⅠ+AAG

60.98

MseⅠ+CTG/EcoRⅠ+ACT

90.48

MseI+CTT/EcoRⅠ+ACG

73.68

Total

315

254

80.63

Pop. ID	Indiv. No.	PL	PPL /%	N_a	N_e	H	I_m
T1	3	4	1.27	1.012 7	1.005 4	0.003 8	0.006 1
T2	3	3	0.95	1.009 5	1.005 7	0.003 5	0.005 2
K1	5	14	4.44	1.044 4	1.028 5	0.016 1	0.023 9
K2	15	12	3.81	1.038 1	1.019 6	0.012 3	0.018 8
G1	15	29	9.21	1.092 1	1.049 1	0.028 1	0.042 2
G2	15	37	11.75	1.117 5	1.068 8	0.038 8	0.057 9
T3	5	18	5.71	1.057 1	1.034 8	0.020 0	0.029 9
T4	3	3	0.95	1.009 5	1.005 7	0.003 5	0.005 2
T5	15	20	6.35	1.063 5	1.031 2	0.018 8	0.028 7
S1	10	50	15.87	1.158 7	1.099 8	0.056 7	0.084 2
S2	15	19	6.03	1.060 3	1.030 6	0.018 2	0.027 7
Y1	15	46	14.6	1.146 0	1.082 2	0.048 7	0.073 3
Y2	5	13	4.13	1.041 3	1.024 3	0.014 4	0.021 7
Y3	4	38	12.06	1.120 6	1.072 0	0.042 2	0.063 6
Q	3	5	1.09	1.015 9	1.006 8	0.004 8	0.007 6
T6	10	23	7.30	1.073 0	1.037 7	0.022 0	0.033 4
T7	8	7	2.22	1.022 2	1.014 9	0.008 2	0.012 0
T8	6	11	3.49	1.034 9	1.013 7	0.009 0	0.014 5
T9	5	20	6.35	1.063 5	1.041 0	0.023 2	0.034 4
Y4	3	61	19.37	1.193 7	1.126 2	0.073 6	0.109 2

Pop.
ID

Indiv.
No.

PPL
/%

N_a

N_e

I_m

1.27

1.012 7

1.005 4

0.003 8

0.006 1

0.95

1.009 5

1.005 7

0.003 5

0.005 2

4.44

1.044 4

1.028 5

0.016 1

0.023 9

3.81

1.038 1

1.019 6

0.012 3

0.018 8

9.21

1.092 1

1.049 1

0.028 1

0.042 2

11.75

1.117 5

1.068 8

0.038 8

0.057 9

5.71

1.057 1

1.034 8

0.020 0

0.029 9

0.95

1.009 5

1.005 7

0.003 5

0.005 2

6.35

1.063 5

1.031 2

0.018 8

0.028 7

15.87

1.158 7

1.099 8

0.056 7

0.084 2

6.03

1.060 3

1.030 6

0.018 2

0.027 7

14.6

1.146 0

1.082 2

0.048 7

0.073 3

4.13

1.041 3

1.024 3

0.014 4

0.021 7

12.06

1.120 6

1.072 0

0.042 2

0.063 6

1.09

1.015 9

1.006 8

0.004 8

0.007 6

7.30

1.073 0

1.037 7

0.022 0

0.033 4

2.22

1.022 2

1.014 9

0.008 2

0.012 0

3.49

1.034 9

1.013 7

0.009 0

0.014 5

6.35

1.063 5

1.041 0

0.023 2

0.034 4

19.37

1.193 7

1.126 2

0.073 6

0.109 2

Variation source	df	Est.Var.	Percentage /%	PhiPT	P value
Among population	19	27.305	87	0.866	< 0.001
Within population	143	4.232	13
Total variation	162	31.536	100

Variation source

Est.Var.

Percentage
/%

PhiPT

P value

Among population

27.305

0.866

< 0.001

Within population

143

4.232

Total variation

162

31.536

100

Pop.ID	psbI-trnS (GCU) 76/104^*	trnS(GCU)- trnG (UCC) 149/539	rpoB-trnC (GCA) 1157/1264	ycf3 intron1 1825/1984
S1a (n=3)	10A	11A	9A	10A
S1b (n=7)	9A	12A	9A	11A
S2(n=3)	9A	12A	10A	11A

Pop.ID

psbI-trnS
(GCU)
76/104^*

trnS(GCU)-
trnG (UCC)
149/539

rpoB-trnC
(GCA)
1157/1264

ycf3 intron1
1825/1984

S1a (n=3)

10A

11A

10A

S1b (n=7)

12A

11A

S2(n=3)

12A

10A

11A

Pop.ID	ccsA 978/984^*	petA-psbJ 90/840	rpl16 intron 167/1170	rpl33 176/201
S1a (n= 3)	T	C	A	C
S1b (n= 7)	G	A	G	A
S2(n= 3)	G	A	G	A

Pop.ID

ccsA
978/984^*

petA-psbJ
90/840

rpl16
intron 167/1170

rpl33
176/201

S1a (n= 3)

S1b (n= 7)

S2(n= 3)