药学学报

No.	City or district	Haplotype	Syringin/%		No.	City or district	Haplotype	Syringin/%
1	Harbin, Heilongjiang	H2	0.038 5		41	Luoyang, Henan	H2	0.062 3
2	Harbin, Heilongjiang	H8	0.426 2	42	Xinxiang, Henan	H2	0.095 2
3	Daqing, Heilongjiang	H2	0.090 1	43	Xuchang, Henan	H10	0.094 9
4	Shenyang, Liaoning	H5	0.064 0	44	Xuchang, Henan	H2	0.199 4
5	Shenyang, Liaoning	H23	0.122 5	45	Xuchang, Henan	H2	0.173 8
6	Shenyang, Liaoning	H10	0.084 7	46	Changge, Henan	H8	0.100 8
7	Shenyang, Liaoning	H2	0.094 5	47	Dingxi, Gansu	H2	0.120 3
8	Dalian, Liaoning	H2	0.009 7	48	Qingyang, Gansu	H2	0.108 3
9	Hohhot, Inner Mongolia	H23	0.411 0	49	Shizuishan, Ningxia	H2	0.165 7
10	Shijingshan District, Beijing	H12	0.524 5	50	Shizuishan, Ningxia	H2	0.152 6
11	Shijingshan District, Beijing	H8	0.178 0	51	Linyi, Ningxia	H2	0.091 3
12	Shijingshan District, Beijing	H10	0.205 6	52	Zibo, Shandong	H10	0.149 3
13	Fengtai District, Beijing	H2	0.150 8	53	Zibo, Shandong	H2	0.004 0
14	Fengtai District, Beijing	H23	0.387 9	54	Liaocheng, Shandong	H2	0.223 5
15	Fengtai District, Beijing	H2	0.462 7	55	Lu'an, Anhui	H2	0.150 2
16	Fengtai District, Beijing	H23	0.256 0	56	Fuyang, Anhui	H2	0.027 3
17	Fengtai District, Beijing	H2	0.145 6	57	Huangshan, Anhui	H14	0.119 2
18	Fangshan District, Beijing	H2	0.155 2	58	Wuhan, Hubei	H8	0.077 3
19	Fangshan District, Beijing	H8	0.256 9	59	Bishan District, Chongqing	H2	0.202 4
20	Fangshan District, Beijing	H10	0.038 3	60	Chengdu, Sichuan	H2	0.017 6
21	Fangshan District, Beijing	H10	0.277 9	61	Chengdu, Sichuan	H2	0.158 7
22	Fangshan District, Beijing	H2	0.154 3	62	Suzhou, Jiangsu	H10	0.167 7
23	Fangshan District, Beijing	H10	0.094 9	63	Nantong, Jiangsu	H2	0.239 8
24	Tongzhou District, Beijing	H2	0.087 9	64	Pudong New Area, Shanghai	H2	0.048 1
25	Dongcheng District, Beijing	H14	0.007 7	65	Wenzhou, Zhejiang	H2	0.025 5
26	Wuqing District, Tianjin	H2	0.134 7	66	Lishui, Zhejiang	H8	0.189 4
27	Cangzhou, Hebei	H2	0.103 9	67	Ningbo, Zhejiang	H10	0.052 9
28	Cangzhou, Hebei	H2	0.102 7	68	Ganzhou, Jiangxi	H8	0.016 5
29	Cangzhou, Hebei	H23	0.106 0	69	Yichun, Jiangxi	H8	0.006 8
30	Shijiazhuang, Hebei	H2	0.177 5	70	Xiamen, Fujian	H2	0.063 8
31	Baoding, Hebei	H2	0.073 1	71	Fuzhou, Fujian	H2	0.038 5
32	Baoding, Hebei	H23	0.176 1	72	Foshan, Guangdong	H2	0.112 2
33	Baoding, Hebei	H12	0.060 8	73	Zhuhai, Guangdong	H2	0.086 2
34	Baoding, Hebei	H2	0.346 6	74	Zhuhai, Guangdong	H10	0.047 2
35	Baoding, Hebei	H12	0.104 6	75	Shenzhen, Guangdong	H2	0.003 7
36	Handan, Hebei	H2	0.052 4	76	Jieyang, Guangdong	H8	0.010 2
37	Datong, Shanxi	H2	0.005 4	77	Nanning, Guangxi	H8	0.203 0
38	Zhengzhou, Henan	H2	0.104 8	78	Dali, Yunnan	H8	0.006 4
39	Zhengzhou, Henan	H2	0.061 4	79	Guiyang, Guizhou	H2	0.063 3
40	Zhengzhou, Henan	H2	0.060 7		80	Haikou, Hainan	H2	0.095 4

No.	City or district	Haplotype	Syringin/%		No.	City or district	Haplotype	Syringin/%
1	Harbin, Heilongjiang	H2	0.038 5		41	Luoyang, Henan	H2	0.062 3
2	Harbin, Heilongjiang	H8	0.426 2	42	Xinxiang, Henan	H2	0.095 2
3	Daqing, Heilongjiang	H2	0.090 1	43	Xuchang, Henan	H10	0.094 9
4	Shenyang, Liaoning	H5	0.064 0	44	Xuchang, Henan	H2	0.199 4
5	Shenyang, Liaoning	H23	0.122 5	45	Xuchang, Henan	H2	0.173 8
6	Shenyang, Liaoning	H10	0.084 7	46	Changge, Henan	H8	0.100 8
7	Shenyang, Liaoning	H2	0.094 5	47	Dingxi, Gansu	H2	0.120 3
8	Dalian, Liaoning	H2	0.009 7	48	Qingyang, Gansu	H2	0.108 3
9	Hohhot, Inner Mongolia	H23	0.411 0	49	Shizuishan, Ningxia	H2	0.165 7
10	Shijingshan District, Beijing	H12	0.524 5	50	Shizuishan, Ningxia	H2	0.152 6
11	Shijingshan District, Beijing	H8	0.178 0	51	Linyi, Ningxia	H2	0.091 3
12	Shijingshan District, Beijing	H10	0.205 6	52	Zibo, Shandong	H10	0.149 3
13	Fengtai District, Beijing	H2	0.150 8	53	Zibo, Shandong	H2	0.004 0
14	Fengtai District, Beijing	H23	0.387 9	54	Liaocheng, Shandong	H2	0.223 5
15	Fengtai District, Beijing	H2	0.462 7	55	Lu'an, Anhui	H2	0.150 2
16	Fengtai District, Beijing	H23	0.256 0	56	Fuyang, Anhui	H2	0.027 3
17	Fengtai District, Beijing	H2	0.145 6	57	Huangshan, Anhui	H14	0.119 2
18	Fangshan District, Beijing	H2	0.155 2	58	Wuhan, Hubei	H8	0.077 3
19	Fangshan District, Beijing	H8	0.256 9	59	Bishan District, Chongqing	H2	0.202 4
20	Fangshan District, Beijing	H10	0.038 3	60	Chengdu, Sichuan	H2	0.017 6
21	Fangshan District, Beijing	H10	0.277 9	61	Chengdu, Sichuan	H2	0.158 7
22	Fangshan District, Beijing	H2	0.154 3	62	Suzhou, Jiangsu	H10	0.167 7
23	Fangshan District, Beijing	H10	0.094 9	63	Nantong, Jiangsu	H2	0.239 8
24	Tongzhou District, Beijing	H2	0.087 9	64	Pudong New Area, Shanghai	H2	0.048 1
25	Dongcheng District, Beijing	H14	0.007 7	65	Wenzhou, Zhejiang	H2	0.025 5
26	Wuqing District, Tianjin	H2	0.134 7	66	Lishui, Zhejiang	H8	0.189 4
27	Cangzhou, Hebei	H2	0.103 9	67	Ningbo, Zhejiang	H10	0.052 9
28	Cangzhou, Hebei	H2	0.102 7	68	Ganzhou, Jiangxi	H8	0.016 5
29	Cangzhou, Hebei	H23	0.106 0	69	Yichun, Jiangxi	H8	0.006 8
30	Shijiazhuang, Hebei	H2	0.177 5	70	Xiamen, Fujian	H2	0.063 8
31	Baoding, Hebei	H2	0.073 1	71	Fuzhou, Fujian	H2	0.038 5
32	Baoding, Hebei	H23	0.176 1	72	Foshan, Guangdong	H2	0.112 2
33	Baoding, Hebei	H12	0.060 8	73	Zhuhai, Guangdong	H2	0.086 2
34	Baoding, Hebei	H2	0.346 6	74	Zhuhai, Guangdong	H10	0.047 2
35	Baoding, Hebei	H12	0.104 6	75	Shenzhen, Guangdong	H2	0.003 7
36	Handan, Hebei	H2	0.052 4	76	Jieyang, Guangdong	H8	0.010 2
37	Datong, Shanxi	H2	0.005 4	77	Nanning, Guangxi	H8	0.203 0
38	Zhengzhou, Henan	H2	0.104 8	78	Dali, Yunnan	H8	0.006 4
39	Zhengzhou, Henan	H2	0.061 4	79	Guiyang, Guizhou	H2	0.063 3
40	Zhengzhou, Henan	H2	0.060 7		80	Haikou, Hainan	H2	0.095 4

DNA barcode	Primer name	The sequence of primers (5′-3′)
atpI	atpI-F	TATTCGGATTGGCTGACC
atpI-R	TGCTTTGGCTTTACTCACG
atpB_rbcL	atpB_rbcL-F	ATAAGAAATGGGAGTTAGCACG
atpB_rbcL-R	AAGAAGTCTCACAACAACAAGG

DNA barcode	Primer name	The sequence of primers (5′-3′)
atpI	atpI-F	TATTCGGATTGGCTGACC
atpI-R	TGCTTTGGCTTTACTCACG
atpB_rbcL	atpB_rbcL-F	ATAAGAAATGGGAGTTAGCACG
atpB_rbcL-R	AAGAAGTCTCACAACAACAAGG

Combined haplotype	Nucleotide position	atpI haplotype	Nucleotide position	atpB_rbcL haplotype
H2	C	G	G	H2	-	-	H2
H5	*	*	*	H2	A	A	H4
H8	T	*	*	H6	*	*	H2
H10	*	*	*	H2	A	*	H1
H12	*	*	A	H1	*	*	H2
H14	*	C	*	H5	*	*	H2
H23	T	*	*	H6	A	*	H1

Combined haplotype	Nucleotide position	atpI haplotype	Nucleotide position	atpB_rbcL haplotype
H2	C	G	G	H2	-	-	H2
H5	*	*	*	H2	A	A	H4
H8	T	*	*	H6	*	*	H2
H10	*	*	*	H2	A	*	H1
H12	*	*	A	H1	*	*	H2
H14	*	C	*	H5	*	*	H2
H23	T	*	*	H6	A	*	H1

Market sample number	Haplotype	Origin
1, 3, 7, 8, 13, 15, 17, 18, 22,	H2	Heilongjiang: Fenglin, Yichun; Shangzhi, Harbin;
24, 26-28, 30, 31, 34, 36-42,	Mingshui, Suihua
44, 45, 47-51, 53-56, 59-61,	Liaoning: Qingyuan, Fushun; Benxi
63-65, 70-73, 75, 79, 80	Jilin: Changbai and Jingyu of Baishan
4	H5	Shangzhi, Harbin, Heilongjiang; Qingyuan, Fushun, Liaoning
2, 11, 19, 46, 58, 66, 68, 69,	H8	Heilongjiang: Raohe, Shuangyashan; Hailin, Mudanjiang
76-78	Qingyuan, Fushun, Liaoning; Changbai, Baishan, Jilin
6, 12, 20, 21, 23, 43, 52, 62,	H10	Heilongjiang: Shangzhi, Harbin; Mingshui, Suihua;
67, 74	Baoqing, Shuangyashan; Hailin and Linkou of Mudanjiang
	Jilin: Changbai and Jingyu of Baishan; Panshi
10, 33, 35	H12	Heilongjiang: Raohe, Shuangyashan; Hailin, Mudanjiang
Qingyuan, Fushun, Liaoning; Changbai, Baishan, Jilin
25, 57	H14	Raohe, Shuangyashan, Heilongjiang
5, 9, 14, 16, 29, 32	H23	Mingshui, Suihua, Heilongjiang

Market sample number	Haplotype	Origin
1, 3, 7, 8, 13, 15, 17, 18, 22,	H2	Heilongjiang: Fenglin, Yichun; Shangzhi, Harbin;
24, 26-28, 30, 31, 34, 36-42,	Mingshui, Suihua
44, 45, 47-51, 53-56, 59-61,	Liaoning: Qingyuan, Fushun; Benxi
63-65, 70-73, 75, 79, 80	Jilin: Changbai and Jingyu of Baishan
4	H5	Shangzhi, Harbin, Heilongjiang; Qingyuan, Fushun, Liaoning
2, 11, 19, 46, 58, 66, 68, 69,	H8	Heilongjiang: Raohe, Shuangyashan; Hailin, Mudanjiang
76-78	Qingyuan, Fushun, Liaoning; Changbai, Baishan, Jilin
6, 12, 20, 21, 23, 43, 52, 62,	H10	Heilongjiang: Shangzhi, Harbin; Mingshui, Suihua;
67, 74	Baoqing, Shuangyashan; Hailin and Linkou of Mudanjiang
	Jilin: Changbai and Jingyu of Baishan; Panshi
10, 33, 35	H12	Heilongjiang: Raohe, Shuangyashan; Hailin, Mudanjiang
Qingyuan, Fushun, Liaoning; Changbai, Baishan, Jilin
25, 57	H14	Raohe, Shuangyashan, Heilongjiang
5, 9, 14, 16, 29, 32	H23	Mingshui, Suihua, Heilongjiang

基于DNA条形码及HPLC对市售刺五加的种质资源鉴定和质量评价

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刘珊瑚 ¹ , 张志飞 ¹ , 黄钰莹 ¹ , 刘子齐 ⁴ , 陈文沁 ¹ , 阿木拉哈 ¹ , 王馨 ¹ , 石玥 ¹ , 张晓芹 ¹ , 何高洁 ¹ , 安克露 ¹ , 王晓晖 ²^,^* , 魏胜利 ¹^,³^,^*

药学学报 | 研究论文 2024,59(7): 2171-2178

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药学学报 | 研究论文 2024, 59(7): 2171-2178

基于DNA条形码及HPLC对市售刺五加的种质资源鉴定和质量评价

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刘珊瑚¹, 张志飞¹, 黄钰莹¹, 刘子齐⁴, 陈文沁¹, 阿木拉哈¹, 王馨¹, 石玥¹, 张晓芹¹, 何高洁¹, 安克露¹, 王晓晖²^,^*, 魏胜利¹^,³^,^*

作者信息

1.北京中医药大学中药学院, 北京 102488

2.北京中医药大学北京中医药研究院中药现代研究中心, 北京 102488

3.中药材规范化生产教育部工程研究中心, 北京 100102

4.黑龙江北草堂中药材有限责任公司, 黑龙江哈尔滨 150900

通讯作者:

^*王晓晖, Tel: 15201166873, E-mail: wangxhui2014@163.com;

魏胜利, Tel: 136833336930, E-mail: wsl7491@126.com

Identification and quality evaluation of germplasm resources of commercial Acanthopanax senticosus based on DNA barcodes and HPLC

Shan-hu LIU¹, Zhi-fei ZHANG¹, Yu-ying HUANG¹, Zi-qi LIU⁴, Wen-qin CHEN¹, La-ha AMU¹, Xin WANG¹, Yue SHI¹, Xiao-qin ZHANG¹, Gao-jie HE¹, Ke-lu AN¹, Xiao-hui WANG²^,^*, Sheng-li WEI¹^,³^,^*

Affiliations

1. School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China

2. Modern Research Center for Traditional Chinese Medicine, Beijing Institute of Traditional Chinese Medicine, Beijing University of Chinese Medicine, Beijing 102488, China

3. Engineering Research Center of Good Agricultural Practice for Chinese Crude Drugs of Ministry of Education, Beijing 100102, China

4. Heilongjiang Beicaotang Traditional Chinese Medicine Co., Ltd., Harbin 150900, China

出版时间: 2024-07-12 doi: 10.16438/j.0513-4870.2023-1407

文章导航

摘要

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刺五加Acanthopanax senticosus (Rupr. et Maxim.) Harms是我国东北道地药材之一, 本研究根据之前的叶绿体基因组测序结果, 选取atpI和atpB_rbcL基因片段对市售刺五加样品进行种质资源鉴定, 并利用HPLC对其紫丁香苷含量进行测定, 以期确定市售刺五加的主流种质和质量。本研究从24省47市收集80份刺五加样品并提取DNA, 通过PCR扩增atpI和atpB_rbcL基因片段, 分析结果表明, 2个基因片段联合分析形成7个单倍型(H2、H5、H8、H10、H12、H14、H23), 其中来自黑龙江伊春、哈尔滨尚志、黑龙江绥化、辽宁抚顺、辽宁本溪、吉林长白和吉林靖宇的单倍型H2是主流单倍型, 占总样本量的58.75%。H14和H23为产地独有单倍型, 分别来自黑龙江省双鸭山市饶河县和黑龙江省绥化市明水县。通过HPLC检测市售刺五加样品中的紫丁香苷含量, 结果表明73.96%样品紫丁香苷含量符合药典标准。样品间的紫丁香苷含量差异显著, 在0.003 7%~0.524 5%之间, 相差0.520 8%, 说明刺五加市场样品质量参差不齐, 但是不同单倍型之间市售刺五加紫丁香苷含量没有显著性差异。市场样品中单倍型H23中的紫丁香苷含量比较高, 因此可能是质量比较好的种质。本文对刺五加市场样品的种质资源和药材质量进行研究, 有利于指导刺五加市场样品流通、临床合理用药和后续优良品种筛选。

关键词

刺五加 / DNA条形码 / 分子鉴定 / 紫丁香苷 / 质量评价

Abstract

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Acanthopanax senticosus is one of the genuine regional herb in Northeast China. In this study, we identified the germplasm resources of commercial A. senticosus samples based on atpI and atpB_rbcL according to the previous chloroplast genome sequencing results, and determinated the content of syringin by HPLC to evaluated the quality of commercial samples. A total of 80 A. senticosus samples were collected from 47 cities in 24 provinces. DNA was extracted to amplify the products of atpI and atpB_rbcL by PCR. The results showed that 7 haplotypes (H2, H5, H8, H10, H12, H14, H23) were formed by the combined analysis of the two gene fragments. H2 from Yichun in Heilongjiang, Shangzhi in Harbin, Suihua in Heilongjiang, Fushun in Liaoning, Benxi in Liaoning, Changbai in Jilin and Jingyu in Jilin were the dominant genotype, representing 58.75% of the total samples. H14 and H23 were the unique haplotypes of the producing area. It is speculated that the commercial A. senticosus samples with haplotypes of H14 and H23 are from Raohe, Shuangyashan, Heilongjiang and Mingshui, Suihua, Heilongjiang, respectively. HPLC analysis indicated that the content of syringin in 73.96% of the samples met the Pharmacopoeia standards. There was a significant difference in the content of syringin among the samples, ranging from 0.003 7% to 0.524 5%, with a difference of 0.520 8%, indicating that the quality of the samples in the market of A. senticosus was uneven. However, there were no significant differences in the contents of syringin in the commercial A. senticosus among different haplotypes. The content of syringin in the haplotype H23 in the market sample is relatively high, so it may be a germplasm with better quality. This study researched the germplasm resources and medicinal materials quality of commercial A. senticosus samples and will help guide the commercial circulation, reasonable medication of A. senticosus, and the screening of excellent germplasm.

Key words

Acanthopanax senticosus / DNA barcode / molecular identification / syringin / quality evaluation

引用本文

刘珊瑚, 张志飞, 黄钰莹, 刘子齐, 陈文沁, 阿木拉哈, 王馨, 石玥, 张晓芹, 何高洁, 安克露, 王晓晖, 魏胜利. 基于DNA条形码及HPLC对市售刺五加的种质资源鉴定和质量评价. 药学学报, 2024 , 59 (7) : 2171 -2178 . DOI: 10.16438/j.0513-4870.2023-1407

Shan-hu LIU, Zhi-fei ZHANG, Yu-ying HUANG, Zi-qi LIU, Wen-qin CHEN, La-ha AMU, Xin WANG, Yue SHI, Xiao-qin ZHANG, Gao-jie HE, Ke-lu AN, Xiao-hui WANG, Sheng-li WEI. Identification and quality evaluation of germplasm resources of commercial Acanthopanax senticosus based on DNA barcodes and HPLC[J]. Acta Pharmaceutica Sinica, 2024 , 59 (7) : 2171 -2178 . DOI: 10.16438/j.0513-4870.2023-1407

正文

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刺五加为五加科植物刺五加Acanthopanax senticosus (Rupr. et Maxim.) Harms的干燥根和根茎或茎, 具有补肾强骨、益气安神、活血通络的功效^[1], 是我国东北地区道地药材之一^[2]。现代药理研究表明刺五加具有保护中枢神经系统和心血管系统、提高机体免疫力、抗肿瘤、抗衰老等作用^[3]。近年有研究发现其药效与同科植物人参具有一定相似性^[4], 以刺五加为主要原料的药品、保健品、食品数量日益增多, 刺五加需求量逐年增长^[2]。因此对市售刺五加进行种质资源鉴定和质量评价, 有利于指导刺五加优良种质筛选和临床用药。

DNA条形码的概念由加拿大学者Hebert等^[5]首次提出, 是指利用标准化的、较短的DNA序列作为条形码, 以实现对物种进行快速、准确的鉴定, 目前已广泛应用于中药材的物种鉴定。叶绿体基因通过母系遗传给后代, 相对分子质量小, 结构简单, 序列保守, 因此被广泛应用于中药材物种基原鉴定和种质资源鉴定。Chen等^[6]的研究结果表明以psbA_trnH+matK+trnL组合序列为DNA条形码可鉴别野菊和药用菊。Zhang等^[7]发现采用联合片段psbK_psbI+trnP_psaJ可对龙血竭基原及同属植物进行准确的分子鉴定。Wang等^[8]通过比较叶绿体基因组学筛选到的特异性DNA条形码petN_psbM和ndhF_rpl32可用于北柴胡种内种质资源鉴定。课题组前期通过比较叶绿体基因组分析得到潜在的特异性DNA片段atpI和atpB_rbcL, 能够将13个产地的刺五加样品鉴定为23个单倍型^[9]。为鉴定市场刺五加样品的产地来源, 根据课题组以前的实验结果, 利用atpI和atpB_rbcL两个片段对市场样品进行种质资源鉴定, 并追溯其产地来源。

化学成分是中药质量的物质基础, 2020版《中华人民共和国药典》中明确规定刺五加药材中紫丁香苷含量应不少于0.050%^[1]。现代研究表明紫丁香苷具有抗肿瘤、免疫抑制等作用^[10]。因此, 本研究从24省47市收集80份刺五加样品, 通过检测atpI和atpB_rbcL单倍型确定市场样品的种质资源, 推测其产地, 利用HPLC检测市售刺五加样品中的紫丁香苷含量, 以确定刺五加质量, 为后续指导刺五加市场样品流通、刺五加临床用药、优良种质筛选奠定基础, 同时也对其他中药材的市场样品检测及临床合理用药具有重要指导意义。

材料与方法

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材料本研究使用的市售刺五加样品共计80份, 自2021年5月至2022年11月从全国24省份47市收集而得, 具体样品信息见表 1。

仪器粉碎机(LG-01, 瑞安市百信制药机械有限公司); 移液枪(大龙兴创实验仪器北京有限公司); 涡旋混合器(GL-88B, 海门市其林贝尔仪器制造有限公司); 电热恒温水浴锅(HH-S4A, 北京科伟永兴仪器有限公司); 冷冻高速离心机(TGL-16, 湘仪离心机仪器有限公司); 超微量紫外分光光度计(NanoDrop One, 美国赛默飞世尔科技有限公司); PCR仪(T100, 美国Bio-Rad公司); 电子天平(PL602E, 瑞士Mettler Toledo公司); 电泳仪(JY 300 E, 北京君意东方电泳设备有限公司); 凝胶成像分析仪(WD-9413B, 北京六一生物科技有限公司); 超声波清洗机(KQ-500E, 昆山市超声波清洗器有限公司); 高效液相色谱仪(LC-20A, 日本岛津公司)。

试剂植物基因组提取试剂盒, 产物纯化试剂盒(南京诺唯赞生物科技股份有限公司); Taq DNA聚合酶, DL 15000 DNA Marker, DL 1000 DNA Marker (TaKaRa); atpI和atpB_rbcL引物(北京六合华大基因科技有限公司); 琼脂糖和GelRed染料(北京百瑞极生物科技有限公司); 紫丁香苷标准品(B21684, 上海源叶生物科技有限公司); 甲醇和乙腈(AR, 美国赛默飞世尔科技有限公司); 磷酸(AR, 天津市大茂化学试剂厂)。

DNA提取和检测 用粉碎机将市售刺五加样品粉碎, 取20 mg粉末, 采用植物基因组DNA提取试剂盒进行总DNA提取。利用超微量紫外分光光度计检测DNA浓度, 同时用1%的琼脂糖凝胶电泳检测DNA的质量。

PCR扩增与测序 本研究选择2个刺五加的DNA条形码atpI和atpB_rbcL进行扩增, 引物序列见表 2^[9]。PCR扩增体系: 10×Taq buffer 5 μL、dNTP Mix 4 μL、正反向引物各1 μL, 0.5 μL Taq酶, 10 ng DNA模板, 加ddH₂O至50 μL。PCR扩增程序: 95 ℃预变性5 min; 95 ℃变性30 s, 复性30 s (atpI、atpB_rbcL退火温度分别为44、50.9 ℃), 72 ℃延伸1 min (35个循环); 72 ℃、10 min; 4 ℃保存。1.0%琼脂糖凝胶中检测PCR扩增产物, 目标片段经纯化回收后(产物纯化试剂盒) 送至北京六合华大基因科技有限公司测序。测序后的序列导入DANMAN软件, 进行拼接、多序列比对分析。

对照品溶液的制备 精密称定紫丁香苷对照品, 加入50%甲醇溶液溶解, 得到紫丁香苷340 μg·mL^-1的标准品溶液。

供试品溶液的制备 市售刺五加样品紫丁香苷提取方法参考Shang等^[11]的研究。精密称定刺五加粗粉1.0 g, 置于具塞锥形瓶中, 精密加入50 mL 50%甲醇溶液溶解, 称定质量。超声处理40 min, 室温放至恒温, 用50%甲醇补足失重, 摇匀, 静置, 取上清液过0.45 μm微孔滤膜, 续滤液作为供试品溶液。

色谱条件 十八烷基硅烷键合硅胶反相色谱柱(YMC-Pack-ODS-A, 250 mm × 4.6 mm, 5 μm), 流动相为乙腈(A)-0.1%磷酸水(B), 梯度洗脱(0~10 min, 9% A; 10~20 min, 10% A; 20~30 min, 15% A; 30~40 min, 15% A; 40~70 min, 30% A); 流速为1.0 mL·min^-1; 进样体积10 μL; 柱温30 ℃; 检测波长214 nm。

HPLC数据分析 利用IBM SPSS Statistical 20.0统计软件中的Kruskal-Wallis检验, 对市售刺五加中的紫丁香苷含量进行统计学分析, 从而对不同单倍型市售刺五加样品的质量进行评价。

结果与分析

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1 市售刺五加DNA提取和atpI、atpB_rbcL基因片段扩增

成功提取80份市售刺五加样品基因组的DNA, 提取的DNA浓度为1.1~65.2 ng·μL^-1, 电泳检测结果如图 1所示。利用atpI和atpB_rbcL基因片段的特异性引物进行PCR扩增, 80份样品均扩增到600 bp左右的片段, 如图 2、3所示, PCR产物纯化后进行双向测序, 测序结果与NCBI中刺五加叶绿体基因组(GenBank登录: JN637765.1) 中的相应基因片段进行比对分析, 确定其分别为刺五加atpI和atpB_rbcL基因片段。

2 市售刺五加DNA条形码比对分析

本课题组前期研究结果表明, 将来自13个产地的184份刺五加样品利用atpI和atpB_rbcL特异性DNA片段进行扩增分析, atpI和atpB_rbcL分别能鉴定9、8个单倍型, 联合分析形成23个单倍型(H1~H23)^[9]。对80份市售刺五加样品测序结果进行分析, atpI、atpB_rbcL分别可鉴定4、3个单倍型, 2个基因片段联合形成7个单倍型, 分别为H2、H5、H8、H10、H12、H14、H23, 联合分析的单倍型变异位点如表 3所示。其中单倍型H2数量最多, 共47份样品, 占总数的58.75%。总体来说, 单倍型数量由高到低排列如下: H2 > H8 > H10 > H23 > H12 > H14 > H5。市售刺五加中单倍型H2分布在21个省(市), 包括黑龙江省、辽宁省、内蒙古自治区、河北省、北京市、天津市、山西省、河南省、甘肃省、宁夏回族自治区、山东省、安徽省、重庆市、四川省、江苏省、上海市、浙江省、福建省、广东省、贵州省、海南省; 之前的研究结果表明, 单倍型H2来自黑龙江伊春、哈尔滨尚志、黑龙江绥化、辽宁抚顺、辽宁本溪、吉林长白和吉林靖宇等7个产地(表 4), 其中黑龙江伊春、哈尔滨尚志、辽宁抚顺、辽宁本溪和吉林靖宇5个产地为野生刺五加, 黑龙江绥化和吉林长白2个产地为栽培刺五加; 说明产自黑龙江伊春、哈尔滨尚志、辽宁抚顺、辽宁本溪和吉林靖宇的野生刺五加、黑龙江绥化和吉林长白的栽培刺五加可能销售到21个省(市)。课题组前期研究表明, 单倍型H14和H23只在一个产地出现, H14在黑龙江省双鸭山市饶河县被发现, H23出现在黑龙江省绥化市明水县(表 4); H14和H23对应市场样品分别来源于北京东城区、安徽黄山和辽宁沈阳、内蒙古呼和浩特、北京市丰台区、河北沧州、保定; 可以推测单倍型为H14和H23的市售刺五加样品分别产自黑龙江省双鸭山市饶河县和黑龙江省绥化市明水县。

3 市售刺五加的质量评价

刺五加中的紫丁香苷的线性回归方程为y = 1 075 564.353 7 x - 5 154.184 7, 相关系数R² > 0.999, 符合分析要求。对80份市售刺五加饮片的紫丁香苷含量进行测定, 结果表明, 80份样品中62份市售刺五加饮片紫丁香苷含量符合药典标准, 合格率为73.96%。黑龙江省、辽宁省、北京市、山西省、山东省、安徽省、重庆市、四川省、江西省、福建省、广东省、云南省的市售刺五加样品存在紫丁香苷含量不合格情况。市售刺五加样品中紫丁香苷含量在0.003 7%~0.524 5%之间, 含量最高的省份是内蒙古自治区, 含量最低的省份是山西省, 以上结果表明市售刺五加样品紫丁香苷含量参差不齐。对市售刺五加不同单倍型的紫丁香苷的含量进行分析, 如图 4所示, 紫丁香苷平均含量最高的单倍型为H23, 平均含量最低的单倍型为H14。采用Kruskal-Wallis检验对不同单倍型刺五加中紫丁香苷的含量进行相关性分析, 结果表明各单倍型之间刺五加紫丁香苷含量无显著性差异。对各单倍型紫丁香苷含量合格情况进行分析, 发现H5、H12和H23对应样品的紫丁香苷含量均合格。但是H5和H12的样品比较少, 存在偶然性, 而单倍型H23的质量远远超过中国药典规定的紫丁香苷的含量, 因此H23可能是质量相对稳定而且比较好的种质, 可用于后续的优良种质资源筛选。

讨论

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刺五加是重要的中成药原料, 市场需求较大, 艾迪注射液、舒肝解郁胶囊、心脑联通胶囊、刺五加脑灵合剂和刺五加注射液等中成药均含有刺五加^[12-16], 因此对市售刺五加进行种质资源鉴定有利于刺五加市场流通和临床用药。传统的药材鉴定方法难以实现对中药材种质的鉴别, 目前DNA条形码技术广泛应用于中药材分子鉴定领域。一方面, 已有学者利用DNA条形码区分中药材产地。Cai等^[17]利用ITS2序列区分了东北地区的10个人参居群。Shen等^[18]以trnI和trnN为DNA条形码对6个产地的21份三叶青样品进行分析, 可区分四川和浙江与其他产地。ccsA和trnC-GCA_petN可作为特异性DNA条形码对不同产地的北苍术进行种质资源鉴定^[19]。基于特异性DNA条形码trnG-UCC和ndhG_ndhF对9省15个产地305份桔梗样品进行分析, 结果表明, 3个产地拥有的特异单倍型, 可作为该产地的分子标签与其他产地进行区分^[20]。另一方面, 在利用DNA条形码区分中药材产地的基础上, Man等^[21]利用matK基因对市售大黄的种质资源和产地来源进行研究, 发现收集的市售大黄样品都是掌叶大黄, 其中来自甘肃、四川和云南省的Rp4和Rp6是主要流通单倍型。Zhang等^[22]的研究表明trnH_psbA片段为鉴定市场样品种质资源最佳片段, 并且THap2为市场样品主要流通单倍型。以上研究结果说明DNA条形码可用于中药种质资源鉴定和产地溯源。本课题组前期利用atpI和atpB_rbcL对不同产地的刺五加样品进行种质资源鉴定, atpI和atpB_rbcL分别能鉴定9、8个单倍型, 联合分析形成23个单倍型^[9]。然而由于炮制加工后的刺五加饮片DNA不易提取、PCR片段不易扩增等原因, 目前对市售刺五加的种质资源鉴定研究较少。本研究首次从全国24省份47市收集80份刺五加药材提取DNA扩增得到atpI和atpB_rbcL序列, 联合分析发现7个单倍型, 其中H2占样品总体的58.75%。之前的研究结果表明, 单倍型H2来自黑龙江伊春、哈尔滨尚志、黑龙江绥化、辽宁抚顺、辽宁本溪、吉林长白和吉林靖宇等7个产地, 其中黑龙江伊春、哈尔滨尚志、辽宁抚顺、辽宁本溪和吉林靖宇5个产地为野生刺五加, 黑龙江绥化和吉林长白2个产地为栽培刺五加。前期研究表明H14和H23为产地独有单倍型, 推测单倍型为H14和H23的市售刺五加样品分别产自黑龙江省双鸭山市饶河县和黑龙江省绥化市明水县。以上结果说明市场上流通的刺五加单倍型集中, 根据产地独有单倍型一定程度上能做到产地溯源。

中药处方和制剂的临床疗效与中药质量密切相关^[23], 而化学成分是中药质量的物质基础。紫丁香苷具有抗肿瘤、抗抑郁、保护心肌缺血、降血脂等作用^[10], 《中华人民共和国药典》(2020年版) 中明确规定刺五加药材中紫丁香苷含量应不少于0.050%^[1]。已有文献^{[11, 24]}报道不同产地的刺五加质量存在差异, 但是目前对市售刺五加的质量研究比较少。本研究利用HPLC测定80份市售刺五加饮片中的紫丁香苷含量, 结果表明73.96%的样品紫丁香苷含量符合药典标准, 样品间的紫丁香苷含量差异显著, 最高含量为0.524 5%, 最低含量为0.003 7%, 相差0.520 8%。对不同单倍型的刺五加市售样品的紫丁香苷含量进行分析, 结果表明相同单倍型含量差异大, 比如单倍型H2对应的样品, 紫丁香苷最高含量是0.462 7%, 最低含量是0.003 7%, 但是不同单倍型之间的市售刺五加的紫丁香苷含量没有显著差异, 这与课题组前期研究结果类似, 可能生长年限、采收加工和环境对中药质量的影响更加显著^[23]。对各单倍型紫丁香苷含量合格情况进行分析, H23的刺五加市场样品的紫丁香苷含量均显著超过中国药典规定的紫丁香苷含量, 单倍型H23可能是质量相对比较好的种质, 可以用于后续优良种质筛选和培育。

作者贡献: 刘珊瑚负责实验、数据分析和论文撰写; 张志飞负责实验; 石玥、黄钰莹、张晓芹、王馨参与论文数据分析; 何高洁、安克露、陈文沁、阿木拉哈、刘子齐参与样品收集; 王晓晖和魏胜利负责设计论文的实验思路, 指导学生实验和论文撰写及修改。

利益冲突: 无任何利益冲突。

基金

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兴安盟科技计划项目(基于分子防伪技术的精准药材北柴胡选育及栽培技术研究)(20221156)
企业横向课题(桔梗等4种精准药材批次分子防伪技术研究项目)(2020110031009385)
国家重点研发计划项目(中医药现代化专项)(2022YFC3501505)

参考文献

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文献

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参考文献引证文献

排序方式：

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2024年第59卷第7期

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doi: 10.16438/j.0513-4870.2023-1407

接收时间：2023-12-17
首发时间：2025-11-26
出版时间：2024-07-12

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出版历史

收稿日期：2023-12-17
修回日期：2024-03-19

基金

兴安盟科技计划项目(基于分子防伪技术的精准药材北柴胡选育及栽培技术研究)(20221156)

企业横向课题(桔梗等4种精准药材批次分子防伪技术研究项目)(2020110031009385)

国家重点研发计划项目(中医药现代化专项)(2022YFC3501505)

作者信息

1.北京中医药大学中药学院, 北京 102488

2.北京中医药大学北京中医药研究院中药现代研究中心, 北京 102488

3.中药材规范化生产教育部工程研究中心, 北京 100102

4.黑龙江北草堂中药材有限责任公司, 黑龙江哈尔滨 150900

通讯作者:

^*王晓晖, Tel: 15201166873, E-mail: wangxhui2014@163.com;

魏胜利, Tel: 136833336930, E-mail: wsl7491@126.com

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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

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No.	City or district	Haplotype	Syringin/%		No.	City or district	Haplotype	Syringin/%
1	Harbin, Heilongjiang	H2	0.038 5		41	Luoyang, Henan	H2	0.062 3
2	Harbin, Heilongjiang	H8	0.426 2	42	Xinxiang, Henan	H2	0.095 2
3	Daqing, Heilongjiang	H2	0.090 1	43	Xuchang, Henan	H10	0.094 9
4	Shenyang, Liaoning	H5	0.064 0	44	Xuchang, Henan	H2	0.199 4
5	Shenyang, Liaoning	H23	0.122 5	45	Xuchang, Henan	H2	0.173 8
6	Shenyang, Liaoning	H10	0.084 7	46	Changge, Henan	H8	0.100 8
7	Shenyang, Liaoning	H2	0.094 5	47	Dingxi, Gansu	H2	0.120 3
8	Dalian, Liaoning	H2	0.009 7	48	Qingyang, Gansu	H2	0.108 3
9	Hohhot, Inner Mongolia	H23	0.411 0	49	Shizuishan, Ningxia	H2	0.165 7
10	Shijingshan District, Beijing	H12	0.524 5	50	Shizuishan, Ningxia	H2	0.152 6
11	Shijingshan District, Beijing	H8	0.178 0	51	Linyi, Ningxia	H2	0.091 3
12	Shijingshan District, Beijing	H10	0.205 6	52	Zibo, Shandong	H10	0.149 3
13	Fengtai District, Beijing	H2	0.150 8	53	Zibo, Shandong	H2	0.004 0
14	Fengtai District, Beijing	H23	0.387 9	54	Liaocheng, Shandong	H2	0.223 5
15	Fengtai District, Beijing	H2	0.462 7	55	Lu'an, Anhui	H2	0.150 2
16	Fengtai District, Beijing	H23	0.256 0	56	Fuyang, Anhui	H2	0.027 3
17	Fengtai District, Beijing	H2	0.145 6	57	Huangshan, Anhui	H14	0.119 2
18	Fangshan District, Beijing	H2	0.155 2	58	Wuhan, Hubei	H8	0.077 3
19	Fangshan District, Beijing	H8	0.256 9	59	Bishan District, Chongqing	H2	0.202 4
20	Fangshan District, Beijing	H10	0.038 3	60	Chengdu, Sichuan	H2	0.017 6
21	Fangshan District, Beijing	H10	0.277 9	61	Chengdu, Sichuan	H2	0.158 7
22	Fangshan District, Beijing	H2	0.154 3	62	Suzhou, Jiangsu	H10	0.167 7
23	Fangshan District, Beijing	H10	0.094 9	63	Nantong, Jiangsu	H2	0.239 8
24	Tongzhou District, Beijing	H2	0.087 9	64	Pudong New Area, Shanghai	H2	0.048 1
25	Dongcheng District, Beijing	H14	0.007 7	65	Wenzhou, Zhejiang	H2	0.025 5
26	Wuqing District, Tianjin	H2	0.134 7	66	Lishui, Zhejiang	H8	0.189 4
27	Cangzhou, Hebei	H2	0.103 9	67	Ningbo, Zhejiang	H10	0.052 9
28	Cangzhou, Hebei	H2	0.102 7	68	Ganzhou, Jiangxi	H8	0.016 5
29	Cangzhou, Hebei	H23	0.106 0	69	Yichun, Jiangxi	H8	0.006 8
30	Shijiazhuang, Hebei	H2	0.177 5	70	Xiamen, Fujian	H2	0.063 8
31	Baoding, Hebei	H2	0.073 1	71	Fuzhou, Fujian	H2	0.038 5
32	Baoding, Hebei	H23	0.176 1	72	Foshan, Guangdong	H2	0.112 2
33	Baoding, Hebei	H12	0.060 8	73	Zhuhai, Guangdong	H2	0.086 2
34	Baoding, Hebei	H2	0.346 6	74	Zhuhai, Guangdong	H10	0.047 2
35	Baoding, Hebei	H12	0.104 6	75	Shenzhen, Guangdong	H2	0.003 7
36	Handan, Hebei	H2	0.052 4	76	Jieyang, Guangdong	H8	0.010 2
37	Datong, Shanxi	H2	0.005 4	77	Nanning, Guangxi	H8	0.203 0
38	Zhengzhou, Henan	H2	0.104 8	78	Dali, Yunnan	H8	0.006 4
39	Zhengzhou, Henan	H2	0.061 4	79	Guiyang, Guizhou	H2	0.063 3
40	Zhengzhou, Henan	H2	0.060 7		80	Haikou, Hainan	H2	0.095 4

No.

City or district

Haplotype

Syringin/%

No.

City or district

Haplotype

Syringin/%

Harbin, Heilongjiang

0.038 5

Luoyang, Henan

0.062 3

Harbin, Heilongjiang

0.426 2

Xinxiang, Henan

0.095 2

Daqing, Heilongjiang

0.090 1

Xuchang, Henan

H10

0.094 9

Shenyang, Liaoning

0.064 0

Xuchang, Henan

0.199 4

Shenyang, Liaoning

H23

0.122 5

Xuchang, Henan

0.173 8

Shenyang, Liaoning

H10

0.084 7

Changge, Henan

0.100 8

Shenyang, Liaoning

0.094 5

Dingxi, Gansu

0.120 3

Dalian, Liaoning

0.009 7

Qingyang, Gansu

0.108 3

Hohhot, Inner Mongolia

H23

0.411 0

Shizuishan, Ningxia

0.165 7

Shijingshan District, Beijing

H12

0.524 5

Shizuishan, Ningxia

0.152 6

Shijingshan District, Beijing

0.178 0

Linyi, Ningxia

0.091 3

Shijingshan District, Beijing

H10

0.205 6

Zibo, Shandong

H10

0.149 3

Fengtai District, Beijing

0.150 8

Zibo, Shandong

0.004 0

Fengtai District, Beijing

H23

0.387 9

Liaocheng, Shandong

0.223 5

Fengtai District, Beijing

0.462 7

Lu'an, Anhui

0.150 2

Fengtai District, Beijing

H23

0.256 0

Fuyang, Anhui

0.027 3

Fengtai District, Beijing

0.145 6

Huangshan, Anhui

H14

0.119 2

Fangshan District, Beijing

0.155 2

Wuhan, Hubei

0.077 3

Fangshan District, Beijing

0.256 9

Bishan District, Chongqing

0.202 4

Fangshan District, Beijing

H10

0.038 3

Chengdu, Sichuan

0.017 6

Fangshan District, Beijing

H10

0.277 9

Chengdu, Sichuan

0.158 7

Fangshan District, Beijing

0.154 3

Suzhou, Jiangsu

H10

0.167 7

Fangshan District, Beijing

H10

0.094 9

Nantong, Jiangsu

0.239 8

Tongzhou District, Beijing

0.087 9

Pudong New Area, Shanghai

0.048 1

Dongcheng District, Beijing

H14

0.007 7

Wenzhou, Zhejiang

0.025 5

Wuqing District, Tianjin

0.134 7

Lishui, Zhejiang

0.189 4

Cangzhou, Hebei

0.103 9

Ningbo, Zhejiang

H10

0.052 9

Cangzhou, Hebei

0.102 7

Ganzhou, Jiangxi

0.016 5

Cangzhou, Hebei

H23

0.106 0

Yichun, Jiangxi

0.006 8

Shijiazhuang, Hebei

0.177 5

Xiamen, Fujian

0.063 8

Baoding, Hebei

0.073 1

Fuzhou, Fujian

0.038 5

Baoding, Hebei

H23

0.176 1

Foshan, Guangdong

0.112 2

Baoding, Hebei

H12

0.060 8

Zhuhai, Guangdong

0.086 2

Baoding, Hebei

0.346 6

Zhuhai, Guangdong

H10

0.047 2

Baoding, Hebei

H12

0.104 6

Shenzhen, Guangdong

0.003 7

Handan, Hebei

0.052 4

Jieyang, Guangdong

0.010 2

Datong, Shanxi

0.005 4

Nanning, Guangxi

0.203 0

Zhengzhou, Henan

0.104 8

Dali, Yunnan

0.006 4

Zhengzhou, Henan

0.061 4

Guiyang, Guizhou

0.063 3

Zhengzhou, Henan

0.060 7

Haikou, Hainan

0.095 4

DNA barcode	Primer name	The sequence of primers (5′-3′)
atpI	atpI-F	TATTCGGATTGGCTGACC
atpI-R	TGCTTTGGCTTTACTCACG
atpB_rbcL	atpB_rbcL-F	ATAAGAAATGGGAGTTAGCACG
atpB_rbcL-R	AAGAAGTCTCACAACAACAAGG

DNA barcode

Primer name

The sequence of primers (5′-3′)

atpI

atpI-F

TATTCGGATTGGCTGACC

atpI-R

TGCTTTGGCTTTACTCACG

atpB_rbcL

atpB_rbcL-F

ATAAGAAATGGGAGTTAGCACG

atpB_rbcL-R

AAGAAGTCTCACAACAACAAGG

Combined haplotype	Nucleotide position	atpI haplotype	Nucleotide position	atpB_rbcL haplotype
H2	C	G	G	H2	-	-	H2
H5	*	*	*	H2	A	A	H4
H8	T	*	*	H6	*	*	H2
H10	*	*	*	H2	A	*	H1
H12	*	*	A	H1	*	*	H2
H14	*	C	*	H5	*	*	H2
H23	T	*	*	H6	A	*	H1

Combined haplotype

Nucleotide position

atpI haplotype

Nucleotide position

atpB_rbcL haplotype

201

293

472

398

399

H10

H12

H14

H23

Market sample number	Haplotype	Origin
1, 3, 7, 8, 13, 15, 17, 18, 22,	H2	Heilongjiang: Fenglin, Yichun; Shangzhi, Harbin;
24, 26-28, 30, 31, 34, 36-42,	Mingshui, Suihua
44, 45, 47-51, 53-56, 59-61,	Liaoning: Qingyuan, Fushun; Benxi
63-65, 70-73, 75, 79, 80	Jilin: Changbai and Jingyu of Baishan
4	H5	Shangzhi, Harbin, Heilongjiang; Qingyuan, Fushun, Liaoning
2, 11, 19, 46, 58, 66, 68, 69,	H8	Heilongjiang: Raohe, Shuangyashan; Hailin, Mudanjiang
76-78	Qingyuan, Fushun, Liaoning; Changbai, Baishan, Jilin
6, 12, 20, 21, 23, 43, 52, 62,	H10	Heilongjiang: Shangzhi, Harbin; Mingshui, Suihua;
67, 74	Baoqing, Shuangyashan; Hailin and Linkou of Mudanjiang
	Jilin: Changbai and Jingyu of Baishan; Panshi
10, 33, 35	H12	Heilongjiang: Raohe, Shuangyashan; Hailin, Mudanjiang
Qingyuan, Fushun, Liaoning; Changbai, Baishan, Jilin
25, 57	H14	Raohe, Shuangyashan, Heilongjiang
5, 9, 14, 16, 29, 32	H23	Mingshui, Suihua, Heilongjiang

Market sample number

Haplotype

Origin

1, 3, 7, 8, 13, 15, 17, 18, 22,

Heilongjiang: Fenglin, Yichun; Shangzhi, Harbin;

24, 26-28, 30, 31, 34, 36-42,

Mingshui, Suihua

44, 45, 47-51, 53-56, 59-61,

Liaoning: Qingyuan, Fushun; Benxi

63-65, 70-73, 75, 79, 80

Jilin: Changbai and Jingyu of Baishan

Shangzhi, Harbin, Heilongjiang; Qingyuan, Fushun, Liaoning

2, 11, 19, 46, 58, 66, 68, 69,

Heilongjiang: Raohe, Shuangyashan; Hailin, Mudanjiang

76-78

Qingyuan, Fushun, Liaoning; Changbai, Baishan, Jilin

6, 12, 20, 21, 23, 43, 52, 62,

H10

Heilongjiang: Shangzhi, Harbin; Mingshui, Suihua;

67, 74

Baoqing, Shuangyashan; Hailin and Linkou of Mudanjiang

Jilin: Changbai and Jingyu of Baishan; Panshi

10, 33, 35

H12

Heilongjiang: Raohe, Shuangyashan; Hailin, Mudanjiang

Qingyuan, Fushun, Liaoning; Changbai, Baishan, Jilin

25, 57

H14

Raohe, Shuangyashan, Heilongjiang

5, 9, 14, 16, 29, 32

H23

Mingshui, Suihua, Heilongjiang