基于cpSSR标记的山药种质资源DNA指纹图谱构建及遗传多样性分析

doi:10.3969/j.issn.1004-1524.2021.07.08

浙江农业学报 ›› 2021, Vol. 33 ›› Issue (7): 1222-1233.DOI: 10.3969/j.issn.1004-1524.2021.07.08

基于cpSSR标记的山药种质资源DNA指纹图谱构建及遗传多样性分析

张静珍¹^,²(), 王连军², 雷剑², 柴沙沙², 杨新笋²^,^*(), 张文英¹^,^*()

1.长江大学农学院,湖北荆州 434025
2.湖北省农业科学院粮食作物研究所,湖北省甘薯工程技术研究中心,粮食作物种质创新与遗传改良湖北省重点实验室,湖北武汉 430064

收稿日期:2020-07-21 出版日期:2021-07-25 发布日期:2021-08-06
通讯作者: 杨新笋,张文英
作者简介:张文英,E-mail: wyzhang@yangtzeu.edu.cn
*杨新笋,E-mail: yangxins013@163.com;
张静珍(1998—),女,浙江宁波人,硕士研究生,主要从事山药遗传多样性研究。E-mail: jingzhen0118@163.com
基金资助:
粮食作物种质创新与遗传改良湖北省重点实验室开放课题;湖北省农业科学院特色学科;湖北省农业科技创新中心资助项目(2007-620-001-03);湖北省农业科学院青年拔尖人才培养计划

Genetic diversity analysis and construction of DNA fingerprint of yam (Dioscorea oppositeac Thunb.) germplasm by cpSSR marker

ZHANG Jingzhen¹^,²(), WANG Lianjun², LEI Jian², CHAI Shasha², YANG Xinsun²^,^*(), ZHANG Wenying¹^,^*()

1. College of Agriculture, Yangtze University, Jingzhou 434025, China
2. Hubei Key Laboratory of Food Crops Germplasm and Genetic Improvement, Institute of Food Crops, Hubei Sweet Potato Engineering and Technology Research Centre, Hubei Academy of Agricultural Sciences, Wuhan 430064, China

Received:2020-07-21 Online:2021-07-25 Published:2021-08-06
Contact: YANG Xinsun,ZHANG Wenying

摘要/Abstract

摘要：

采用叶绿体SSR(cpSSR)分子标记对从国内外引进的64份山药种质资源进行遗传多样性分析,构建DNA指纹图谱,旨在为山药品种亲本选择,山药种质创新及品种改良提供依据。结果显示:从21对cpSSR引物中筛选出10对扩增稳定、多态性高的引物,在64份山药种质材料中扩增出69个条带,多态性条带59个,多态性比率高达85.51%,平均观测等位基因数(Na)为2,平均有效等位基因数(Ne)为1.569 3(1.231 1~1.916 0),平均Shannon信息指数为0.560 6(0.329 0~0.671 1),平均Nei’s基因多样性指数(He)为0.378 1(0.184 8~0.478 1),表明64份山药种质资源具有丰富的遗传多样性。聚类分析结果表明,遗传相似系数变幅为 0.55~0.93,在相似系数为 0.63时,可将64份山药种质资源分为4类,结果表明,基于cpSSR 分子标记的山药种质资源的分类在种间具有较好的区分能力,但是cpSSR 分类表现出较大的地理分布的相关性,而与形态特征关系不密切。选用较少的引物区分较多的品种为原则,采用2对引物成功构建供试种质的DNA指纹图谱,为山药种质资源鉴定和品种选育亲本的选择提供理论依据。

关键词: 山药, 种质资源, cpSSR标记, DNA指纹图谱, 遗传多样性

Abstract:

In order to provide molecular foundation for the innovative parental selection and the varieties improvement of Dioscorea oppositeac Thunb. germplasm, in present study, the molecular fingerprints of 64 accessions of D. oppositeac from home and abroad were mapped and the genetic diversity was analyzed using cpSSR molecular markers technology. The results showed that 10 pairs of primers were selected from 21 pairs of cpSSR primers.The genome DNA of 64 accessions of D. oppositeac germplasm were amplified by 10 pairs of cpSSR primers with stable amplification, high polymorphism selected from 21 pairs of primers. Sixty-nine bands were got totally, of which 59 were polymorphic bands, and the rate of polymorphic bands reached as high as 85.51%. The average number of observed alleles (Na) was 2, the mean effective allele (Ne) was 1.569 3 (1.231 1-1.916 0), and the average Shannon’s information index was 0.560 6(0.329 0-0.671 1), the average Nei’s genetic diversity index (He) was 0.378 1 (0.184 8-0.478 1),indicating a rich genetic diversity. Based on genetic similarity coefficient (GS), which ranged from 0.55 to 0.93, the phylogenetic relationships of 64 D. oppositeac germplasm resources were analyzed. When the threshold value of genetic similarity coefficient was 0.63, all the tested materials could be clustered into 4 classes. With the principle of identifying 64 D. oppositeac germplasm resources with a minimum number of primers, 2 pairs of primers were used to successfully construct DNA fingerprint.

Key words: Dioscorea oppositeac Thunb., germplasm resources, cpSSR molecular marker, DNA fingerprint, genetic diversity

中图分类号:

S632.1

张静珍, 王连军, 雷剑, 柴沙沙, 杨新笋, 张文英. 基于cpSSR标记的山药种质资源DNA指纹图谱构建及遗传多样性分析[J]. 浙江农业学报, 2021, 33(7): 1222-1233.

ZHANG Jingzhen, WANG Lianjun, LEI Jian, CHAI Shasha, YANG Xinsun, ZHANG Wenying. Genetic diversity analysis and construction of DNA fingerprint of yam (Dioscorea oppositeac Thunb.) germplasm by cpSSR marker[J]. Acta Agriculturae Zhejiangensis, 2021, 33(7): 1222-1233.

图/表 6

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编号 Code	种质名称 Accession name	编号 Code	种质名称 Accession name	编号 Code	种质名称 Accession name	编号 Code	种质名称 Accession name
1	太谷山药 Taigushanyao	17	河北小白嘴山药 Hebeixiaobaizuishanyao	33	寿阳山药 Shouyangshanyao	49	山东铁棍 Shandongtiegun
2	垣曲采山药 Yuanqucaishanyao	18	野山药 Yeshanyao	34	高阳紫药 Gaoyangziyao	50	芹峰淮山药 Qingfenghuaishanyao
3	高阳小白嘴 Gaoyangxiaobaizui	19	襄汾城关刘存山药 Xiangfenchengguanliucun shanyao	35	山西永济山药 Shanxiyongji shanyao	51	山西山药 Shanxishanyao
4	沁阳太谷山药 Qinyangtaigushanyao	20	日本青森山药 Japan Qinsengshanyao	36	南京采山药2号 Nanjingcaishanyao No. 2	52	嘉祥细长毛山药 Jiaxiangxichangmaoshanyao
5	沁野山药 Qinyeshanyao	21	丽江2号 Lijiang No.2	37	温引1号 Wenying No.1	53	白山药 Baishanyao
6	山东牛腿 Shandongniutui	22	晋城采 Jingchengcai	38	铁棍山药 Tiegunshanyao	54	自然薯蓣 Ziranshuyu
7	灌南山药 Guannanshanyao	23	河北棒药 Hebeibangyao	39	滨海山药 Binhaishanyao	55	日本白山药 Japan Baishanyao
8	上海毛山药 Shanghaimaoshanyao	24	武当山山药2号 Wudangshanshanyao No.2	40	广西农科院山药 Guangxinongkeyuan shanyao	56	南召山药 Nanzhaoshanyao
9	武当山山药1号 Wudangshanshanyao No.1	25	冀城2号 Jicheng No.2	41	沙滩园山药 Shantanyuanshanyao	57	安砂小叶薯 Anshaxiaoyeshu
10	青源山药 Qingyuanshanyao	26	栾川野山药 Luanchuanyeshanyao	42	神农山4号 Shennongshan No.4	58	明淮1号 Minghuai No.1
11	太和山药 Taiheshanyao	27	孝义梧桐山药 Xiaoyiwutongshanyao	43	日本大和山药 Japan Daheshanyao	59	永丰淮山药 Yongfenghuaishanyao
12	温县2号 Wenxian No.2	28	佛手山药 Foshoushanyao	44	广西玉林山药 Guangxiyulinshanyao	60	瑞昌山药 Ruichangshanyao
13	山西太原山药 Shanxitaiyuanshanyao	29	勒家岭2号 Jinjialing No.2	45	紫玉淮山药 Ziyuhuaishanyao	61	阳明山山药 Yangmingshanshanyao
14	日本山药 Japan Shanyao	30	怀山药 Huaishanyao	46	太谷县南阳村山药 Taiguxiannanyangcunshanyao	62	明淮3号 Minghuai No.3
15	南京采山药 Nanjingcaishanyao	31	水晶山药 Shuijingshanyao	47	台湾紫山药 Taiwanzishanyao	63	苏蓣4号 Suyu No.4
16	惠楼山药 Huiloushanyao	32	晋城1号 Jincheng No.1	48	毛山药 Maoshanyao	64	苏蓣6号 Suyu No.6

编号 Code	种质名称 Accession name	编号 Code	种质名称 Accession name	编号 Code	种质名称 Accession name	编号 Code	种质名称 Accession name
1	太谷山药 Taigushanyao	17	河北小白嘴山药 Hebeixiaobaizuishanyao	33	寿阳山药 Shouyangshanyao	49	山东铁棍 Shandongtiegun
2	垣曲采山药 Yuanqucaishanyao	18	野山药 Yeshanyao	34	高阳紫药 Gaoyangziyao	50	芹峰淮山药 Qingfenghuaishanyao
3	高阳小白嘴 Gaoyangxiaobaizui	19	襄汾城关刘存山药 Xiangfenchengguanliucun shanyao	35	山西永济山药 Shanxiyongji shanyao	51	山西山药 Shanxishanyao
4	沁阳太谷山药 Qinyangtaigushanyao	20	日本青森山药 Japan Qinsengshanyao	36	南京采山药2号 Nanjingcaishanyao No. 2	52	嘉祥细长毛山药 Jiaxiangxichangmaoshanyao
5	沁野山药 Qinyeshanyao	21	丽江2号 Lijiang No.2	37	温引1号 Wenying No.1	53	白山药 Baishanyao
6	山东牛腿 Shandongniutui	22	晋城采 Jingchengcai	38	铁棍山药 Tiegunshanyao	54	自然薯蓣 Ziranshuyu
7	灌南山药 Guannanshanyao	23	河北棒药 Hebeibangyao	39	滨海山药 Binhaishanyao	55	日本白山药 Japan Baishanyao
8	上海毛山药 Shanghaimaoshanyao	24	武当山山药2号 Wudangshanshanyao No.2	40	广西农科院山药 Guangxinongkeyuan shanyao	56	南召山药 Nanzhaoshanyao
9	武当山山药1号 Wudangshanshanyao No.1	25	冀城2号 Jicheng No.2	41	沙滩园山药 Shantanyuanshanyao	57	安砂小叶薯 Anshaxiaoyeshu
10	青源山药 Qingyuanshanyao	26	栾川野山药 Luanchuanyeshanyao	42	神农山4号 Shennongshan No.4	58	明淮1号 Minghuai No.1
11	太和山药 Taiheshanyao	27	孝义梧桐山药 Xiaoyiwutongshanyao	43	日本大和山药 Japan Daheshanyao	59	永丰淮山药 Yongfenghuaishanyao
12	温县2号 Wenxian No.2	28	佛手山药 Foshoushanyao	44	广西玉林山药 Guangxiyulinshanyao	60	瑞昌山药 Ruichangshanyao
13	山西太原山药 Shanxitaiyuanshanyao	29	勒家岭2号 Jinjialing No.2	45	紫玉淮山药 Ziyuhuaishanyao	61	阳明山山药 Yangmingshanshanyao
14	日本山药 Japan Shanyao	30	怀山药 Huaishanyao	46	太谷县南阳村山药 Taiguxiannanyangcunshanyao	62	明淮3号 Minghuai No.3
15	南京采山药 Nanjingcaishanyao	31	水晶山药 Shuijingshanyao	47	台湾紫山药 Taiwanzishanyao	63	苏蓣4号 Suyu No.4
16	惠楼山药 Huiloushanyao	32	晋城1号 Jincheng No.1	48	毛山药 Maoshanyao	64	苏蓣6号 Suyu No.6

引物名称 Primers	引物正向序列 Forward sequence (5’-3’)	引物反向序列 Reverse sequence (5’-3’)	重复 Repeat	退火温度 Annealing temperature/℃
SY69	TGGTCGTGCTCGAAAAGTTA	TGAGTCCTCGAAAAGGGATG	(AT)₅	54
SY104	TGAGTAGATCCGAATAGTCTTTCC	GACCAAGCATTGCCTGAACT	(TA)₆	52
SY108	ATTGTCCTTGAATGAGTGCAT	CGGATTTGAACCGATGACTT	(TA)₉	53
SY158	AATCGTGAGGGTTCAAGTCC	TGGTGACACGAGGATTTTCA	(AGAT)₃	55
SY161	TGCTCCCTCTTTCTCCTTTG	GAAATGAAAAGGGATCGGATT	(TA)₆	53
SY196	AACGATTTTGGATTGGCTCA	CGAGAGATCAAATGAGTTCTTCA	(AATG)₃	53
SY216	GATGCTGGTTCGAAAGCAAT	GGCGCATTCCAAAATAACTC	(TTC)₄	52
SY280	GCGATTCTTCCACAATGGAC	GAGGAACTCCCGAACATTTG	(TCT)₄	52
SY302	CGAGTCGGGTTCTTCTCCTA	TAAGGCAAAGGGTCGAGAAA	(ATA)₅	52
SY326	CCATATCGCGGAACAATAAAA	CCGATTCCCCATCAAGTAAA	(TA)₅	53

引物名称 Primers	引物正向序列 Forward sequence (5’-3’)	引物反向序列 Reverse sequence (5’-3’)	重复 Repeat	退火温度 Annealing temperature/℃
SY69	TGGTCGTGCTCGAAAAGTTA	TGAGTCCTCGAAAAGGGATG	(AT)₅	54
SY104	TGAGTAGATCCGAATAGTCTTTCC	GACCAAGCATTGCCTGAACT	(TA)₆	52
SY108	ATTGTCCTTGAATGAGTGCAT	CGGATTTGAACCGATGACTT	(TA)₉	53
SY158	AATCGTGAGGGTTCAAGTCC	TGGTGACACGAGGATTTTCA	(AGAT)₃	55
SY161	TGCTCCCTCTTTCTCCTTTG	GAAATGAAAAGGGATCGGATT	(TA)₆	53
SY196	AACGATTTTGGATTGGCTCA	CGAGAGATCAAATGAGTTCTTCA	(AATG)₃	53
SY216	GATGCTGGTTCGAAAGCAAT	GGCGCATTCCAAAATAACTC	(TTC)₄	52
SY280	GCGATTCTTCCACAATGGAC	GAGGAACTCCCGAACATTTG	(TCT)₄	52
SY302	CGAGTCGGGTTCTTCTCCTA	TAAGGCAAAGGGTCGAGAAA	(ATA)₅	52
SY326	CCATATCGCGGAACAATAAAA	CCGATTCCCCATCAAGTAAA	(TA)₅	53

引物 Primer	扩增条带总数 Total number of amplified bands	多态性条带总数 Total number of polymorphic bands	PPB/%	Na	Ne	He	I
SY69	3	2	66.67	2	1.911 3	0.475 7	0.668 4
SY104	4	3	75.00	2	1.688 7	0.394 9	0.579 8
SY108	8	6	75.00	2	1.664 4	0.386 8	0.571 8
SY158	6	5	83.33	2	1.415 5	0.252 2	0.393 5
SY161	13	12	92.31	2	1.529 9	0.337 1	0.516 6
SY196	3	2	66.67	2	1.916 0	0.478 1	0.671 1
SY216	3	2	66.67	2	1.487 6	0.327 8	0.509 3
SY280	5	4	80.00	2	1.231 1	0.184 8	0.329 0
SY302	14	14	100.00	2	1.524 9	0.310 3	0.474 6
SY326	10	9	90.00	2	1.323 9	0.221 5	0.363 8
平均Mean	6.9	5.9	79.57	2	1.569 3	0.378 1	0.560 6
合计Total	69	59

基于cpSSR标记的山药种质资源DNA指纹图谱构建及遗传多样性分析

Genetic diversity analysis and construction of DNA fingerprint of yam (Dioscorea oppositeac Thunb.) germplasm by cpSSR marker

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编号No.	SY161	SY326	编号No.	SY161	SY326
1	011110000001	000001000	33	010001101001	001110000
2	010110010111	010010110	34	010000000001	000000000
3	011110011001	010001110	35	000000010001	000000001
4	110001101101	010010100	36	011110010001	000000000
5	110001101101	011011100	37	111000000001	001000000
6	000000000001	011000101	38	011110010011	001000000
7	011110010001	001010100	39	011110010011	101010000
8	010110010001	001110010	40	110001101001	010000100
9	110001101001	001110000	41	011110010011	010001100
10	110001101001	001110100	42	011110010001	001010000
11	010000000001	000000100	43	010001001011	000010100
12	110001101001	011010110	44	011110010011	000000000
13	110001101001	011110100	45	000000010001	000000000
14	110001101001	011110100	46	000000000000	000000000
15	110001101001	001110000	47	011110010001	000000000
16	011110010011	000000001	48	000000000110	000000000
17	011110010011	001110100	49	011110010001	000001000
18	110001101001	000000100	50	011000011001	010001100
19	011110010001	001000000	51	110111111101	011010100
20	010000000001	001000000	52	110110110101	001110100
21	110001101001	010011100	53	110110000101	001010100
22	011110010001	011110100	54	110110000101	000010000
23	011110010011	000000010	55	110111110111	001010000
24	010000000001	001000000	56	110111000101	000000000
25	100000000000	000000000	57	011100001001	000001000
26	001000010001	000000001	58	110011110101	001001100
27	111110010011	000000000	59	000000000100	000010100
28	010000011001	000000000	60	110110000101	110001100
29	010110011001	101000000	61	110111100101	100110100
30	110000101101	010001100	62	110111100101	000100100
31	010000000001	001010000	63	110111110101	010110100
32	010110010011	001010000	64	111110010001	010001100

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