应用拮抗和ISSR方法综合鉴定灰树花菌株

袁滨; 柯丽娜; 黄艺宁; 张志鸿; 连燕萍; 张丹凤

doi:10.19303/j.issn.1008-0384.2019.04.003

应用拮抗和ISSR方法综合鉴定灰树花菌株

doi: 10.19303/j.issn.1008-0384.2019.04.003

1.
福建省漳州市农业科学研究所, 福建漳州 363005
2.
漳州职业技术学院, 福建漳州 363000
3.
闽南师范大学, 福建漳州 363000

基金项目:

国家现代农业产业技术体系建设专项 CARS-20

详细信息

作者简介:
袁滨(1984-), 男, 硕士, 助理研究员, 研究方向:食用菌育种与栽培(E-mail:yuanbin07031@163.com)

通讯作者:
张丹凤(1979-), 女, 教授, 研究方向:食药用菌(E-mail:zhdanfeng@163.com)

中图分类号: S646
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- 文章访问数: 1078
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- 被引次数: 0
出版历程
- 收稿日期: 2018-11-28
- 修回日期: 2019-03-19
- 刊出日期: 2019-04-28

Identification of Grifola frondosa by Antagonistic Test and ISSR

1.
Zhangzhou Institute of Agricultural Science, Zhangzhou, Fujian 363005, China
2.
Zhangzhou Vocational and Technical College, Zhangzhou, Fujian 363000, China
3.
Minnan Normal University, Zhangzhou, Fujian 363000, China

摘要

摘要: 目的准确、科学地鉴定灰树花菌株，明确其亲缘关系。方法采用拮抗和ISSR分子标记对供试的11个灰树花菌株进行鉴定和遗传多样性分析。结果拮抗反应表明，供试菌株之间拮抗作用有一定差异，根据拮抗反应的有无，供试菌株分为4组。ISSR分子标记分析表明，当相似性系数达到0.87水平时，供试菌株聚成5组，第1组包括菌株1（石家庄灰树花）、2（台灰）、10（XG-W）、5（Gr0001+2）、6（CN）、7（雪圆）；第2组为菌株11（CN-9）；第3组为菌株4（Gr0003）；第4组包括菌株8（H）、9（Hokto）；第5组为菌株3（Gr20）。结论拮抗反应与ISSR两种方法结论较一致，推测石家庄灰树花与台灰，Gr0001+2、CN与雪圆，H与Hokto这3组菌株分别来源于同一个菌株，属于同种异名现象Gr0003、H和Hokto亲缘关系较远；Gr20与其他菌株亲缘关系最远。
- 拮抗 /
- ISSR技术 /
- 灰树花 /
- 鉴定 /
- 聚类分析
Abstract: Objective To reliably identify strains of Grifola frondosa and precisely determine the genetic relationship among them. Method The identification and genetic diversity analysis on 11 strains of G. frondosa were carried out by the combined use of the antagonistic challenging test and ISSR. Result The antagonistic test on the strains demonstrated apparent differences on their responses. Presence or absence of antagonism allowed the classification of the strains into 4 distinct groups. Meanwhile, from the ISSR analysis, when the similarity coefficient reached 0.87, the strains were clustered into 5 groups. They were Group 1 that included Strain #1 (Shijiazhuang ash tree flower), Strain #2 (stage ash), Strain #10 (XG-W), Strain #5 (Gr0001+2), Strain #6 (CN), and Strain #7 (snow circle); Group 2, Strain #11 (CN-9); Group 3, Strain #4 (Gr0003); Group 4, Strain #8 (H) and Strain #9 (Hokto); and, Group 5, Strain #3 (Gr20). Conclusion It appeared that Strain #1 and Strain #2 belonged to a same species; so were Strains #5, Strain #6 and Strain #7, and Strains #8 and Strain #9. At the similarity coefficient of 0.8, Strain #4 could be clustered with Strain #8 and Strain #9 with slight differences indicating a distance on genetic relationship among them. Strain #3 had a lowest coefficient of 0.34, therefore, it was most remotely related to all.
- antagonistic effect /
- ISSR /
- Grifola frondosa /
- identification /
- cluster analysis

HTML全文

图 1 灰树花菌丝拮抗类型

注：A为隆起拮抗图谱，B为拮抗线图谱，C为沟状拮抗图谱。

Figure 1. Antagonistic classes of G. frondosa mycelia

Note: A:Uplift antagonistic map, B:Antagonistic line map, C:Groove antagonistic map.

下载: 全尺寸图片幻灯片

图 2 引物P1、P2对供试菌株扩增的ISSR

注：指纹图谱从左到右菌株编号依次为1~11，DNA Marker为DL2000。

Figure 2. ISSR fingerprints amplified by primers P1 and P2

Note: Fingerprint of strains from left to right are 1-11, Marker is DL2000.

下载: 全尺寸图片幻灯片

图 3 供试灰树花菌株ISSR多态性的聚类

Figure 3. Dendrogram based on ISSR of G. frondosa strains

下载: 全尺寸图片幻灯片

表 1 供试的灰树花菌株

Table 1. G. frondosa strains tested

编号 No.	菌株名称 Strain	来源 Origin
1	石家庄灰树花	河北省农业科学院
2	台灰	台湾引进
3	Gr20	制种户赠送
4	Gr0003	福建农林大学
5	Gr0001+2	福建农林大学
6	CN	三明真菌所
7	雪圆	上海雪榕公司
8	H	日本产品分离
9	Hokto	日本产品分离
10	XG-W	三明真菌所
11	CN-9	三明真菌所

下载: 导出CSV

表 2 供试菌株鉴定所用ISSR引物

Table 2. ISSR primers for strain identification

引物名称 Primer No.	引物序列 Primer sequence(5′-3′)	退火温度 Annealing temperature/℃
P1	TGCACACACACACAC	46
P2	GTGACACACACACAC	46
P4	GGATGCAACACACACACAC	50
P5	CGTGTGTGTGTGTGT	46
P6	AGTGTGTGTGTGTGT	44
P7	CCAGTGGTGGTGGTG	50
P8	GGAGTGGTGGTGGTG	50
P9	AGAGAGAGAGAGAGAGG	48
P10	GAGAGAGAGAGAGAGAC	48
P11	GAGAGAGAGAGAGAGAAC	48
P12	AGAGAGAGAGAGAGAGGC	50
P16	TGTGTGTGTGTGTGTGGA	48
P17	ACACACACACACACAC	46
P18	ACACACACACACACACC	48
P19	ACACACACACACACACCT	48
P20	ACACACACACACACACCTG	50
P23	GAGAGAGAGAGAGAGACT	48
P25	GAGAGAGAGAGAGAGACC	50
P26	CACCACACACACACACA	48
P28	GTATGTATGTATGTATGC	48

下载: 导出CSV

表 3 PCR反应扩增体系

Table 3. PCR reaction amplification

组分 Component	移取量 Volume
重蒸水ddH₂O	17.2 μL
模板Template(100 ng)	10 ng
脱氧核糖核苷三磷酸dNTP(2.5 mmol·L^-1)	2.5 μL
缓冲液10×PCR Buffer(含1.5μL MgCl₂25 mmmol·L^-1)	2.5 μL
镁离子Mg²⁺(25 mmmol·L^-1)	0.5 μL
引物Primer(10 μmmol·L^-1)	1 μL
Taq酶Taq polymerase(5 U·μL^-1)	0.3 μL
总体积Total volume	25 μL

下载: 导出CSV

表 4 供试菌株间的拮抗反应

Table 4. Antagonistic responses of strains

No.	1	2	3	4	5	6	7	8	9	10	11
1	-
2	-	-
3	+	+	-
4	+	+	+	-
5	-	-	+	+	-
6	-	-	+	+	-	-
7	-	-	+	+	-	-	-
8	+	+	+	+	+	+	+	-
9	+	+	+	+	+	+	+	-	-
10	-	-	+	+	-	-	-	+	+	-
11	-	-	+	+	-	-	-	+	+	-	-
注:‘+’表示拮抗性明显；‘－’表示没有拮抗。 Note:‘+’indicates apparent antagonism; ‘-’indicates absence of antagonism.

下载: 导出CSV

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