鉴别紫芝菌株的PCR引物筛选及其序列比对验证

钟礼义; 陈体强; 刘新锐; 应正河; 杨驰

doi:10.19303/j.issn.1008-0384.2020.07.005

鉴别紫芝菌株的PCR引物筛选及其序列比对验证

doi: 10.19303/j.issn.1008-0384.2020.07.005

1.
福建武平县食用菌技术推广服务站，福建　武平　364300
2.
福建省农业科学院食用菌研究所，福建　福州　350014
3.
福建农林大学菌物研究中心，福建　福州　350002

基金项目: 福建省财政专项－福建省农业科学院科技创新团队建设项目（STIT2017-2-8，STIT2017-1-6）

详细信息

作者简介:
钟礼义（1968−），男，推广研究员，研究方向：食药用菌技术推广应用（E-mail：fjzly99@163.com）

通讯作者:
陈体强（1968−），男，研究员，研究方向：药用菌与闽产中药材（E-mail：chen_tiqiang@189.cn）

中图分类号: S 646.2
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出版历程
- 收稿日期: 2020-01-16
- 修回日期: 2020-06-03
- 刊出日期: 2020-07-31

Selection and Sequence Alignment of PCR Primers for Identifying Zizhi Strain

1.
Fujian Wuping Mushroom Extension Station, Wuping, Fujian 364000, China
2.
Edible & Medicinal Fungi Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350014, China
3.
Mycological Research Center, Fujian Agriculture and Forestry University, Fuzhou, Fujian 350002, China

摘要

摘要: 目的紫芝S2（品种名：武芝2号）是近年来在福建及周边省份推广应用的紫芝栽培新品种，为了避免与遗传背景不同的栽培菌株混杂而建立有效的分子标记。方法采用PCR扩增筛选条带清晰稳定、呈现多态性的引物，根据菌株间UPMGA聚类构建系统发生树，通过遗传距离确定主要栽培菌株间的亲缘关系，并与菌株间的拮抗反应结果相映证，进而与‘紫芝S2’基因组序列进行比对验证。结果筛选得到能清晰且稳定地扩增出特异性或多态性条带的2个RAPD-PCR引物和3个ISSR-PCR引物，比对发现这5个引物短序列与‘紫芝S2’基因组序列完全匹配上的位点与数目不同。结论基于紫芝S2基因组序列比对，确认其中3个引物（ISSR13、S1326和S1506）可以有效地用于紫芝栽培菌株鉴定。
- 紫芝 /
- 栽培菌株 /
- 分子标记 /
- PCR引物 /
- 序列比对
Abstract: Objective The molecular markers of the cultivated strain of Ganoderma sinense, Zizhi S2 (aka Wu-Zhi No. 2), recently popularized in Fujian and surrounding provinces were studied to facilitate the authentication of the medicinal fungus. Method Relevant primers of Zizhi S2 showing clear and stable bands and polymorphism were screened using PCR. Phylogenetic tree of UPMGA clustering analysis on the verified authentic cultivars was constructed to determine their relationship by genetic distance as well as antagonistic reaction. Subsequently, sequences of the selected primers were blasted on the genome of Zizhi S2 to validate the methodology. Result There were 2 RAPD-PCR and 3 ISSR-PCR primers found to clearly and stably amplify the specific or polymorphic bands. However, the sites and numbers on the scaffolds of the Zizhi S2 genome that matched the sequences of the 5 primers were not same. Conclusion It was confirmed that three primers(ISSR13, S1326, and S1506)could be effectively used for identification of Zizhi cultivated strains based on sequences alignment with the genome of G. sinense strain Zizhi S2.
- Ganoderma sinense /
- cultivated strain /
- molecular markers /
- PCR primers /
- sequence alignment

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图 1 紫芝菌株间的拮抗现象

注：A：紫芝S2-紫芝S2；B：紫芝S2-紫芝X5；C：紫芝S2-紫芝1；D：紫芝S2-紫芝2；E：紫芝S2-紫芝3；F：紫芝S2-紫芝4。

Figure 1. Antagonism between different Zizhi strains

Note: A: Zizhi S2 vs. Zizhi S2; B: Zizhi S2 vs. Zizhi X5; C: Zizhi S2 vs. Zizhi 1; D: Zizhi S2 vs. Zizhi 2; E: Zizhi S2 vs. Zizhi 3; and, F: Zizhi S2 vs. Zizhi 4.

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图 2 RAPD-PCR扩增图谱

注：泳道1～6：紫芝S2、紫芝X5、紫芝-1、紫芝-2、紫芝-3和紫芝-4, M为分子标记DL2000。

Figure 2. RAPD-PCR amplifications

Note: Electrophoretic lines 1-6: Zizhi S2, Zizhi X5, Zizhi 1, Zizhi 2, Zizhi 3, and Zizhi 4; M: molecular marker DL2000.

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图 3 ISSR -PCR扩增图谱

注：泳道1～6：紫芝S2、紫芝X5、紫芝-1、紫芝-2、紫芝-3和紫芝-4, M为分子标记物DL2000。

Figure 3. ISSR-PCR amplifications

Note: Line 1-6: Electrophoretic results of Zizhi S2, Zizhi X5, Zizhi 1, Zizhi 2, Zizhi 3, Zizhi 4 and the M is the molecular marker DL2000.

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图 4 紫芝菌株的遗传聚类图

Figure 4. Clustering dendrogram of Zizhi strains

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表 1 供试紫芝菌株信息

Table 1. Strains of cultivated Zizhi used in study

编号 No.	菌株名 Strain name	来源 Source
1	紫芝 S2	福建省农作物品种审定委员会认定品种（武芝2号）
	Zizhi S2	New varieties identified by Fujian Crop Variety Approval Committee（Wuzhi No.2）
2	紫芝 X5	福建省武平县食用菌技术推广服务站
	Zizhi X5	Fujian Wuping County Edible Fungi Technology Extension Service Statio
3	紫芝1	华中农业大学菌种实验中心
	Zizhi 1	Microbial Species Laboratory Center of Huazhong Agricultural Universit
4	紫芝2	上海市农业科学院食用菌研究所
	Zizhi 2	Institute of Edible Fungi, Shanghai Academy of Agricultural Sciences
5	紫芝3	江苏省江都天达食用菌研究所
	Zizhi 3	Jiangdu Tianda Institute of Edible Fungi, Jiangsu Province
6	紫芝4	辽宁省锦州市亚泰食用菌研究所
	Zizhi4	Yatai Institute of Edible Fungi, Jinzhou City, Liaoning Province

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表 2 PCR引物与基因组比对结果

Table 2. Blast result of PCR primer sequences on genomes

引物名称_碱基数 Primer name _Base number	比对上引物序列的 Scaffold 数目 All scaffold number	与引物匹配的位点数 All primer number	比对相似度 Identity（%）
不允许错配（空位为0）No mismatches allowed（Gap=0）
ISSR13_14	5	6	100
S1326_10	48	158	100
S1506_10	47	172	100
允许一个错配（空位为0）Allow one mismatch（Gap=0）
ISSR2_17	11	14	93.75～94.12
ISSR13_14	18	31	100
S1326_10	48	158	100
S1506_10	47	172	100
注：引物ISSR13短序列出现在Scaffold_1上2个位点，引物S1326出现在Scaffold_1上15个位点；S1506引物出现在Scaffold_1上17个位点，出现在Scaffold_60上5个位点。 Note: Short sequence of ISSR 13 appeared at two sites on Scaffold No. 1; S1326 at 15 sites on Scaffold No. 1; and S1506 at 17 sites on Scaffold No. 1 and 5 sites on Scaffold No. 60.

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