Identification of Grifola frondosa by Antagonistic Test and ISSR
-
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。 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. -
Key words:
- antagonistic effect /
- ISSR /
- Grifola frondosa /
- identification /
- cluster analysis
-
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
Origin1 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 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 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 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. -
[1] 刘佳, 包海鹰, 图力古尔.灰树花化学成分及药理活性研究进展[J].菌物研究, 2018, 16(3):150-157. http://d.old.wanfangdata.com.cn/Periodical/jwyj201803004LIU J, BAO H Y, BAU Tolgor. Chemical Constituents and Pharmacological Activities of Grifola frondosa[J]. Journal of Fungal Research, 2018, 16(3):150-157. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/jwyj201803004 [2] 郭健.灰树花标准菌株库的建立及优良菌株筛选[D].长春: 吉林农业大学, 2016.GUO J.Establishment of standard strain Bank of Grifola frondosa and screening of excellent strains[D]. Changchun: Jilin Agricultural University, 2016. (in Chinese) [3] 杨杨.灰树花EST-SSR引物开发及遗传多样性研究[D].长春: 吉林农业大学, 2016.YANG Y.Development of EST-SSR Primers and Study on Genetic Diversity of Grifola frondosa[D]. Changchun: Jilin Agricultural University, 2016. (in Chinese) [4] 曹鸿雁.黑木耳等9种食用菌的种质资源分子鉴定[D].福州: 福建农林大学, 2009.CAO H Y. Molecular Identification of Germplasm Resources of 9 Edible Fungi such as Auricularia auricula[D]. Fuzhou: Fujian Agricultural and Forestry University, 2009. (in Chinese) [5] 田君琪, 韩晓伟.灰树花对免疫系统的影响[J].吉林中医药, 2018, 38(10):1203-1205. http://d.old.wanfangdata.com.cn/Periodical/jlzyy201810026TIAN J Q, HAN X W. Effect of Grifola frondosa on immune system[J].Jilin Traditional Chinese Medicine, 2018, 38(10):1203-1205. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/jlzyy201810026 [6] 刘佳, 包海鹰, 图力古尔, 等.灰树花提取物的体内抗肿瘤及免疫活性研究[J/OL].西北农林科技大学学报(自然科学版), 2019(4): 1-3[2018-11-27].https://doi.org/10.13207/j.cnki.jnwafu.2019.04.014.LIU J, BAO H Y, BAU Tolgor, et al. Anti-tumor and Immune Activity of Grifola frondosa Extract in Vitro[J/OL].Journal of Northwest A & F University(Natural Science Edition), 2019(4): 1-3[2018-11-27].https://doi.org/10.13207/j.cnki.jnwafu.2019.04.014. (in Chinese) [7] 胡卫红.灰树花工厂液体菌种生产工艺的研究[J].科学中国人, 2016(20):12. http://epub.cnki.net/grid2008/detail.aspx?filename=KXZG201620008&dbname=CJFN2016HU W H. Study on Production Technology of Liquid Strains in Grifola frondosa Factory[J]. Scientific Chinese, 2016(20):12. (in Chinese) http://epub.cnki.net/grid2008/detail.aspx?filename=KXZG201620008&dbname=CJFN2016 [8] 张立霞, 林绪涛, 李军, 等.灰树花多糖及其复合多糖对肺纤维化大鼠的作用[J].中国老年学杂志, 2018, 38(17):4242-4245. doi: 10.3969/j.issn.1005-9202.2018.17.054ZHANG L X, LIN X T, LI J, et al.Effects of Grifola frondosa polysaccharide and its compound polysaccharide on pulmonary fibrosis in rats[J].Chinese Journal of Gerontology, 2018, 38(17):4242-4245. (in Chinese) doi: 10.3969/j.issn.1005-9202.2018.17.054 [9] 陈沛, 刘会平, 孙娜新, 等.灰树花多糖的分离纯化及其体外抗肿瘤活性[J].现代食品科技, 2018, 34(6):107-114. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=gzspgykj201806015CHEN P, LIU H P, SUN N X, et al. Isolation and Purification of Polysaccharide from Grifola frondosa and Its in vitro Antitumor Activity[J]. Modern Food Science and Technology, 2018, 34(6):107-114. (in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=gzspgykj201806015 [10] 张医芝.灰树花菌丝体液体深层发酵工艺优化及降血糖和抗肿瘤活性研究[D].长春: 吉林大学, 2017.ZHANG Y Z. Study on Optimization of submerged fermentation of Grifola frondosa mycelium and its hypoglycemic and antineoplastic activities[D]. Changchun: Jilin University, 2017. (in Chinese) [11] 郭家瑞.灰树花多糖高产菌株的选育及其发酵特性研究[D].郑州: 河南工业大学, 2011.GUO J R. Breeding and Fermentation Characteristics of Grifola frondosa Polysaccharide High-yielding Strain[D].Zhengzhou: Henan University of Technology, 2011. (in Chinese) [12] 刘振伟, 史秀娟.灰树花杂交良种的选育[J].山东农业科学, 2005(1):27-28, 32. doi: 10.3969/j.issn.1001-4942.2005.01.008LIU Z W, S X J. Breeding of improved hybrid Grifola frondosa[J]. Shandong Agricultural Science, 2005(1):27-28, 32. (in Chinese) doi: 10.3969/j.issn.1001-4942.2005.01.008 [13] 陈世通, 李荣春.食用菌育种方法的研究现状、存在的问题及展望[J].安徽农业科学, 2012, 40(10):5850-5852. doi: 10.3969/j.issn.0517-6611.2012.10.049CHEN S T, LI R C.Present Research on Breeding Methods of Edible Fungi and the Problems in It as Well as the Prosipects[J]. Anhui Agricultural Science, 2012, 40(10):5850-5852. (in Chinese) doi: 10.3969/j.issn.0517-6611.2012.10.049 [14] 刘盛荣, 张维瑞, 柯斌榕, 等.基于拮抗试验的杏鲍菇菌株分类研究[J].食药用菌, 2015, 23(1):33-36.LIU S R, ZHANG W R, KE B R, et al.Study on classification of Pleurotus eryngii strains based on antagonism test[J].Edible and Medicinal Mushrooms, 2015, 23(1):33-36. (in Chinese) [15] 李维焕, 蔡德华, 郑芳, 等.秀珍菇菌株的亲缘关系分析[J].食品科学, 2010, 31(17):267-271. http://d.old.wanfangdata.com.cn/Periodical/spkx201017060LI W H, CAI D H, ZHENG F, et al.Analysis of the Phylogenetic Relationships of Pleurotus pulmonarius Strains[J]. Food Science, 2010, 31(17):267-271. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/spkx201017060 [16] 唐传红, 张劲松, 陈明杰, 等.利用拮抗试验和RAPD对灵芝属菌株进行分类研究[J].微生物学通报, 2005(5):72-76. doi: 10.3969/j.issn.0253-2654.2005.05.015TANG C H, ZHANG J S, CHEN M J, et al.Study on Classification of Strains of Ganotterma by Anatagonistic Effect and Rapd[J]. Microbiology China, 2005(5):72-76. (in Chinese) doi: 10.3969/j.issn.0253-2654.2005.05.015 [17] 郑亚凤.高多糖的姬松茸和灰树花品种及其生物活性的研究[D].福州: 福建农林大学, 2008.ZHENG Y F. Studies on the Varieties of Agaricus blazei Murrill and Grifola frondosa with High Polysaccharides and Their Biological Activities[D]. Fuzhou: Fujian Agricultural and Forestry University, 2008. (in Chinese) [18] 李辉平.ISSR在食用菌遗传多样性研究中的应用[D].北京: 中国农业科学院, 2007.LI H P. Application of ISSR in the Study of Genetic Diversity of Edible Fungi[D]. Beijing: Chinese Academy of Agricultural Sciences, 2007. (in Chinese) [19] 孟虎, 孙国琴, 睢韡, 等.ISSR技术在食用菌研究上的应用[J].北方园艺, 2016(5):207-210. http://d.old.wanfangdata.com.cn/Periodical/bfyany201605053MENG H, SUN G Q, SUI W, et al.The Application of ISSR in Edible Fungi Research[J]. Northern Horticulture, 2016(5):207-210. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/bfyany201605053 [20] 贾媛, 李忠海, 黎继烈, 等.ISSR指纹图谱技术在种质资源研究上的应用[J].河北农业科学, 2013, 17(5):63-68. doi: 10.3969/j.issn.1088-1631.2013.05.019JIA Y, LI Z H, LI J L, et al.The Application of ISSR Fingerprinting Technology in Germplasm Resource Research[J]. Journal of Hebei Agricultural Sciences, 2013, 17(5):63-68. (in Chinese) doi: 10.3969/j.issn.1088-1631.2013.05.019 [21] 王春晖, 尹永刚, 胡汝晓.基于ISSR和RAPD标记的八株灰树花栽培菌株遗传多样性分析[J].食用菌学报, 2013, (4):1-5. doi: 10.3969/j.issn.1005-9873.2013.04.001WANG C H, YIN Y G, HU R X, et al.Analysis of genetic diversity of eight Grifola frondosa cultivated strains based on ISSR and RAPD markers[J].Journal of Edible Fungi, 2013, (4):1-5. (in Chinese) doi: 10.3969/j.issn.1005-9873.2013.04.001 [22] 温志强, 熊芳, 陈吉娜, 等.分子标记鉴别灰树花种质资源的研究[J].热带作物学报, 2011, 32(7):1330-1336. doi: 10.3969/j.issn.1000-2561.2011.07.028WEN Z Q, XIONG F, CHEN J N, et al. Identification of Grifola frondosa Germplasm Resources by Molecular Markers[J].Journal of Tropical Crops, 2011, 32(7):1330-1336. (in Chinese) doi: 10.3969/j.issn.1000-2561.2011.07.028 [23] 顾万春.统计遗传学[M].北京:科学出版社, 2004:179.GU W C. Statistical Genetics[M]. Beijing:Science Press, 2004:179. (in Chinese) [24] 岳万松.云南牛肝菌的分子鉴定及其多糖抗氧化性研究[D].昆明: 云南民族大学, 2015.YUE W S. Molecular identification of Boletus yunnanensis and antioxidant activity of its polysaccharides[D].Kunming: Yunnan Nationalities University, 2015. (in Chinese) [25] 戚元成, 张小强, 高玉千, 等.中国栽培糙皮侧耳品种体细胞不亲和性实验与RAPD分析结果的比较[J].菌物学报, 2010, 29(3):379-388. http://d.old.wanfangdata.com.cn/Periodical/jwxt201003010QI Y C, ZHANG X Q, GAO Y Q, et al.Comparison between somatic incompatibility and RAPD analysis of cultivated Pleurotus ostreatus in China[J]. Mycosystema, 2010, 29(3):379-388. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/jwxt201003010 [26] 宿红艳, 王磊, 明永飞, 等. ISSR分子标记技术在金针菇菌株鉴别中的应用[J].生态学杂志, 2008, 27(10):1725-1728. http://d.old.wanfangdata.com.cn/Periodical/stxzz200810015SU H Y, WANG L, MING Y F, et al.Application of ISSR in identifying Flammulina velutipes strains[J]. Journal of Ecology, 2008, 27(10):1725-1728. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/stxzz200810015 [27] 张介驰, 马庆芳, 张丕奇, 等.用ISSR分子标记鉴别东北地区黑木耳生产菌株的研究[J].菌物学报, 2007(4):534-538. http://d.old.wanfangdata.com.cn/Periodical/jwxt200704008ZHANG J C, MA Q F, ZHANG P Q, et al. Identification of Auricularia auricula producing strains in Northeast China by ISSR molecular markers[J].Journal of Mycology, 2007(4):534-538. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/jwxt200704008 [28] 秦莲花, 宋春艳, 谭琦, 等.用ITS和ISSR分子标记技术鉴别香菇生产用种[J].菌物学报, 2006, 25(1):94-100. http://d.old.wanfangdata.com.cn/Periodical/jwxt200601015QIN L H, SONG C Y, TANG Q, et al. Identification of Lentinus edodes varieties by ITS and ISSR molecular markers[J]. Mycosystema, 2006, 25(1):94-100. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/jwxt200601015 [29] 王守现, 刘宇, 耿小丽, 等.北京地区8个白金针菇主栽菌株的鉴别研究[J].华北农学报, 2009, 24(1):219-222. http://d.old.wanfangdata.com.cn/Periodical/hbnxb200901048WANG S X, LIU Y, GENG X L, et al. Identification of eight main strains of Flammulina velutipes in Beijing[J]. Acta Agriculturae Boreali-Sinica, 2009, 24(1):219-222. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/hbnxb200901048 [30] 王小艳, 王春晖, 姜性坚, 等.杏鲍菇多孢分离菌株ISSR、RAPD分子标记及体细胞不亲和性分析[J].中国食用菌, 2015, 34(3):57-60, 65. http://d.old.wanfangdata.com.cn/Periodical/zgsyj201503014WANG X Y, WANG C H, JIANG X J, et al. Analysis on Multi-spore Isolated Pleurotus eryngii Using ISSR and RAPD Molecular[J]. Edible Fungi of Ching, 2015, 34(3):57-60, 65. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/zgsyj201503014 [31] 周洁. ISSR分子标记在姬菇杂交菌株鉴定中的应用[D].雅安: 四川农业大学, 2010.ZHOU J. Application of ISSR Molecular Markers in Identification of Hybrid Strains of Agaricus blazei[D]. Yaan: Sichuan Agricultural University, 2010. (in Chinese) [32] 林戎斌, 张慧, 林陈强, 等. ISSR、RAPD和SRAP分子标记技术在姬松茸菌株鉴定上的应用比较[J].福建农业学报, 2012, 27(2):149-152. doi: 10.3969/j.issn.1008-0384.2012.02.008LIN R B, ZHANG H, LIN C Q, et al.DNA Fingerprinting and Genetic Diversity of Cultivated Strains of Agaricus blazei Murill[J]. Fujian Journal of Agricultural Sciences, 2012, 27(2):149-152. (in Chinese) doi: 10.3969/j.issn.1008-0384.2012.02.008 [33] 杨军, 徐吉, 宋一鸣, 等.耐高温型灰树花菌株筛选及供试灰树花亲缘关系分析[J].基因组学与应用生物学, 2016, 35(2):396-405. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=jyzxyyyswx201602026YANG J, XU J, SONG Y M, et al.Screen of Grifola frondosa Thermotolerant Strains and Analysis of the Genetic Relationship of Candidate Strains[J]. Genomics and Applied Biology, 2016, 35(2):396-405. (in Chinese) http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=jyzxyyyswx201602026 [34] 王守现, 刘宇, 张英春, 等.六个灰树花菌株遗传多样性分析[J].北方园艺, 2010(1):201-204. http://d.old.wanfangdata.com.cn/Periodical/bfyany201001083WANG S X, LIU Y, ZHANG Y C, et al.The analysis of genetic diversity of six Grifola frondosa Strains[J]. Northern Horticulture, 2010(1):201-204. (in Chinese) http://d.old.wanfangdata.com.cn/Periodical/bfyany201001083