平菇单核体与双核体差异的RAPD分析

张朝辉; 王勋; 陈天航; 梅会元; 韩盼盼; 刘天翔; 王振河; 邱立友

doi:10.19303/j.issn.1008-0384.2022.005.013

平菇单核体与双核体差异的RAPD分析

doi: 10.19303/j.issn.1008-0384.2022.005.013

张朝辉^{1, 2,},
王勋¹,
陈天航²,
梅会元²,
韩盼盼²,
刘天翔¹,
王振河²,
邱立友^1, ,

1.
河南农业大学生命科学学院/农业农村部农业微生物酶工程重点实验室，河南　郑州　450002
2.
河南科技学院生命科技学院，河南　新乡　453003

基金项目: 河南省科技攻关计划项目（202102110019、212102110430）；国家级大学生创新创业训练计划项目（202110467035X）

详细信息

作者简介:
张朝辉（1983−），男，博士，副教授，主要从事食药用菌生理生化研究（E-mail：p686@163.com）

通讯作者:
邱立友（1963−），男，博士，教授，主要从事真菌学研究（E-mail：qliyou@henau.edu.cn）

中图分类号: S 646.1+4
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- 文章访问数: 387
- HTML全文浏览量: 153
- PDF下载量: 16
- 被引次数: 0
出版历程
- 收稿日期: 2022-03-02
- 修回日期: 2022-04-04
- 刊出日期: 2022-05-28

RAPD Analysis on Differences between Monokaryotic and Dikaryotic Pleurotus ostreatus

1.
College of Life Sciences, Henan Agricultural University/Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture and Rural Affairs, Zhengzhou, Henan　450002, China
2.
College of Life Science and Technology, Henan Institute of Science and Technology, Xinxiang, Henan　453003, China

摘要

摘要: 目的探究平菇单双核菌丝体间的差异，为平菇品种培育提供技术理论基础。方法从平菇TD300的担孢子中分离得到两个单核菌株MK13和MK3，通过交配得到优良双核菌株DK300，进行平板试验和出菇试验，测定4个菌株的菌丝生长状况和出菇产量，再利用随机扩增多态性DNA（Randomly Amplified Polymorphic DNA，RAPD）标记技术检测4个菌株之间的遗传差异性。结果 DK300与亲本TD300在菌丝生长状况和产量上没有显著性差异，但MK13、MK3和交配所得的DK300与亲本TD300间存在较大的遗传差异性，而MK13、MK3和DK300间遗传差异性较小，尤其是MK3和DK300间遗传差异性最小。结论经过减数分裂形成担孢子时可能发生有基因重组，而通过两个单核菌株交配得到双核菌株过程则较少发生基因重组。
- 平菇 /
- 单核体 /
- 双核体 /
- 产量 /
- 遗传差异性
Abstract: Objective Differences between uni- and bi-nucleate mycelia of Pleurotus ostreatus were studied to aid the mushroom breeding and cultivation. Method Monokaryons MK13 and MK3 were isolated from basidiospores of P. ostreatus TD300 and mated to obtain the dikaryotic DK300. Conventional plate and fruiting tests were applied to compare the mycelial growth and fruiting body yield of the 4 strains. Genetic differences of the strains were determined by random amplified polymorphic DNA (RAPD) markers. Result There were no significant differences in mycelial growth and fruiting body yield between DK300 and TD300. However, a significant genetic difference existed between the offspring (i.e., MK13, MK3, and DK300) and parent TD300. The differences among MK13, MK3, and DK300 were minute, especially between MK3 and DK300. Conclusion Gene recombination might occur during the basidiospore formation by meiosis, but less likely in the mating of two monokaryotic strains to generate a dikaryotic offspring.
- Pleurotus ostreatus /
- monokaryon /
- dikaryon /
- yield /
- genetic diversity

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图 1 菌株在不同培养基中生长状况

Figure 1. Mycelial growth of strains in different culture media

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图 2 DNA电泳检测结果

M为λ-Hind Ⅲ。

Figure 2. DNA electrophoresis of 4 mushroom strains

M: λ-Hind III.

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图 3 随机引物扩增电泳图谱

Figure 3. PCR electrophoresis using random primers

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图 4 RAPD聚类分析

Figure 4. RAPD clustering on mushroom strains

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表 1 供试随机引物

Table 1. Random primers applied

引物编号 Primer number	引物序列（5′-3′） Primer sequence(5′−3′)
S10	CTGCTGGGAC
S22	TGCCGAGCTG
S23	AGTCAGCCAC
S28	GTGACGTAGG
S36	AGCCAGCGAA
S39	CAAACGTCGG
S43	GTCGCCGTCA
S62	GTGAGGCGTC
S75	GACGGATCAG
S78	TGAGTGGGTG
S79	GTTGCCAGCC
S124	GGTGATCAGG
S126	GGGAATTCGG

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表 2 菌株的菌丝形态

Table 2. Mycelium morphology of tested strains

菌株 Bacterial strain	菌落形态 Colonial morphology	菌丝长势 Mycelium growth vigor	菌丝颜色 Color of mycelium
TD300	半气生型 Semi-air type	+	浅白 Light white
DK300	半气生型 Semi-air type	++	白 White
MK13	匍匐型 Prostrate type	+++	白 White
MK3	匍匐型 Prostrate type	+++	白 White
+++：粗壮浓密；++：较粗壮浓密；+：纤细稀疏。 +++: thick; ++: thick and dense; +: slender sparse.

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表 3 菌株出菇情况及子实体性状

Table 3. Germination and fruiting body properties of mushrooms

菌株 Bacterial strain	出菇情况 Mushroom-budding	子实体形状 Fruit-body shape	子实体颜色 Fruit-body color
TD300	出菇 Producing mushroom	贝壳状 Conchoid type	深褐色 Dark brown
DK300	出菇 Producing mushroom	扇叶状 Fan leaf type	浅褐色 Light brown
MK13	不出菇 No mushrooms	–	–
MK3	不出菇 No mushrooms	–	–
–：不出菇。 –: No mushrooms.

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表 4 菌株出菇产量

Table 4. Yields of mushrooms

菌株 Strain	第一茬均产量 Average yield of the first crop/ （kg·袋⁻¹）	第二茬均产量 Average yield of the second crop/ （kg·袋⁻¹）	平均总产量 Average total output/ （kg·袋⁻¹）
TD300	0.273 a	0.139 a	0.412 a
DK300	0.285 a	0.114 b	0.399 a
MK13	–	–	–
MK3	–	–	–
表中不同字母表示差异显著（P＜0.05）；–：不出菇。 Different letters indicate significant difference at P＜0.05; –: no mushrooms.

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参考文献(25)

[1]	戴玉成, 周丽伟, 杨祝良, 等. 中国食用菌名录 [J]. 菌物学报, 2010, 29(1):1−21. DAI Y C, ZHOU L W, YANG Z L, et al. A revised checklist of edible fungi in China [J]. Mycosystema, 2010, 29(1): 1−21.(in Chinese)
[2]	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. QI 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)
[3]	刘海旺, 梁丽梅, 黄日保, 等. 平菇菌株栽培试验初报 [J]. 食用菌, 2021, 43(4):29−30,33. doi: 10.3969/j.issn.1000-8357.2021.04.008 LIU H W, LIANG L M, HUANG R B, et al. Preliminary report on cultivation test of Pleurotus ostreatus strains [J]. Edible Fungi, 2021, 43(4): 29−30,33.(in Chinese) doi: 10.3969/j.issn.1000-8357.2021.04.008
[4]	GHAREHAGHAJI A N, GOLTAPEH E M, MASIHA S, et al. Hybrid production of oyster mushroom Pleurotus ostreatus (Jacq: Fries) kummer [J]. Pakistan Journal of Biological Sciences, 2007, 10(14): 2334−2340. doi: 10.3923/pjbs.2007.2334.2340
[5]	BOTSTEIN D, WHITE R L, SKOLNICK M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms [J]. Am J Hum Genet, 1980, 32(3): 314−331.
[6]	LI G, QUIROS C F. Sequence-related amplified polymorphism (SRAP), a new marker system based on a simple PCR reaction: Its application to mapping and gene tagging in Brassica [J]. Theoretical and Applied Genetics, 2001, 103(2/3): 455−461.
[7]	WILLIAMS J G K, KUBELIK A R, LIVAK K J, et al. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers [J]. Nucleic Acids Research, 1990, 18(22): 6531−6535. doi: 10.1093/nar/18.22.6531
[8]	ZIETKIEWICZ E, RAFALSKI A, LABUDA D. Genome fingerprinting by simple sequence repeat (SSR)-anchored polymerase chain reaction amplification [J]. Genomics, 1994, 20(2): 176−183. doi: 10.1006/geno.1994.1151
[9]	VOS P, HOGERS R, BLEEKER M, et al. AFLP: a new technique for DNA fingerprinting [J]. Nucleic Acids Research, 1995, 23(21): 4407−4414. doi: 10.1093/nar/23.21.4407
[10]	陈艳秋, 李玉, 郭晓帆. 应用RAPD标记对斜生褐孔菌菌株亲缘关系的分析 [J]. 菌物学报, 2007, 26(1):122−127. CHEN Y Q, LI Y, GUO X F. RAPD analysis of Phaeoporus obliquus strains from different areas of China [J]. Mycosystema, 2007, 26(1): 122−127.(in Chinese)
[11]	詹才新, 朱兰宝, 杨新美. RAPD技术在金针菇杂交育种中的应用 [J]. 食用菌学报, 1995, 2(1):7−11. ZHAN C X, ZHU L B, YANG X M. Application of RAPD technique in crossbreeding of Flammulina velutipes [J]. Acta Edulis Fungi, 1995, 2(1): 7−11.(in Chinese)
[12]	赵光辉, 方洪枫, 陈剑, 等. 20株草菇遗传多样性的分析 [J]. 江苏农业科学, 2020, 48(16):194−197. ZHAO G H, FANG H F, CHEN J, et al. Strains of mushroom genetic diversity analysis [J]. Jiangsu Agricultural Science, 2020, 48(16): 194−197.(in Chinese)
[13]	林范学, 林芳灿. 香菇亲本菌株及其杂交后代的RAPD分析 [J]. 菌物系统, 1999, 18(3):279−283. LIN F X, LIN F C. Rapd analysis of parental strains and their crossing progenies of Lentinula edodes [J]. Mycosystema, 1999, 18(3): 279−283.(in Chinese)
[14]	STAJICH J E, BERBEE M L, BLACKWELL M, et al. The fungi [J]. Curr Biol, 2009, 19(18): R840−845. doi: 10.1016/j.cub.2009.07.004
[15]	LIU T X, LI H R, DING Y T, et al. Genome-wide gene expression patterns in dikaryon of the basidiomycete fungus Pleurotus ostreatus [J]. Brazilian Journal of Microbiology, 2016, 48(2): 380−390.
[16]	王瑞娟, 杨慧, 刘建雨, 等. 肺形侧耳‘申秀1号’的选育报告 [J]. 菌物学报, 2020, 39(12):2389−2391. WANG R J, YANG H, LIU J Y, et al. A new Pleurotus pulmonarius cultivar ‘Shenxiu 1’ [J]. Mycosystema, 2020, 39(12): 2389−2391.(in Chinese)
[17]	李巧珍, 刘建雨, 章炉军, 等. 香菇自交选育新菌株及其亲本子实体的氨基酸特征及蛋白质营养价值 [J]. 食用菌学报, 2019, 26(3):51−57. LI Q Z, LIU J Y, ZHANG L J, et al. Amino acid profile and protein nutritional value of Lentinula edodes huxiang F₂ and its self-breeding progenies [J]. Acta Edulis Fungi, 2019, 26(3): 51−57.(in Chinese)
[18]	张美彦, 于海龙, 章炉军, 等. 香菇‘申香1644’的选育报告[J/OL]. 菌物学报 [2022-04-04]. http://kns.cnki.net/kcms/detail/11.5180.Q.20220106.1217.014.html. ZHANG M Y, YU H L, ZHANG L J, et al. A new Lentinula edodes cultivar ‘Shenxiang 1644’[J/OL]. Mycosystema [2022-04-04]. http://kns.cnki.net/kcms/detail/11.5180.Q.20220106.1217.014.html. (In Chinese)
[19]	郑文妹, 章炉军, 李巧珍, 等. 香菇沪香F2菌株重要农艺性状在F₂和F₃代中的遗传规律 [J]. 菌物学报, 2021, 40(12):3129−3142. ZHENG W M, ZHANG L J, LI Q Z, et al. Genetic law of important agronomic traits of Lentinula edodes in F₂ and F₃ generations [J]. Mycosystema, 2021, 40(12): 3129−3142.(in Chinese)
[20]	KHUSH R S, BECKER E, WACH M. DNA amplification polymorphisms of the cultivated mushroom Agaricus bisporus [J]. Applied and Environmental Microbiology, 1992, 58(9): 2971−2977. doi: 10.1128/aem.58.9.2971-2977.1992
[21]	XU J P, YOELL H J, ANDERSON J B. An efficient protocol for isolating DNA from higher fungi [J]. Trends in Genetics, 1994, 10(7): 226−227.
[22]	YIN Y G, LIU Y, WANG S X, et al. Examining genetic relationships of Chinese Pleurotus ostreatus cultivars by combined RAPD and SRAP markers [J]. Mycoscience, 2013, 54(3): 221−225. doi: 10.1016/j.myc.2012.09.015
[23]	邹锋, 谭晓风, 袁德义, 等. RAPD分子标记及其在油茶遗传育种研究中的应用 [J]. 亚热带植物科学, 2009, 38(1):70−73. doi: 10.3969/j.issn.1009-7791.2009.01.020 ZOU F, TAN X F, YUAN D Y, et al. RAPD marker and its application in Camellia oleifera genetic breeding [J]. Subtropical Plant Science, 2009, 38(1): 70−73.(in Chinese) doi: 10.3969/j.issn.1009-7791.2009.01.020
[24]	张明月, 刘冬忍, 张大飞, 等. 糙皮侧耳单核体和双核体同工酶谱的比较 [J]. 河南农业大学学报, 2013, 47(3):325−329. doi: 10.3969/j.issn.1000-2340.2013.03.017 ZHANG M Y, LIU D R, ZHANG D F, et al. Comparison of isoenzyme patterns between the monokaryons and dikaryons of Pleurotus ostreatus [J]. Journal of Henan Agricultural University, 2013, 47(3): 325−329.(in Chinese) doi: 10.3969/j.issn.1000-2340.2013.03.017
[25]	贺小英, 荆乾鸽, 姜欣颖, 等. 端粒酶与体细胞重编程的最新研究进展 [J]. 南方农业学报, 2019, 50(5):1133−1140. doi: 10.3969/j.issn.2095-1191.2019.05.30 HE X Y, JING Q G, JIANG X Y, et al. Research progress of telomerase and somatic cell reprogramming [J]. Journal of Southern Agriculture, 2019, 50(5): 1133−1140.(in Chinese) doi: 10.3969/j.issn.2095-1191.2019.05.30