荷叶离褶伞遗传差异性分析及菌丝形态鉴定

黄家庆; 陈华; 林怡; 叶菁; 王义祥; 郭月仙; 马立验

doi:10.19303/j.issn.1008-0384.2023.03.007

荷叶离褶伞遗传差异性分析及菌丝形态鉴定

doi: 10.19303/j.issn.1008-0384.2023.03.007

黄家庆^{1, 2,},
陈华^{1, 2},
林怡^{1, 2},
叶菁^{1, 2},
王义祥^{2, 3, ,},
郭月仙⁴,
马立验⁴

1.
福建省农业科学院农业生态研究所，福建　福州　350013
2.
福建省红壤山地农业生态过程重点实验室，福建　福州　350013
3.
福建省农业科学院土壤肥料研究所，福建　福州　350013
4.
福建恒绿生物科技有限公司，福建　宁德　355300

基金项目: 福建省科技计划星火项目（2020S0065）

详细信息

作者简介:
黄家庆（1979−），男，博士，助理研究员，主要从事环境微生物及菌种资源化利用（ E-mail：hjq19791001@126.com）

通讯作者:
王义祥（1978−），男，博士，研究员，主要从事环境微生物和农业环境修复（ E-mail：sd_wolong@163.com）

中图分类号: S646
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出版历程
- 收稿日期: 2022-07-19
- 修回日期: 2022-10-18
- 网络出版日期: 2023-03-28
- 刊出日期: 2023-03-28

Genetic Differences and Mycelial Morphology of Lyophyllum decastes Strains

HUANG Jiaqing^{1, 2
,},
CHEN Hua^{1, 2},
LIN Yi^{1, 2},
YE Jing^{1, 2},
WANG Yixiang^{2, 3
, ,},
GUO Yuexian⁴,
MA Liyan⁴

1.
Agricultural Ecology Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
2.
Fujian Key Laboratory of Agricultural Ecological Process of Red Soil Mountain, Fuzhou, Fujian　350013, China
3.
Institute of Soil and Fertilizer, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian　350013, China
4.
Fujian Henglv Biotechnology Co., Ltd., Ningde, Fujian　355300, China

摘要

摘要: 目的比较荷叶离褶伞（Lyophyllum decastes，Lyd）外地引进菌株与本地栽培菌株的遗传差异性和菌丝形态特征，为荷叶离褶伞选育提供参考。方法以真菌18S rRNA（V4）和ITS（ITS1–ITS4）（18S rRNA-ITS）序列对外地引进和本地栽培的荷叶离褶伞进行序列同源性分析、多重序列比对和构建系统发育进化树，通过分析碱基序列突变和遗传距离远近明确荷叶离褶伞菌株间的亲缘关系，并通过菌丝体生长及形态变化确定荷叶离褶伞的遗传学差异。结果 18S rRNA-ITS序列比对结果显示，外地荷叶离褶伞菌株（Lyd-LR1、Lyd-LR6、Lyd-LR10、Lyd-LR15和Lyd-LR17）发生较高比例的碱基缺失和碱基替换突变，且ITS序列同源性下降至80.09%～89.72%； Lyd-LR1和Lyd-LR6菌株的碱基突变比例高于Lyd-LR10、Lyd-LR15和Lyd-LR17以及福建本地栽培菌株（Lyd-LRX和Lyd-LRY）。进化树分析结果进一步显示，Lyd-LR1和Lyd-LR6与Lyd-LR10、Lyd-LR15、Lyd-LR17、Lyd-LRX、Lyd-LRY以及NCBI已经登录的部分荷叶离褶伞或离褶伞（Lyophyllum，Ly）菌株有较远的遗传距离和亲缘关系。Lyd-LR1梭子状菌丝生长较慢且不规则生长，菌丝体呈松散放射状；Lyd-LR6凹陷状菌丝生长较快且明显聚合生长，菌丝体呈平铺状且边缘厚度较高。Lyd-LR1和Lyd-LR6菌株的菌丝体生长速度和菌丝形态均不同于Lyd-LR10、Lyd-LR15、Lyd-LR17、Lyd-LRX、Lyd-LRY菌株圆柱形饱满的菌丝和厚度高生长快的突起状菌丝体。而与Lyd-LR1菌株相比，Lyd-LR6菌株的碱基序列突变位置、碱基突变比例和菌丝形态发生的变化更明显。结论外地荷叶离褶伞菌株Lyd-LR6的基因序列碱基突变位置（碱基替换和碱基缺失）、18S rRNA-ITS序列同源性和种属亲缘关系明显区别于其他外地及本地的荷叶离褶伞菌株。高比例的基因序列碱基突变造成Lyd-LR6菌株的菌丝体生长速度和菌丝形态发生明显改变，可作为选育荷叶离褶伞新菌株的材料。
- 荷叶离褶伞 /
- 18S rRNA-ITS /
- 同源性分析 /
- 发育进化树 /
- 菌丝生长
Abstract: Objective Genetic differences and mycelial morphology of imported and locally cultivated Lyophyllum decastes (Lyd) were compared. Methods Sequence homology, multiple sequence alignment, and phylogenetic tree were examined and constructed based on the sequences of the fungal 18S rRNA (V4) and ITS (ITS1-ITS4) (18S rRNA-ITS) of the imported and locally cultivated Lyd strains. Mutation analysis and genetic distance determination were performed to identify the phylogenetic relationship, while mycelium growth and scanning electron microscopic images observed to differentiate the morphology of the strains. Results The 18S rRNA-ITS sequence alignment between the two strains showed the imported Lyd-LR1, Lyd-LR6, Lyd-LR10, Lyd-LR15, and Lyd-LR17 to be higher on the ratio of base deletion and base substitution mutations. The ITS sequence homology between them decreased to 80.09%–89.72% with Lyd-LR1 and Lyd-LR6 being higher than Lyd-LR10, Lyd-LR15, Lyd-LR17 as well as the local Lyd-LRX and Lyd-LRY on the proportions of base mutation. The phylogenetic tree analysis indicated a distant genetic relationship between Lyd-LR1 and Lyd-LR6 and Lyd-LR10, Lyd-LR15, Lyd-LR17, Lyd-LRX, Lyd-LRY, and some Lyd or Lyophyllum (Ly) registered by NCBI. The loose and radial fusiform mycelia of Lyd-LR1 grew slowly and irregularly, whereas the pitted mycelia of Lyd-LR6 tiled with high margin thickness proliferated rapidly and polymerized considerably. The growth rates and morphology of Lyd-LR1 and Lyd-LR6 mycelia differed from those of Lyd-LR10, Lyd-LR15, Lyd-LR17, Lyd-LRX, and Lyd-LRY, which had full cylindrical mycelia and thick and fast-growing protuberant mycelia. Compared to Lyd-LR1, Lyd-LR6 exhibited more apparent changes in gene sequence base mutation location, base mutation proportion, and mycelial morphology. Conclusion The gene sequence base mutation location (base substitution and base deletion), 18S rRNA-ITS sequence homology, and phylogenetic relationship of Lyd-LR6 significantly differed from those of the other imported and the locally cultivated Lyd strains. A high proportion of gene sequence base mutations induced significant changes in the growth and morphology of Lyd-LR6 mycelia indicating a potential use of it in breeding new varieties of Lyd mushrooms.
- Lyophyllum decastes /
- 18S rRNA-ITS /
- homology analysis /
- phylogenetic tree /
- mycelial growth

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图 1 荷叶离褶伞菌株的18S rRNA（V4）区序列多重比对分析

A1–A9为序列比对发生碱基突变的位置。

Figure 1. Multiple alignment of 18S rRNA(V4) sequences in Lyd strains

A1–A9 are the location where base mutations occur in sequence alignment.

下载: 全尺寸图片幻灯片

图 2 荷叶离褶伞菌株的ITS（ITS1–ITS4）序列多重比对分析

B1–B37为序列比对发生碱基突变的位置。

Figure 2. Multiple alignment of ITS (ITS1-ITS4) sequences in Lyd strains

B1–B37 are the location where base mutations occur in sequence alignment.

下载: 全尺寸图片幻灯片

图 3 基于18S rRNA（V4）序列构建的系统进化树

Figure 3. Phylogenetic tree based on 18S rRNA (V4) sequences

下载: 全尺寸图片幻灯片

图 4 基于ITS（ITS1–ITS4）序列构建的系统进化树

Figure 4. Phylogenetic tree based on ITS (ITS1–ITS4) sequences

下载: 全尺寸图片幻灯片

图 5 荷叶离褶伞菌株菌丝在PDA培养基上的生长状况

Figure 5. Mycelial growth of Lyd strains on PDA medium

下载: 全尺寸图片幻灯片

图 6 荷叶离褶伞菌株菌丝的扫描电镜观察

Figure 6. SEM images of mycelia of Lyd strains

下载: 全尺寸图片幻灯片

表 1 荷叶离褶伞菌株的18S rRNA（V4）序列同源性分析

Table 1. Homology of 18S rRNA (V4) sequences in Lyd strains （单位：%）

菌株 Strain	Lyd-LR1	Lyd-LR6	Lyd-LR10	Lyd-LR15	Lyd-LR17	Lyd-LRX	Lyd-LRY
Lyd-PBM3069	98.45	98.22	98.88	99.11	98.48	98.66	98.21
Lyd-JM87/16	98.23	97.55	98.66	98.44	97.83	97.99	97.54
Ly-shimeji（D84555.1）	97.96	97.90	98.59	98.83	97.95	98.13	97.90
Ly-sp. MSG166	96.69	96.44	97.10	97.32	96.96	96.88	96.64
Ly-sp. PBM 2688	96.47	96.21	96.88	97.10	96.75	96.65	96.42
Ly-praslinense 10295	94.92	94.65	95.31	95.54	95.23	95.09	94.85
Ly-leucophaeatum HAe251	94.48	94.21	94.87	95.09	94.79	94.64	94.41
Ly-shimeji（D84553.1）	100.00	93.44	98.36	98.36	100.00	95.08	93.22

下载: 导出CSV

表 2 荷叶离褶伞菌株的ITS（ITS1–ITS4）序列同源性分析

Table 2. Homology of ITS (ITS1–ITS4) sequences in Lyd strains (单位：%)

菌株 Strain	Lyd-LR1	Lyd-LR6	Lyd-LR10	Lyd-LR15	Lyd-LR17	Lyd-LRX	Lyd-LRY
Lyd-dcy2255	89.72	83.52	90.33	84.64	89.38	86.52	82.92
Lyd-ywy1	89.42	82.76	90.05	84.33	89.06	86.06	82.49
Lyd-Ld418	89.27	82.92	89.94	84.45	89.24	85.67	82.58
Lyd-060926	89.30	82.82	90.08	84.07	88.79	85.80	82.24
Lyd-060801	89.15	82.66	89.94	83.94	88.61	85.91	82.06
Lyd-061011	88.98	82.35	89.77	83.33	88.02	85.76	81.65
Lyd-iNAT	88.34	82.37	88.99	83.26	87.95	85.23	81.26
Lyd-22/14DMRJU	88.69	81.74	88.98	83.14	88.69	85.60	80.09

下载: 导出CSV

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