Evaluation on 20 Lentinula edodes Strains Using ISSR and F-MSAP Markers
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摘要: 利用简单序列重复区间扩增多态性（inter-simple sequence repeat，ISSR）和荧光标记甲基化敏感扩增多态性（fluorescence-labeled methylation-sensitive amplified polymorphism，F-MSAP）分子标记技术对收集到的20株香菇Lentinula edodes菌株进行遗传及甲基化多样性分析。ISSR结果表明，8对引物共可扩增出98条稳定清晰可辨的条带，其中多态性条带89条，多态性比率为90.82%，样品间的遗传相似系数范围为0.520 4~0.989 7。F-MSAP结果表明，15对引物共扩增产生13 487个CCGG位点。在全部检测位点中，半甲基化位点为1 704个，平均半甲基化率12.6%；全甲基化位点为1 865个，平均全甲基化率13.8%。进一步将MSAP数据分为甲基化敏感多态性（methylation sensitive polymorphism，MSP）和甲基化不敏感多态性（methylation insensitive polymorphism，MISP）。Mantel检测结果表明，ISSR与MISP分子标记的分析结果有显著的相关性，但ISSR与MSP分子标记的分析结果无显著相关性。说明香菇不同株系间广泛存在DNA甲基化多样性，在育种中具有重要的应用价值。Abstract: The inter-simple sequence repeat (ISSR) and fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) markers were used to evaluate the genetic diversity of 20 Lentinula edodes strains. Ninety-eight bands were detected by 8 ISSR primers, of which 90.82% were polymorphic. The coefficient of pairwise genetic similarity ranged from 0.520 4 to 0.989 7.A total of 13 487 CCGG sites were detected using 15 selected primer pairs. Among the sites, 1 704 were of hemi-methylation with an average rate of 12.63%, and 1 865 of full-methylation with an average rate of 13.83%. The MSAP data were further divided into methylation sensitive polymorphisms (MSP) and methylation insensitive polymorphisms (MISP) groups. A significant correlation between ISSR and MISP was found by the Mantel test, while none between ISSR and MSP. It appeared that there was a significant methylation diversity among various L. edodes which could be of value for the breeding purpose.
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Key words:
- Lentinula edodes /
- ISSR /
- methylation /
- MSAP
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图 1 20株香菇ISSR分析的UPMGA聚类
Figure 1. UPMGA dendrogram based on ISSR data on 20 L. edodes strains
图 2 20株香菇MISP分析的UPMGA聚类
Figure 2. UPMGA dendrogram based on MISP data on 20 L. edodes strains
图 3 20株香菇MSP分析的UPMGA聚类
Figure 3. UPMGA dendrogram based on MSP data on 20 L. edodes strains
表 1 20株香菇DNA甲基化水平分析
Table 1. Analysis of methylation polymorphisms on 20 L. edodes strains
菌株 带型 半甲基化率/% 全甲基化率/% 总甲基化率/% Ⅰ Ⅱ Ⅲ Cr04 516 101 70 14.70 10.19 24.89 Cr66 538 64 93 9.21 13.38 22.59 L01 502 78 101 11.45 14.83 26.28 L04 484 85 106 12.59 15.70 28.30 L135 490 78 78 12.07 12.07 24.15 L18 510 91 70 13.56 10.43 23.99 L20 537 76 88 10.84 12.55 23.40 L2016-1 449 77 89 12.52 14.47 26.99 L2016-2 478 100 82 15.15 12.42 27.58 L2016-5 503 108 94 15.32 13.33 28.65 L236 341 78 242 11.80 36.61 48.41 L241-4 537 77 84 11.03 12.03 23.07 L258 497 74 91 11.18 13.75 24.92 L26 549 79 77 11.21 10.92 22.13 L808 504 74 88 11.11 13.21 24.32 L9015 500 89 70 13.51 10.62 24.13 L921 479 81 91 12.44 13.98 26.42 L939 503 97 54 14.83 8.26 23.09 Wuxiang-1 498 91 114 12.94 16.22 29.16 Zhongxiang-68 503 106 83 15.32 11.99 27.31 注：总扩增位点= Ⅰ+Ⅱ+Ⅲ；甲基化总位点= Ⅱ+Ⅲ；总甲基化率=(Ⅱ+Ⅲ)/(Ⅰ+Ⅱ+Ⅲ)；全甲基化率=Ⅲ/(Ⅰ+Ⅱ+Ⅲ)；半甲基化率= Ⅱ/(Ⅰ+Ⅱ+Ⅲ)。 
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