A Preliminary Report on Resequencing 18 Representative Strains of Agaricus bisporus
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摘要:
目的 通过对双孢蘑菇不同类型核心种质的基因组重测序分析，探讨双孢蘑菇不同类型菌株间基因组存在的差异及开发相关分子标记。 方法 对双孢蘑菇国内外杂交菌株、野生菌株、高产型或优质型传统菌株、棕色菌株、不育菌株等共18株核心种质进行基因组重测序，应用不同的生物信息学处理软件，对测序得到的原始reads序列与双孢蘑菇参考基因组H97序列进行比对，同时基于比对结果进行SNP、SV 检测，通过检测结果对多态性标记分布进行统计并实现DNA水平差异基因挖掘和差异基因功能注释等。 结果 样品测序共获得21.63 G数据量，Q30平均达到89.10%。样品的reads与参考基因组H97的比对效率平均为82.50%，基因组覆盖度为96.32%，平均深度分别在33X左右。基于测序数据与参考基因组的比对结果，共检测获得约813 768个SNP，53 840个InDel，平均每个个体获得924个SV变异。 结论 国内外菌株的亲缘关系表明As2796系列与U1系列是世界上并列的两大双孢蘑菇杂交品系。 Abstract:Objective By resequencing the genomes of representative strains of Agaricus bisporus, the genetic differentiations and molecular markers were investigated for taxonomic and analytical studies on the mushrooms. Method Genes of 18 major strains of A. bisporus including hybrids and wild strains from local and foreign sources, traditional highly productive or good quality varieties, and brown-colored and sterile strains were resequenced. Various bioinformatics software programs were applied to align the readings with the reference A. bisporus genome data. From the aligned results, SNP and SV were obtained to statistically analyze the distribution of the genetic differentiations in search for DNA-based molecular markers and to decipher the functions of the genomes. Result From the resequencing, 21.63 G data were obtained with an average Q30 reaching 89.10%. A matching rate of 82.50%, a coverage of 96.32%, and an average depth of 33X were found between the readings and sequence of the H97 reference genome. From the alignment results, 813 768 SNPs and 53 840 InDels were obtained with an average of 924 SV variations for each strain. Conclusion Of the various A. bisporus strains in the world, the Chinese As2796 and Dutch U1 lines seemed to be the two major commercial hybrids on the market. -
Key words:
- edible fungus /
- genome /
- SNP /
- InDel /
- structure variation
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表 1 供试菌株及其来源
Table 1. Strains and origins of A. bisporus studied
序号 No. 菌株 Strain 来源 Origin 菌株类型 Strain type 1 As2796 1989年福建省农科院食用菌所杂交（02和8213)
Crossbreeding of 02 and 8213 by IEF-FAAS in 1989国内杂交菌株
Domestic hybrid2 W192 2008年福建省农科院食用菌所杂交（02和As2796)
Crossbreeding of 02 and As2796 by IEF-FAAS in 2008国内杂交菌株
Domestic hybrid3 192-38 2015年福建省农科院食用菌所单孢分离自W192
Isolated from W192 by IEF-FAAS in 2015国内杂交菌株
Domestic hybrid4 U1 1980年荷兰Horst蘑菇试验站杂交
Crossbred by Horst Station of the Netherland in 1980国外杂交菌株
Imported hybrid5 U3 1980年荷兰Horst蘑菇试验站杂交
Crossbred by Horst Station of the Netherland in 1980国外杂交菌株
Imported hybrid6 A15 1989年美国Sylvan公司选育，U1衍生品种
Bred from U1 by Sylvan America in 1989国外杂交菌株
Imported hybrid7 02 1980年轻工业部食品发酵所引自荷兰
Introduced from the Netherland by Food Fermentation Institute of Light Industry Department高产传统菌株
High-production traditional strain8 176 1979年中科院微生物所，张树庭教授引自法国
From Microbiology Institute of CAS, introduced from France by Prof.S.T.Chang in 1979高产传统菌株
High-production traditional strain9 8213 1982年分离自龙溪地区，013多孢子代
Multi-spore offspring derived from 013 at Longxi in 1982优质传统菌株
Good-quality traditional strain10 8211 1982年分离自浙江瑞安老法国品种
Old French variety separated from Zhejiang Ruian in 1982优质传统菌株
Good-quality traditional strain11 C1 1980年引自上海市农科院食用菌研究所
Introduced from Edible Fungi Institute of SAAS in 1980优质传统菌株
Good-quality traditional strain12 SA6 1987年上海市农科院食用菌研究所，引自波兰
Introduced from Poland by Edible Fungi Institute of SAAS in 1987中间类型菌株
Middle-type strain13 MC441 2005年引自美国滨州大学
Introduced from The Pennsylvania State University in 2005棕色菌株 Brown strain 14 Ag78331 2007年四川省农科院和福建省农科院采自西藏
Collected from Tibet by SAAS and FAAS in 2007国内野生菌株
Domestic wild strain15 AgLH830 2008年四川省农科院和福建省农科院采自四川
Collected from Sichuan by SAAS and FAAS in 2008国内野生菌株
Domestic wild strain16 ARP159 2002年引自美国Sylvan公司的四孢变种
Tetraspore strain from Sylvan America in 2002国外野生菌株
US wild strain17 02-S5 2014年福建省农科院分离自02的同核体
Homokaryon isolated from 02 by FAAS in 2014不育菌株
Sterile strain18 8213-S2 2014年福建省农科院食用菌所分离自8213的同核体
Homokaryon isolated from 8213 by FAAS in 2014不育菌株
Sterile strain注：福建省农科院食用菌所 (IEF-FAAs)，中科院微生物所 (CAS)，上海市农科院 (SAAS)，四川省农科院 (SAAS)，福建省农业科学院（FAAS) 。
Note: The Institute of Edible Fungi, Fujian Academy of Agricultural Sciences (IEF-FAAS), Chinese Acadenmy of Sciences (CAS), Shanghai Academy of Agricultural Sciences (SAAS), Sichuan Academy of Agricultural Sciences (SAAS), Fujian Academy of Agricultural Sciences （FAAS)．
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表 2 18个双孢蘑菇菌株测序数据评估及与参考基因组H97比对结果
Table 2. Genetic sequences of 18 A. bisporus strains and their alignments with H97 reference genome
菌株
Strain总片段数目
Total Reads≥Q30的百分比
Percentage of ≥Q30/%覆盖深度
Depth匹配片段百分比
Mapped Reads/%基因组覆盖率
Coverage/%As2796 10 832 772 91.51 29 78.53 95.75 02 9 969 928 91.60 26 84.91 99.82 8213 11 279 190 91.68 27 76.44 95.98 W192 11 529 394 91.54 30 78.52 95.80 A15 10 590 252 91.39 31 90.34 99.90 U1 11 492 098 91.56 33 89.20 99.92 U3 10 838 354 83.40 26 73.97 99.42 MC441 11 032 666 86.19 30 83.33 97.05 SA6 11 180 630 85.31 28 73.92 96.20 176 11 244 788 86.68 30 84.28 99.55 Ag78331 10 903 678 86.12 31 80.44 92.85 C1 11 037 982 85.44 30 81.65 96.02 192-38 10 066 128 90.38 36 83.93 95.60 8211 8 255 514 90.19 30 87.16 95.74 ARP159 11 103 174 89.19 42 82.31 90.20 AgLH830 9 167 040 90.30 32 77.27 93.43 02-S5 9 930 026 90.42 37 90.32 97.33 8213-S2 17 432 836 90.88 67 88.52 93.16
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表 3 18个双孢蘑菇菌株与参考基因组H97比较的SNP和SV统计
Table 3. Statistics on SNP and SV of 18 A. bisporus strains as compared with H97 reference genome
菌株 Strain 多态性位点 SNP 结构变异 SV 插入类型 INS 删除类型 DEL 倒位类型 INV 染色体内易位 ITX 染色体间易位 CTX As2796 510 425 1 000 70 459 80 43 348 W192 510 122 1 111 216 441 79 53 322 192-38 553 782 932 18 398 91 62 363 U1 258 738 755 214 290 44 29 178 U3 286 594 596 73 281 51 28 163 A15 256 674 704 145 300 39 28 192 02 248 100 609 105 238 37 28 201 176 266 232 701 189 242 41 25 204 ARP159 326 622 828 31 343 74 66 314 Ag78331 574 890 1 001 108 438 70 60 325 AgLH830 396 488 880 26 366 77 66 345 8213 386 625 1 798 995 418 68 43 274 8211 426 241 1 283 521 324 73 56 309 C1 404 756 956 99 417 71 55 314 MC441 407 661 1 015 213 405 72 34 291 SA6 417 898 845 46 398 74 55 272 02-S5 102 187 419 141 110 28 21 119 8213-S2 300 352 1 195 421 327 72 51 324
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表 4 18个双孢蘑菇菌株与H97相比产生的3种差异基因的分类统计
Table 4. Differentially expressed genes of 18 A. bisporus strains as compared with H97 reference genome
菌株
Strain非同义突变的
差异基因
Genes with Non-
synonymous SNP存在InDel的
差异基因
Genes with
InDel存在SV的
差异基因
Genes with
SVAs2796 25 480 5 578 357 W192 25 433 5 551 461 192-38 25 487 5 606 330 U1 16 742 3 156 325 U3 17 404 3 262 241 A15 16 621 3 148 293 02 16 902 3 089 240 176 16 925 3 064 284 ARP159 21 112 3 311 299 Ag78331 22 670 3 998 383 AgLH830 22 611 4 002 304 8213 22 260 4 511 832 8211 22 331 4 585 624 C1 22 246 4 531 348 MC441 20 963 3 840 427 SA6 22 554 4 564 301 02-S5 6 137 1 269 175 8213-S2 17 014 3 349 557
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表 5 2个菌株之间的SNP与InDel比较
Table 5. Comparison between SNP and InDel of 2 strains of A. bisporus
菌株比较
Strains comparisonSNP数目
SNP numberInDel 数目
InDel numberAs2796 与 192 31 630 2 568 W192 与 192-38 35 232 2 806 A15 与 U1 20 071 1 594 A15 与 U3 60 822 4 711 As2796 与 A15 490 998 35 292 As2796 与 U1 490 201 35 176 As2796 与 U3 488 391 34 978 As2796 与 02 524 359 37 764 As2796 与 8213 405 421 29 926 ARP159 与 AgLH830 295 927 18 801 02 与 02-S5 268 304 18 514 8213 与 8213-S2 265 151 19 039
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