生物技术通报 ›› 2021, Vol. 37 ›› Issue (11): 57-64.doi: 10.13560/j.cnki.biotech.bull.1985.2021-1091
• 食用菌生物技术专题(专题主编: 黄晨阳) • 上一篇 下一篇
收稿日期:
2021-08-27
出版日期:
2021-11-26
发布日期:
2021-12-03
作者简介:
陈卫民,男,博士,研究员,研究方向:食用菌遗传育种;E-mail: 基金资助:
CHEN Wei-min(), CHAI Hong-mei, YANG Wei-xian, MA Yuan-hao, ZHAO Yong-chang()
Received:
2021-08-27
Published:
2021-11-26
Online:
2021-12-03
摘要:
柱状田头菇(茶树菇)Cyclocybe aegerita在我国广泛种植,其菌株资源分为多个类群且遗传背景多样。多种命名和复杂的交配特征给柱状田头菇及近缘种的育种研究带来障碍。以两个物种柱状田头菇和杨柳田头菇C. salicacola菌株群体为研究材料,通过基因组测序及序列分析,设计特异引物扩增获取菌株群体的交配区域片段,用于菌株鉴定及系统构建。克隆获得了两个柱状田头菇菌株的B交配位点,其中约8 kb的片段序列非常保守,包括信息素受体rcb1、rcb2和部分非编码区。基于该区域含有变异区的片段设计特异引物,经对35个菌株扩增及克隆测序后,每个实验菌株中均获得1-2类型约3.2 kb片段(cbmr)。使用该片段与线粒体小亚基构建的聚类树契合度较高,但前者在类群的划分上更为精细。此外,该片段的变异区也为类群和交配型鉴定提供了靶位点。依据B交配型部分位点设计的引物在所有的实验菌株中均获得相应的片段,可以作为分子标记用于柱状田头菇和杨柳田头菇菌株及B交配型分子鉴定。
陈卫民, 柴红梅, 杨玮娴, 马渊浩, 赵永昌. 柱状田头菇和杨柳田头菇B交配型位点特征分析及其应用[J]. 生物技术通报, 2021, 37(11): 57-64.
CHEN Wei-min, CHAI Hong-mei, YANG Wei-xian, MA Yuan-hao, ZHAO Yong-chang. Characterization of B Mating Loci Identification and Application in Cyclocybe aegerita and C. salicacola[J]. Biotechnology Bulletin, 2021, 37(11): 57-64.
菌株 Strain | 种类 Species | 采集地 Collection site | 线粒体小亚基MtSSU rDNA variable domain sequence | Cbmr类型 Cbmr type Type-1/type-2 | |||||
---|---|---|---|---|---|---|---|---|---|
V4/nt | 序列号 Sequence No. | V6/nt | 序列号 Sequence No. | V9/nt | 序列号 Sequence No. | ||||
YAASM0566 | C. aegerita | 云南 | 194 | HQ916212 | 157 | HQ916219 | 246 | HQ916226 | MG742437/MG742438 |
YAASM0567 | C. aegerita | 云南 | 194 | HQ916213 | 157 | HQ916220 | 246 | HQ916227 | MG742434/MG742439 |
YAASM0594 | C. aegerita | 云南 | 194 | HQ916214 | 157 | HQ916221 | 246 | HQ916228 | MG742435/MG742440 |
YAASM0625 | C. aegerita | 云南 | 194 | HQ916215 | 157 | HQ916222 | 246 | HQ916229 | MG742436/- |
YAASM1024 | C. aegerita | 云南 | 194 | HQ916216 | 157 | HQ916223 | 246 | HQ916230 | MG742433/MG742441 |
YAASM2135 | C. aegerita | 云南 | 193 | MF477939 | 157 | MF477941 | 246 | MF477943 | MG742432/MG737355 |
YAASM2136 | C. aegerita | 云南 | 193 | MF477940 | 157 | MF477942 | 246 | MF477944 | MG737353/MG737356 |
XW01 | C. aegerita | 云南 | 194 | HQ916217 | 157 | HQ916224 | 246 | HQ916231 | MG742459/MG737352 |
YUNCHA | C. aegerita | 云南 | 194 | MG737328 | 157 | MG737338 | 245 | MG737347 | MG737354/- |
CS45 | C. aegerita | 云南 | 195 | HQ916218 | 157 | HQ916225 | 246 | HQ916232 | MG742445/MG742457 |
AS-1 | C. aegerita | 江西 | 195 | MF461694 | 164 | MF461707 | 246 | MF461720 | MG742451/MG742458 |
AS-2 | C. aegerita | 江西 | 194 | MF461693 | 164 | MF461706 | 246 | MF461719 | MG742454/MG742455 |
CHA3 | C. aegerita | 江西 | 194 | MF461689 | 165 | MF461702 | 246 | MF461715 | MG742450/- |
C45 | C. aegerita | 江西 | 194 | MF461692 | 164 | MF461705 | 246 | MF461718 | -/MG742456 |
C89 | C. aegerita | 江西 | 194 | MF461691 | 164 | MF461704 | 246 | MF461717 | MG742447/- |
YAASM0473 | C. aegerita | 江西 | 194 | MG737329 | 164 | MG737336 | 246 | MG737346 | MG742449/ MG742453 |
YSD1H | C. aegerita | 江西 | 194 | MF461690 | 164 | MF461703 | 246 | MF461716 | MG742443/- |
CHA5 | C. aegerita | 江西 | 277 | MF461687 | 157 | MF461700 | 246 | MF461713 | MG737357/- |
YSG | C. aegerita | 江西 | 282 | MF461686 | 157 | MF461699 | 246 | MF461712 | MG737351/- |
BAICHA | C. aegerita | 江西 | 282 | MF461685 | 157 | MF461698 | 246 | MF461711 | MG737350/- |
AC0001 | C. aegerita | 福建 | 282 | MF461684 | 157 | MF461697 | 246 | MF461710 | MG742442/- |
AC0021 | C. aegerita | 福建 | 193 | MF461683 | 156 | MF461696 | 246 | MF461709 | MG742444/- |
AC0005 | C. aegerita | 福建 | 194 | MF461695 | 164 | MF461708 | 246 | MF461721 | MG742446/- |
AC0007 | C. aegerita | 福建 | 194 | MF461688 | 164 | MF461701 | 246 | MF461714 | MG742448/MG742460 |
CSJL | C. aegerita | 福建 | 195 | MG737327 | 164 | MG737337 | 246 | MG737345 | MG742452/MG742461 |
YAASM0711 | C. salicacola | 云南 | 170 | HQ916233 | 170 | HQ916249 | 221 | HQ916265 | MG742467/MG742478 |
YAASM0967 | C. salicacola | 云南 | 170 | HQ916238 | 170 | HQ916254 | 221 | HQ916270 | MG742462/MG742479 |
YAASM0971 | C. salicacola | 云南 | 170 | HQ916241 | 170 | HQ916257 | 221 | HQ916273 | MG742472/MG742469 |
YAASM0894 | C. salicacola | 云南 | 170 | HQ916234 | 170 | HQ916250 | 221 | HQ916266 | MG742465/MG742475 |
YAASM2120 | C. salicacola | 云南 | 170 | MG737321 | 170 | MG737330 | 221 | MG737343 | MG742464/MG742474 |
YAASM2544 | C. salicacola | 云南 | 170 | MG737322 | 170 | MG737331 | 221 | MG737339 | MG742466/- |
YAASM2595 | C. salicacola | 云南 | 170 | MG737323 | 170 | MG737332 | 220 | MG737340 | -/ MG742476 |
YAASM2722 | C. salicacola | 云南 | 170 | MG737324 | 170 | MG737333 | 221 | MG737341 | MG742470/MG742473 |
YAASM5262 | C. salicacola | 云南 | 170 | MG737325 | 170 | MG737334 | 220 | MG737342 | MG742468/MG742471 |
YAASMYXC | C. salicacola | 云南 | 170 | MG737326 | 170 | MG737335 | 221 | MG737344 | MG742463 /MG742477 |
表1 实验菌株及序列
Table 1 Strains and sequences used in this study
菌株 Strain | 种类 Species | 采集地 Collection site | 线粒体小亚基MtSSU rDNA variable domain sequence | Cbmr类型 Cbmr type Type-1/type-2 | |||||
---|---|---|---|---|---|---|---|---|---|
V4/nt | 序列号 Sequence No. | V6/nt | 序列号 Sequence No. | V9/nt | 序列号 Sequence No. | ||||
YAASM0566 | C. aegerita | 云南 | 194 | HQ916212 | 157 | HQ916219 | 246 | HQ916226 | MG742437/MG742438 |
YAASM0567 | C. aegerita | 云南 | 194 | HQ916213 | 157 | HQ916220 | 246 | HQ916227 | MG742434/MG742439 |
YAASM0594 | C. aegerita | 云南 | 194 | HQ916214 | 157 | HQ916221 | 246 | HQ916228 | MG742435/MG742440 |
YAASM0625 | C. aegerita | 云南 | 194 | HQ916215 | 157 | HQ916222 | 246 | HQ916229 | MG742436/- |
YAASM1024 | C. aegerita | 云南 | 194 | HQ916216 | 157 | HQ916223 | 246 | HQ916230 | MG742433/MG742441 |
YAASM2135 | C. aegerita | 云南 | 193 | MF477939 | 157 | MF477941 | 246 | MF477943 | MG742432/MG737355 |
YAASM2136 | C. aegerita | 云南 | 193 | MF477940 | 157 | MF477942 | 246 | MF477944 | MG737353/MG737356 |
XW01 | C. aegerita | 云南 | 194 | HQ916217 | 157 | HQ916224 | 246 | HQ916231 | MG742459/MG737352 |
YUNCHA | C. aegerita | 云南 | 194 | MG737328 | 157 | MG737338 | 245 | MG737347 | MG737354/- |
CS45 | C. aegerita | 云南 | 195 | HQ916218 | 157 | HQ916225 | 246 | HQ916232 | MG742445/MG742457 |
AS-1 | C. aegerita | 江西 | 195 | MF461694 | 164 | MF461707 | 246 | MF461720 | MG742451/MG742458 |
AS-2 | C. aegerita | 江西 | 194 | MF461693 | 164 | MF461706 | 246 | MF461719 | MG742454/MG742455 |
CHA3 | C. aegerita | 江西 | 194 | MF461689 | 165 | MF461702 | 246 | MF461715 | MG742450/- |
C45 | C. aegerita | 江西 | 194 | MF461692 | 164 | MF461705 | 246 | MF461718 | -/MG742456 |
C89 | C. aegerita | 江西 | 194 | MF461691 | 164 | MF461704 | 246 | MF461717 | MG742447/- |
YAASM0473 | C. aegerita | 江西 | 194 | MG737329 | 164 | MG737336 | 246 | MG737346 | MG742449/ MG742453 |
YSD1H | C. aegerita | 江西 | 194 | MF461690 | 164 | MF461703 | 246 | MF461716 | MG742443/- |
CHA5 | C. aegerita | 江西 | 277 | MF461687 | 157 | MF461700 | 246 | MF461713 | MG737357/- |
YSG | C. aegerita | 江西 | 282 | MF461686 | 157 | MF461699 | 246 | MF461712 | MG737351/- |
BAICHA | C. aegerita | 江西 | 282 | MF461685 | 157 | MF461698 | 246 | MF461711 | MG737350/- |
AC0001 | C. aegerita | 福建 | 282 | MF461684 | 157 | MF461697 | 246 | MF461710 | MG742442/- |
AC0021 | C. aegerita | 福建 | 193 | MF461683 | 156 | MF461696 | 246 | MF461709 | MG742444/- |
AC0005 | C. aegerita | 福建 | 194 | MF461695 | 164 | MF461708 | 246 | MF461721 | MG742446/- |
AC0007 | C. aegerita | 福建 | 194 | MF461688 | 164 | MF461701 | 246 | MF461714 | MG742448/MG742460 |
CSJL | C. aegerita | 福建 | 195 | MG737327 | 164 | MG737337 | 246 | MG737345 | MG742452/MG742461 |
YAASM0711 | C. salicacola | 云南 | 170 | HQ916233 | 170 | HQ916249 | 221 | HQ916265 | MG742467/MG742478 |
YAASM0967 | C. salicacola | 云南 | 170 | HQ916238 | 170 | HQ916254 | 221 | HQ916270 | MG742462/MG742479 |
YAASM0971 | C. salicacola | 云南 | 170 | HQ916241 | 170 | HQ916257 | 221 | HQ916273 | MG742472/MG742469 |
YAASM0894 | C. salicacola | 云南 | 170 | HQ916234 | 170 | HQ916250 | 221 | HQ916266 | MG742465/MG742475 |
YAASM2120 | C. salicacola | 云南 | 170 | MG737321 | 170 | MG737330 | 221 | MG737343 | MG742464/MG742474 |
YAASM2544 | C. salicacola | 云南 | 170 | MG737322 | 170 | MG737331 | 221 | MG737339 | MG742466/- |
YAASM2595 | C. salicacola | 云南 | 170 | MG737323 | 170 | MG737332 | 220 | MG737340 | -/ MG742476 |
YAASM2722 | C. salicacola | 云南 | 170 | MG737324 | 170 | MG737333 | 221 | MG737341 | MG742470/MG742473 |
YAASM5262 | C. salicacola | 云南 | 170 | MG737325 | 170 | MG737334 | 220 | MG737342 | MG742468/MG742471 |
YAASMYXC | C. salicacola | 云南 | 170 | MG737326 | 170 | MG737335 | 221 | MG737344 | MG742463 /MG742477 |
引物 Primer | 序列 Sequence(5'-3') | 用途 Function |
---|---|---|
Cs1f | CCTCCAACGCTATTTGCCTACG | 验证 M67菌株B交配位点 Verified the B mating locus of M67 |
Cs1r | GCACCACCATCCTCACTTCACC | |
Cs2f | CGGCGTGTTTTCGTTTTTCT | |
Cs2r | ATGGCGGAGCGGTAGTTCTT | |
Cs3f | TAGCCTGGAACACTGGAACT | |
Cs3r | GAATGGAGCGAGGAAATAGA | |
Cs4f | AGCAAGGCGACAGTGGAAGAT | |
Cs4r | CGAGTGACATGAGGCGGAAGT | |
Cs5f | ATTCGCCTTCATTTCCTTCAT | |
Cs5r | TCAGCGTTCTCCCATTCTCAC | |
Cs6f | CACGGTAGCAAAGCAGAATGA | |
Cs6r | AGGAGCGTAGAGAAAGCAGGA | |
Ysg1f | GCTGTGCTCCTCCAACTCTATT | 验证 M2菌株B交配位点 Verified the B mating locus of M2 |
Ysg1r | ACTTCTCGTCTGCCTTCTTCTC | |
Ysg2f | GTCTCGGTCTGCCCGTCTTGGA | |
Ysg2r | AGGTGGGATTAGGCCGTTTGCG | |
Ysg3f | CGACACCGACATCTTCTACTGC | |
Ysg3r | TCCCCCTCTTGTTTTCATCCTA | |
Ysg4f | TCCCATCTCCCACCCACGAATC | |
Ysg4r | CACACACCCCACCAACACCCAA | |
Ysg5f | ACGCAGAGATGGAGGTGTGTAT | |
Ysg5r | AGGAAATGATTGGTGAATGGAG | |
Ysg6f | TGGAGATAAGATAAAGCCTGGG | |
Ysg6r | ATGCGTGCGTGTAGTGTAGAAA | |
Cbmrf | CTTATCCCAACGAAGTCTTTACC | 扩增cbmr片段 |
Cbmrr | CTCTGCCTGACGAACAACAT | Amplified for cbmr fragment |
SPf | ATCTGCCTATCTGCTCGTCC | 扩增YUN-3和SE-1/2/3群体 |
SPr | ATGAGTCTGTTTTGCTGGGC | Amplified for groups of YUN-3 and SE-1/2/3 |
表2 实验引物及序列
Table 2 Primers and sequences used in this study
引物 Primer | 序列 Sequence(5'-3') | 用途 Function |
---|---|---|
Cs1f | CCTCCAACGCTATTTGCCTACG | 验证 M67菌株B交配位点 Verified the B mating locus of M67 |
Cs1r | GCACCACCATCCTCACTTCACC | |
Cs2f | CGGCGTGTTTTCGTTTTTCT | |
Cs2r | ATGGCGGAGCGGTAGTTCTT | |
Cs3f | TAGCCTGGAACACTGGAACT | |
Cs3r | GAATGGAGCGAGGAAATAGA | |
Cs4f | AGCAAGGCGACAGTGGAAGAT | |
Cs4r | CGAGTGACATGAGGCGGAAGT | |
Cs5f | ATTCGCCTTCATTTCCTTCAT | |
Cs5r | TCAGCGTTCTCCCATTCTCAC | |
Cs6f | CACGGTAGCAAAGCAGAATGA | |
Cs6r | AGGAGCGTAGAGAAAGCAGGA | |
Ysg1f | GCTGTGCTCCTCCAACTCTATT | 验证 M2菌株B交配位点 Verified the B mating locus of M2 |
Ysg1r | ACTTCTCGTCTGCCTTCTTCTC | |
Ysg2f | GTCTCGGTCTGCCCGTCTTGGA | |
Ysg2r | AGGTGGGATTAGGCCGTTTGCG | |
Ysg3f | CGACACCGACATCTTCTACTGC | |
Ysg3r | TCCCCCTCTTGTTTTCATCCTA | |
Ysg4f | TCCCATCTCCCACCCACGAATC | |
Ysg4r | CACACACCCCACCAACACCCAA | |
Ysg5f | ACGCAGAGATGGAGGTGTGTAT | |
Ysg5r | AGGAAATGATTGGTGAATGGAG | |
Ysg6f | TGGAGATAAGATAAAGCCTGGG | |
Ysg6r | ATGCGTGCGTGTAGTGTAGAAA | |
Cbmrf | CTTATCCCAACGAAGTCTTTACC | 扩增cbmr片段 |
Cbmrr | CTCTGCCTGACGAACAACAT | Amplified for cbmr fragment |
SPf | ATCTGCCTATCTGCTCGTCC | 扩增YUN-3和SE-1/2/3群体 |
SPr | ATGAGTCTGTTTTGCTGGGC | Amplified for groups of YUN-3 and SE-1/2/3 |
图1 B交配位点结构特征 a, b:C. salicacola YAASM0711菌株B交配位点;c:M2菌株B交配位点;d:M67菌株B交配位点;e:序列相似性比较
Fig. 1 Characterization of B mating locus a, b:B mating locus of C. salicacola YAASM0711;c:B mating locus of M2;d:B mating locus of M67;e:comparison of sequence similarity
图3 基于mtSSU V4-6-9序列构建的邻接树 SE:东南地区菌株群体;YSG:YSG菌株群体;YUN:云南地区菌株群体;YS:杨柳田头菇菌株群体
Fig.3 Neighbor-joining tree constructed by mtSSU V4-6-9 sequences SE:Strain groups from southwest of China;YSG:strain groups of YSG;YUN:strain groups from Yunnan;YS:strain groups of C. salicacola
图4 基于cbmr序列构建的邻接树 SE-1/2/3/4:东南地区菌株群体;YSG:YSG菌株群体;YUN-1/2/3:云南地区菌株群体;YS-1/2/3:杨柳田头菇菌株群体
Fig.4 Neighbor-joining tree constructed by cbmr sequences SE-1/2/3/4:Strain groups from southwest of China;YSG:strain groups of YSG;YUN-1/2/3:strain groups from Yunnan;YS-1/2/3:strain groups of C. salicacola
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