ISSR-PCR Optimization and Primer Selection for Analyzing Exserohilum turcicum Isolates Collected in Fujian
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摘要:
目的 明确适用于福建省玉米大斑病菌ISSR分子标记的反应体系和引物。 方法 采用单因素水平优化法对ISSR-PCR扩增反应中的Taq聚合酶用量、dNTPs浓度、Mg2+浓度、模板DNA浓度、PCR反应循环数以及引物的最佳退火温度等重要参数进行优化。 结果 适合福建省玉米大斑病菌群体遗传多样性分析的ISSR-PCR反应体系（25 μL）为：Taq聚合酶0.55 U、dNTPs 0.30 mmol·L-1、Mg2+ 1.30 mmol·L-1、DNA模板100 ng、引物10 pmol。ISSR-PCR扩增程序为：94℃预变性4 min；94℃变性45 s，51.2~56.0℃退火45 s，72℃延伸1.5 min，35个循环；72℃延伸10 min。利用优化的反应体系从56条ISSR引物中筛选出稳定性好、多态性高的引物10条：UBC117、UBC118、UBC808、UBC835、UBC847、UBC855、UBC856、UBC857、UBC866和UBC887，其最佳退火温度分别为55.6、53.1、51.2、51.2、56.0、53.1、53.1、51.2、51.2和51.2℃。利用优化的ISSR-PCR反应体系对21株供试菌株进行PCR扩增，结果表明，相同地理来源以及不同地理来源菌株间的DNA多态性均不同，表明福建省玉米大斑病菌群体存在丰富的遗传多样性。 结论 本研究优化的ISSR-PCR反应体系和筛选的引物可用于福建省玉米大斑病菌群体遗传多样性和遗传结构的研究。 -
关键词:
- 玉米大斑病菌 /
- 反应体系 /
- 简单序列重复区间扩增多态性 /
- 玉米大斑病 /
- 遗传多样性
Abstract:Objective To determine the optimal reaction conditions and primers for the inter-simple sequence repeat (ISSR) PCR in analyzing isolates of Exserohilum turcicum, a pathogen of the northern corn leaf blight in Fujian. Method Conditions for the ISSR-PCR amplification including Taq polymerase dosage, concentrations of dNTPs, Mg2+ and template DNA and reaction cycle as well as annealing temperatures for selected primers were optimized using a single-factor method. Result For a 25 μL ISSR-PCR genetic diversity analysis on E. turcicum, the following conditions were applied:0.55 U of Taq polymerase, 0.30 mmoL·L-1 of dNTPs, 1.30 mmoL·L-1 of Mg2+, 100 ng of template DNA, and 10 pmol of primer under 94℃ for 4 min followed by 35 cycles of 45 s at 94℃, 45 s at 51.2-56.0℃, and 1.5 min at 72℃, and finally, at 72℃ for 10 min. Ten stable, polymorphic primers, i.e., UBC117, UBC118, UBC808, UBC835, UBC847, UBC855, UBC856, UBC857, UBC866, and UBC887, were selected from 56 ISSR primers, with their optimized annealing temperatures determined to be 55.6, 53.1, 51.2, 51.2, 56.0, 53.1, 53.1, 51.2, 51.2, and 51.2℃, respectively. Regardless of their geographical origins, the 21 E. turcicum isolates differed on DNA polymorphism indicating an abundance on genetic diversity among them. Conclusion The optimized ISSR-PCR reaction conditions and primers could be applied for genetic studies on E. turcicum in Fujian. -
图 1 引物UBC847的反应体系优化
注：M：5-kb DNA Marker；(A) 1~8：Taq酶用量分别为0.25、0.35、0.45、0.55、0.65(推荐剂量)、0.75、0.85和0.95 U；(B) 1~8：dNTPs浓度分别为0.07、0.10、0.14、0.17、0.20(推荐剂量)、0.23、0.26和0.30 mmol·L-1；(C) 1~8：Mg2+浓度分别为0.5、0.8、1.0、1.3、1.5(推荐剂量)、1.7、2.0和2.2 mmol·L-1；(D) 50~200：DNA模板量分别为50、100、150和200 ng；(E) 25~40：循环数分别为25、30、35和40次；CK：空白对照。
Figure 1. Reaction condition optimization for primer UBC847
Note:M:5-kb DNA Marker; (A) 1-8:the amount of Taq polymerase 0.25, 0.35, 0.45, 0.55, 0.65 (recommended dosages), 0.75, 0.85 and 0.95 U, respectively; (B) 1-8:the concentration of dNTPs 0.07, 0.10, 0.14, 0.17, 0.20 (recommended dosages), 0.23, 0.26 and 0.30 mmol·L-1, respectively; (C) 1-8:the concentration of Mg2+ 0.5, 0.8, 1.0, 1.3, 1.5 (recommended dosages), 1.7, 2.0 and 2.2 mmol·L-1, respectively; (D) 50-200:the concentration of template DNA 50, 100, 150 and 200 ng, respectively; (E) 25-40:the cycle number 25, 30, 35 and 40, respectively; CK:blank control.
图 2 引物UBC847的最佳退火温度筛选
注：M：5-kb DNA Marker；1~8：退火温度梯度依次为56.0、55.6、54.7、53.1、51.2、49.6、48.5和48.0℃；CK：空白对照。
Figure 2. Selection of annealing temperature for primer UBC847
Note:M:5-kb DNA Marker; 1-8:the annealing temperature 56.0, 55.6, 54.7, 53.1, 51.2, 49.6, 48.5 and 48.0℃, respectively; CK:blank control.
图 3 使用引物UBC847和UBC887对福建省21个玉米大斑病菌株进行ISSR-PCR扩增
注：M：5-kb DNA Marker；1~21：福建省玉米大斑病菌的菌株序号与表 1对应；(A)和(B)：引物UBC847和UBC887分别对21株玉米大斑病菌的ISSR-PCR扩增结果；CK：空白对照。
Figure 3. ISSR-PCR amplification of 21 E. turcicum isolates using primer UBC847 and UBC887
Note:M:5-kb DNA Marker; 1-21:isolates of E. turcicum in Fujian Province corresponding with Table 1; (A) and (B):ISSR-PCR amplification results of 21 E. turcicum isolates in Fujian Province using primer UBC847 and UBC887, respectively; CK:blank control.
表 1 供试的福建省玉米大斑病菌菌株
Table 1. E. turcicum isolates collected in Fujian
序号
Number菌株编号
Isolate code采集地
Location年份
Year1 JO1801 建瓯市东游镇Dongyou town of Jian′ou city 2018 2 JO1809 建瓯市东游镇Dongyou town of Jian′ou city 2018 3 JODB1701 建瓯市东游镇Dongyou town of Jian′ou city 2017 4 JODB1702 建瓯市东游镇Dongyou town of Jian′ou city 2017 5 DFDB1901 建瓯市东峰镇Dongfeng town of Jian′ou city 2019 6 DFDB1902 建瓯市东峰镇Dongfeng town of Jian′ou city 2019 7 DFDB1903 建瓯市东峰镇Dongfeng town of Jian′ou city 2019 8 DFDB1905 建瓯市东峰镇Dongfeng town of Jian′ou city 2019 9 SX1801 松溪县Songxi county 2018 10 SX1802 松溪县Songxi county 2018 11 SX1804 松溪县Songxi county 2018 12 PNDB1602a 屏南县Pingnan county 2016 13 PNDB1604b 屏南县Pingnan county 2016 14 PNDB1606a 屏南县Pingnan county 2016 15 PNDB1608a 屏南县Pingnan county 2016 16 PNDB1609a 屏南县Pingnan county 2016 17 NJDB1805 南靖县Nanjing county 2018 18 NJDB1809 南靖县Nanjing county 2018 19 NJDB1810 南靖县Nanjing county 2018 20 NJDB1816 南靖县Nanjing county 2018 21 NJDB1830 南靖县Nanjing county 2018 
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表 2 适宜福建省玉米大斑病菌ISSR分子标记的引物及最佳退火温度
Table 2. Primers and annealing temperatures for ISSR molecular marker of E. turcicum isolates
引物名称
Primer name引物序列(5′-3′)
Primer sequence最适退火温度(℃)
Optimal Tm引物来源
Primer origin是否适合福建种群
Whether suitable for Fujian populationUBC112 (AG)8TA (52) UBC, [15] 不适合unsuitability UBC113 (AG)8TC (54) (52.7) UBC, [15], [16] 不适合unsuitability UBC117 (AGA)7 55.6 (56) (52.7) UBC, [15], [16] 适合suitability UBC118 (GTC)6 53.1 (59.5) (53) UBC, [15], [16] 适合suitability UBC121 (GTGC)4 (61.8) (49) UBC, [15], [16] 不适合unsuitability UBC808 (AG)8C 51.2 (52.7) UBC, [16] 适合suitability UBC835 (AG)8YC 51.2 (53) UBC, [16] 适合suitability UBC847 (CA)8RT 56.0 (49.1) UBC, [16] 适合suitability UBC855 (AC)8YT 53.1 (49.4) UBC, [16] 适合suitability UBC856 (AC)8YA 53.1 UBC 适合suitability UBC857 (AC)8YG 51.2 UBC 适合suitability UBC866 (CTC)6 51.2 UBC 适合suitability UBC887 DVD(TC)7 51.2 UBC 适合suitability 注：① Tm：退火温度，小括弧内的数字为文献中报道的引物最佳退火温度；② UBC：加拿大哥伦比亚大学植物病原真菌基因组DNA重复序列ISSR引物库(http:/www.michaelsmith.ubc.ca)。
Note：①Tm：annealing temperature, numbers in parenthesis are the optimal annealing temperatures for primers reported in the literatures; ② UBC：Primer database for genomic DNA repeat sequence of phytopathogenic fungi established by University of Columbia, Canada (http:/www.michaelsmith.ubc.ca).
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