Development of a molecular marker for sex identification of Spodoptera frugiperda
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摘要:目的
开发用于草地贪夜蛾Spodoptera frugiperda性别鉴定的分子标记引物。
方法依据草地贪夜蛾雌、雄虫性信息素结合蛋白(Pheromone-binding protein, PBP)基因片段序列的差异设计特异性性别鉴定引物；利用引物对经过形态学鉴定的草地贪夜蛾雌、雄样本进行PCR验证。
结果2对引物分别对对应的雌、雄虫样本均扩增出450 bp左右的特异性条带。
结论开发的分子标记引物可以快速有效地对各个时期的草地贪夜蛾进行性别鉴定，为研究草地贪夜蛾雌、雄个体差异提供一种有效工具。为制定基于性别鉴定的草地贪夜蛾的有效治理防控策略提供参考。
Abstract:ObjectiveTo develop molecular marker primers for sexual identification of Spodoptera frugiperda.
MethodBased on the difference in pheromone-binding protein (PBP) gene sequences of male and female S. frugiperda, the primers were designed for distinguishing sexual distinction specifically. Using the primers, the male and female S. frugiperda samples already identified by morphology were further verified by PCR.
ResultSpecific bands of about 450 bp for the corresponding female and male samples were amplified by two pairs of primers respectively.
ConclusionThe molecular marker primers can be used to discriminate the sex of S. frugiperda in various developmental stages efficiently, and provide an effective tool for the study of the individual distinction between different sexual insects. This study provide a reference for formulating the strategy to control S. frugiperda effectively based on sexual distinction.
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图 1 草地贪夜蛾性别鉴定引物设计
深蓝色表示相同的核苷酸序列，浅蓝色表示差异位点，黑点表示缺失位点；Sf-male-R：雄虫基因差异区段；Sf-female-R：雌虫基因差异区段；Sf-F：正向引物；Sf-R：反向引物
Figure 1. Primers design for sexual identification of Spodoptera frugiperda
Dark blue represents the same nucleotide sequence, light blue represents the differencial sites and black dots represent the missing sites; SF-male-R: Differential gene segment of male; Sf-female-R: Differential gene segment of female; Sf-F: Forward primer; Sf-R: Reverse primer
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图 2 草地贪夜蛾蛹期雌、雄虫腹部末端差异对比
a：臀刺；b：肛门；c：第10腹节；d：第9腹节e：半圆形瘤状突起；f：第8腹节；g：产卵孔；h：生殖孔
Figure 2. Distinction between abdomen ends of male and female of Spodoptera frugiperda at pupal stage
a: Buttocks stab; b: Anus; c: The 10th abdominal segment; d: The 9th abdominal segment; e: Semicircular tumor-like protrusion; f: The 8th abdominal segment; g: Spawning hole; h: Genital hole
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图 3 3对引物对草地贪夜蛾雌、雄虫样本的扩增
M：2000 DNA marker; m1: Sf-F/ Sf-male-R-1; m2: Sf-F/Sf-male-R-2; m3: Sf-F/Sf-male-R-3; f1: Sf-F/Sf-female-R-1; f2: Sf-F/Sf-female-R-2; f3: Sf-F/Sf-female-R-3
Figure 3. Amplification of different sexual samples of Spodoptera frugiperdaby three pairs of primers
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图 4 基于PCR扩增对草地贪夜蛾雌、雄虫蛹性别鉴定
F1~F5：雌虫蛹DNA；M1~M5：雄虫蛹DNA；a、c：雌虫标记引物对 Sf-F/Sf-female-R-2；b、d：雄虫标记引物对Sf- F/Sf-male-R-2
Figure 4. Sex identification of male and female pupae of Spodoptera frugiperdabased on PCR amplification
F1−F5: DNA of female pupae; M1−M5: DNA of male pupae; a and c: Pair of female marker primers of Sf-F/Sf-female-R-2; b and d: Pair of male marker primers of Sf-F/Sf-male-R-2
表 1 引物序列表
Table 1 List of primer sequence
引物名称1) Primer name 引物序列(5′→3′) Primer sequence Sf-F TAGCCGTGAGTTTGAATAGGGT Sf-female-R-1 CCTGCCAGTGCCTTATTAATTAA Sf-male-R-1 TTTTGGCAGTGCCTTATTGATTA Sf-female-R-2 CTCAGAGGTTTTTGATATGGTTT Sf-male-R-2 TGTATTCTTCTCAGTGCGAAGAC Sf-female-R-3 TTAACAACGCTCCATAATAACCT Sf-male-R-3 TAAGAACCAGTTCTTATAAACAC 1) F、R分别表示正、反向引物
1) F and R respectively represents forward and reverse primers
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