巴氏新小绥螨转录组SSR位点分析及开发

刘辉, 李霞, 乌恩, 常静, 李志平, 霍志家, 李桂英, 武建华

刘辉, 李霞, 乌恩, 常静, 李志平, 霍志家, 李桂英, 武建华. 2024: 巴氏新小绥螨转录组SSR位点分析及开发. 南方农业学报, 55(3): 689-698. DOI: 10.3969/j.issn.2095-1191.2024.03.010
引用本文: 刘辉, 李霞, 乌恩, 常静, 李志平, 霍志家, 李桂英, 武建华. 2024: 巴氏新小绥螨转录组SSR位点分析及开发. 南方农业学报, 55(3): 689-698. DOI: 10.3969/j.issn.2095-1191.2024.03.010
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LIU Hui, LI Xia, WU En, CHANG Jing, LI Zhi-ping, HUO Zhi-jia, LI Gui-ying, WU Jian-hua. 2024: Analysis and development of SSR loci based on transcriptome of Neoseiulus barkeri. Journal of Southern Agriculture, 55(3): 689-698. DOI: 10.3969/j.issn.2095-1191.2024.03.010
Citation: LIU Hui, LI Xia, WU En, CHANG Jing, LI Zhi-ping, HUO Zhi-jia, LI Gui-ying, WU Jian-hua. 2024: Analysis and development of SSR loci based on transcriptome of Neoseiulus barkeri. Journal of Southern Agriculture, 55(3): 689-698. DOI: 10.3969/j.issn.2095-1191.2024.03.010
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巴氏新小绥螨转录组SSR位点分析及开发

  • 1 内蒙古农业大学园艺与植物保护学院, 内蒙古呼和浩特 010019;

  • 2 内蒙古农牧业技术推广中心, 内蒙古呼和浩特 010020;

  • 3 呼和浩特市农牧技术推广中心, 内蒙古呼和浩特 010020;

  • 4 巴彦淖尔市现代农牧事业发展中心, 内蒙古巴彦淖尔 015001

基金项目: 

内蒙古自然科学基金项目(2023LHMS03016)

内蒙古硕士研究生科研创新项目(S20231111Z)

详细信息
    作者简介:

    刘辉(1996-),https://orcid.org/0009-0001-2019-1241,研究方向为农药学,E-mail:3299248450@qq.com

    通讯作者:

    常静(1978-),https://orcid.org/0009-0002-5186-7950,博士,副教授,主要从事昆虫毒理学研究工作,E-mail:changjing10220@imau.edu.cn

  • 中图分类号: S476.2

Analysis and development of SSR loci based on transcriptome of Neoseiulus barkeri

  • 1 College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010019, China;

  • 2 Agriculture and Animal Husbandry Technology Promotion Center of Inner Mongolia Autonomuous Region, Hohhot, Inner Mongolia 010020, China;

  • 3 Hohhot Agriculture and Animal Husbandry Technology Promotion Center, Hohhot, Inner Mongolia 010020, China;

  • 4 Bayannnur Modern Agriculture and Animal Husbandry Development Center, Bayannnur, Inner Mongolia 015001, China

Funds: 

Natural Science Foundation of Inner Mongolia Autonomuous Region(2023LHMS03016)

Postgraduate Research Innovation Project of Inner Mongolia Autonomuous Region(S20231111Z)

摘要

    摘要
  • 摘要: 【目的】开发巴氏新小绥螨（Neoseiulus barkeri）高效氯氟氰菊酯抗药和敏感品系的微卫星（SSR）分子标记，为其抗药性基因遗传分析和抗药性品系的快速鉴定及筛选提供理论基础。【方法】应用MISA对巴氏新小绥螨转录组已注释的Unigenes数据进行搜索，高通量挖掘该螨的SSR位点。使用Primer 5.0进行SSR引物设计，随机挑选其中25对引物对巴氏新小绥螨高效氯氟氰菊酯抗性和敏感品系SSR位点基因进行PCR扩增，用重复性和稳定性高的12对引物进行实时荧光定量PCR分析基因表达。【结果】2个品系共获得52741条Unigenes，且SSR位点数量和分布密度一致。共筛选出5483个SSR位点，分布在4276条Unigenes中，发生频率为8.11%。主要重复类型为三核苷酸重复，占SSR总数的43.75%。共发现81种重复基元，其中单核苷酸重复基元A/T出现频率最高，占总量的19.65%。基于筛选出的SSR，共设计出4570对SSR引物，随机挑选的25对引物中有23对可扩增出巴氏新小绥螨高效氯氟氰菊酯抗性和敏感品系的目的基因片段。实时荧光定量PCR扩增结果显示，巴氏新小绥螨高效氯氟氰菊酯抗性品系有8个SSR基因位点的表达量高于敏感品系，其中TRINITY_DN24111_c0_g1基因的相对表达量为4.11，极显著高于敏感品系（1.06）（P<0.001，下同）；有4个SSR位点基因的相对表达量低于敏感品系，其中TRINITY_DN20624_c0_g1基因的相对表达量为0.68，极显著低于敏感品系（1.01）。【结论】巴氏新小绥螨转录组SSR位点多态性丰富，已筛选开发出的12对在巴氏新小绥螨抗性和敏感品系中表达量不同的SSR特异性引物可应用于对各地巴氏新小绥螨种群对高效氯氟氰菊酯抗药性的快速检测。
    Abstract: 【Objective】The aim of this study was to develop microsatellite （SSR） molecular markers for Lambdacyhalothrin resistant and susceptible strains of Neoseiulus barkeri， and to provide theoretical basis for genetic analysis of resistance genes and rapid identification and screening of resistant strains. 【Method】All unigenes from the transcriptome of N. barkeri were scanned using MISA software and its microsatellite （SSR） loci was screened by high-throughput. SSR primers were designed using Primer 5.0 software， 25 of these primer pairs were randomly selected for PCR amplification of the SSR loci genes for Lambda-cyhalothrin resistance and susceptible strains， and real-time fluorescence quantitative PCR gene expression analysis was conducted with 12 primer pairs with high reproducibility and stability. 【Result】A total of 52741 unigenes were obtained from the two strains， and the number and density of SSR loci were consistent. A total of 5483 SSR loci were detected from transcriptome， whose occurrence frequency was up to 8.11%， the analysis found that these loci were distributed in 4276 unigenes. SSR repeat types was mainly trinucleotide repeats， which accounted for 43.75% of the total number of SSR. A total of 81 kinds of repeat motifs were found， A/T was the most dominant motif in nucleotide repeats， accounting for 19.65% of the total， and 4570 SSR primers were designed， 23 pairs out of all 25 pairs randomly selected SSR primers could amplify the target gene fragments of both Lambda-cyhalothrin resistant and susceptible strains of N. barkeri. The results of real-time fluorescence quantitative PCR showed that the expression of 8 SSR loci genes of the Lambda-cyhalothrin resistant strains was higher than that of the susceptible strains ， and the relative expression of TRINITY_DN24111_c0_g1 gene was 4.11， extremely significantly higher than that of the susceptible strains （1.06）（P<0.001，the same below）. There were 4 SSR loci genes with lower relative expression than the susceptible strains， the relative expression of TRINITY_DN20624_c0_g1 gene was 0.68， extremely significant smaller than the susceptible strains（1.01）. 【Conclusion】The SSR loci in the transcriptome of N. barkeri are rich in information.The screened and developed 12 SSR specific primer pairs that express at different levels in N. barkeri resistant and sensitive lines can be applied to the rapid detection of Lambda-cyhalothrin resistance of N. barkeri.
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