Analysis and development of SSR loci based on transcriptome of Neoseiulus barkeri
-
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.
-
Keywords:
- Neoseiulus barkeri /
- SSR /
- high-throughput sequencing /
- sequence analysis /
- expression level
-
-
常静,李子奇,白琳,刘喆,董晓涵,孟瑞霞. 2021. 巴氏新小绥螨溴氰菊酯抗性品系的筛选及其抗性遗传方式研究[J]. 应用昆虫学报,58(6):1409-1416.[Chang J,Li Z Q,Bai L,Liu Z,Dong X H,Meng R X. 2021. A deltamethrin resistant strain of Neoseiulus barkeri:Selection for resistance and genetic analysis[J]. Chinese Journal of Applied Entomology,58(6):1409-1416.] doi: 10.7679/j.issn.20951353.2021.142. 崔明明,陶静,宗世祥. 2017. 基于转录组的沙棘木蠹蛾简单重复序列特征分析[J]. 环境昆虫学报,39(3):605-610.[Cui M M,Tao J,Zong S X. 2017. Feature analysis of simple sequence repeats in Eogystia hippophaecolus transcriptome[J]. Journal of Environmental Entomology,39(3):605-610.] doi: 10.3969/j.issn.1674-0858.2017.03.15. 杜炫星,牛敏敏,赵清,蔡波,卢运运,魏久锋,张虎芳. 2023.李白盾蚧转录组分析及SSR 位点开发[J]. 环境昆虫学报,45(5):1318-1322.[Du X X,Niu M M,Zhao Q,Cai B,Lu Y Y,Wei J F,Zhang H F. 2023. Analysis of the transcriptome and development of SSR loci in Pseudaulacaspis prunicola(Maskell)[J]. Journal of Environmental Entomology, 45(5):1318-1322.] doi: 10.969/j/issn.1674-0858.2023.05.18. 段惠生,张安盛,赵传志,于毅,褚栋. 2012. 西花蓟马ESTSSR 信息分析、标记筛选及其与Genomic-SSR 的多态性比较[J]. 昆虫学报,55(6):634-640.[Duan H S,Zhang A S,Zhao C Z,Yu Y,Chu D. 2012. Characterization and molecular marker screening of EST-SSRs and their polymorphism compared with genomic-SSRs in Frankliniella occidentalis (Thysanoptera:Thripidae)[J]. Acta Entomologica Sinica,55(6):634-640.] doi: 10.16380/j.kcxb.2012.06.004. 郭欢,王刚,张树田,黄敏. 2018. 基于RNA-Seq数据的窄足真蚋SSR分子标记开发[J]. 昆虫学报,61(7):815-824.[Guo H,Wang G,Zhang S T,Huang M. 2018. Development of SSR primers for Simulium (Eusimulium) angustipes (Diptera:Simuliidae) based on RNA-Seq dataset[J]. Acta Entomologica Sinica,61(7):815-824.] doi: 10.16380/j.kcxb.2018.07.008. 郭睿,陈华枝,庄天艺,熊翠玲,郑燕珍,付中民,陈恒,陈大福. 2018. 利用转录组数据开发意大利蜜蜂的SSR 分子标记[J]. 安徽农业大学学报,45(3):404-408.[Guo R, Chen H Z,Zhuang T Y,Xiong C L,Zheng Y Z,Fu Z M, Chen H,Chen D F. 2018. Exploitation of SSR markers for Apis mellifera ligustica based on transcriptome data[J]. Journal of Anhui Agricultural University,45(3):404-408.] doi: 10.13610/j.cnki.1672-352x.20180620.015. 侯栋元,丛林,陈飞,杨娟生,周浩楠,成禄艳,于士将,雷双,刘浩强,冉春. 2020. 巴氏新小绥螨甲氰菊酯抗性品系生物学特性及其对常用药剂的交互抗性[J]. 应用昆虫学报,57(3):690-699.[Hou D Y,Cong L,Chen F,Yang J S, Zhou H N,Cheng L Y,Yu S J,Lei S,Liu H Q,Ran C. 2020. Biological characteristics of a fenpropathrin-resistant strain of Neoseiulus barkeri including cross-resistance to commonly used pesticides in orange orchards[J]. Chinese Journal of Applied Entomology,57(3):690-699.] doi: 10.7679/j.issn.2095-1353.2020.070. 柯富士. 2013. 小菜蛾转录组微卫星序列的分析和多态性位点的开发[D]. 福州:福建农林大学.[Ke F S. 2013. Characterization and isolation of microsatellites from the Plutella xylostella transcriptome[D]. Fuzhou:Fujian Agriculture and Forestry University.] 冷春蒙,李引,胡迪,仵均祥,李怡萍. 2018. 梨小食心虫幼虫中肠转录组及SSR分子标记分析[J]. 昆虫学报,61(11):1272-1283.[Leng C M,Li Y,Hu D,Wu J X,Li Y P. 2018. Analysis of the larval midgut transcriptome and SSR markers in Grapholitha molesta (Lepidoptera:Tortricidae)[J]. Acta Entomologica Sinica,61(11):1272-1283.] doi: 10.16380/j.kcxb.2018.11.004. 李斌,夏庆友,鲁成,周泽扬. 2004. 蜜蜂EST中的微卫星分析(英文)[J]. 遗传学报,31(10):1089-1094.[Li B,Xia Q Y,Lu C,Zhou Z Y. 2004. Analysis of microsatellites derived from bee ESTs[J]. Acta Genetica Sinica,31(10):1089-1094.] 黎东海,赵萍. 2019. 基于转录组数据的齿缘刺猎蝽微卫星分子标记开发[J]. 昆虫学报,62(6):694-702.[Li D H,Zhao P. 2019. Development of microsatellite markers based on the transcriptome data of Sclomina erinacea (Heteroptera:Reduviidae)[J]. Acta Entomologica Sinica,62(6):694702.] doi: 10.16380/j.kcxb.2019.06.005. 李冬芝,张美霞,单伊曼,王芳. 2022. 基于转录组的褐软蜡蚧微卫星分子标记开发[J]. 河北师范大学学报(自然科学版),46(5):502-511.[Li D Z,Zhang M X,Shan Y M, Wang F. 2022. Development of microsatellite markers based on the transcriptome data of Coccus hesperidum(Hemiptera:Coccidae)[J]. Journal of Hebei Normal University(Natural Science),46(5):502-511.] doi: 10.13763/j.cnki.jhebun.nse.202204011. 李敏,张丹丽,李荣荣,雷廷,宋鲜梅,卜文俊. 2022. 中华大仰蝽转录组学研究及SSR 新标记开发[J]. 昆虫学报,65(1):31-43.[Li M,Zhang D L,Li R R,Lei T,Song X M, Bu W J. 2022. Analysis of the transcriptome and development of novel SSR markers in Notonecta chinensis (Hemiptera:Notonectidae)[J]. Acta Entomologica Sinica,65(1):31-43.] doi: 10.16380/j.kcxb.2022.01.004. 李微,张蕾,程云霞,罗礼智,江幸福. 2017. 应用转录组测序高通量发掘东方粘虫SSR 标记[J]. 植物保护学报,44(3):377-384.[Li W,Zhang L,Cheng Y X,Luo L Z,Jiang X F. 2017. High-throughput discovery of microsatellite markers based on transcriptome sequencing in the oriental armyworm,Mythimna separata(Walker)[J]. Journal of Plant Protection,44(3):377-384.] doi: 10.13802/j.cnki.zwbhxb.2017.2016058. 刘昭阳,李玉蓉,潘忠玉,郭冰,骆有庆,宗世祥,陶静. 2016.光肩星天牛两型种群表型多样性分析[J]. 应用昆虫学报,53(5):1045-1057.[Liu Z Y,Li Y R,Pan Z Y,Guo B, Luo Y Q,Zong S X,Tao J. 2016. Phenotypic diversity of Anoplophora glabripennis(Motschulsky)[J]. Chinese Journal of Applied Entomology,53(5):1045-1057.] doi: 10.7679/j.issn.2095-1353.2016.129. 罗梅,张鹤,宾淑英,林进添. 2014. 基于转录组数据高通量发掘扶桑绵粉蚧微卫星引物[J]. 昆虫学报,57(4):395400.[Luo M,Zhang H,Bin S Y,Lin J T. 2014. Highthroughput discovery of SSR genetic markers in the mealybug, Phenacoccus solenopsis(Hemiptera:Pseudococcidae), from its transcriptome database[J]. Acta Entomologica Sinica,57(4):395-400.] doi: 10.16380/j.kcxb.2014.04.005. 桑迪,徐叶,王伟亮,向敏,季荣,王晗. 2020. 基于转录组数据的意大利蝗微卫星位点分析与分子标记开发[J]. 应用昆虫学报,57(3):658-666.[Sang D,Xu Y,Wang W L, Xiang M,Ji R,Wang H. 2020. Analysis of microsatellite loci from Calliptamus italicus (Orthopera:Acrididae) based on a transcriptome dataset[J]. Chinese Journal of Applied Entomology,57(3):658-666.] doi:10.7679/j.issn. 20951353.2020.066. 唐培安,陶冶心,薛昊,袁明龙. 2017. 基于转录组数据的印度谷螟微卫星位点分析[J]. 植物保护,43(3):43-48.[Tang P A,Tao Y X,Xue H,Yuan M L. 2017. Analysis of microsatellite loci in Plodia interpunctella based on transcriptome dataset[J]. Plant Protection,43(3):43-48.] doi:10. 3969/j.issn.0529-1542.2017.03.00. 王晨,杜联明,李鹏,杨茗羽,李午佼,沈咏梅,张修月,岳碧松. 2015. 德国小蠊全基因组中微卫星分布规律[J]. 昆虫学报,58(10):1037-1045.[Wang C,Du L M,Li P, Yang M Y,Li W J,Shen Y M,Zhang X Y,Yue B S. 2015. Distribution patterns of microsatellites in the genome of the German cockroach(Blattella germanica)[J]. Acta Entomologica Sinica,58(10):1037-1045.] doi: 10.16380/j.kcxb.2015.10.001. 王定锋,李良德,李慧玲,李金玉,吴光远. 2021. 基于转录组数据高通量发掘灰茶尺蠖微卫星标记[J]. 茶叶学报,62(4):191-197.[Wang D F,Li L D,Li H L,Li J Y,Wu G Y. 2021. High-throughput unveiling of microsatellite markers using Ectropis grisescens transcriptome[J]. Acta Tea Sinica, 62(4):191-197.] doi:10.3969/j.issn.1007-4872.2021. 04.006. 王恩东,吴圣勇,吕佳乐,姜晓环,马兆义,刘亚杰,刘振州,赵宏玉,吴朔,吴霞,徐学农. 2020. 释放巴氏新小绥螨防治温室大棚番茄上的烟粉虱[J]. 植物保护,46(4):234238.[Wang E D,Wu S Y,Lü J L,Jiang X H,Ma Z Y,Liu Y J,Liu Z Z,Zhao H Y,Wu S,Wu X,Xu X N. 2020. Control of whitefly Bemisia tabaci on tomato plants in greenhouse by releasing Neoseiulus barkeri[J]. Plant Protection, 46(4):234-238.] doi: 10.16688/j.zwbh.2019151. 王晖,王敬,高妍夏,杨帆,马宝骏,古苗,张乘云,薛翠翠,谢岩. 2022. 基于家蚕转录组测序的SSR 序列分析[J]. 山东农业科学,54(1);14-20.[Wang H,Wang J,Gao Y X, Yang F,Ma B J,Gu M,Zhang C Y,Xue C C,Xie Y. 2022. SSR sequence analysis based on Bombyx mori transcriptome sequencing[J]. Shandong Agricultural Sciences,54(1):14-20.] doi: 10.14083/j.issn.1001-4942.2022.01.003. 王蔓,李波,黄婕,门兴元,季洁,刘永杰,尹淑艳. 2019. 加州新小绥螨和巴氏新小绥螨对二斑叶螨的捕食能力比较[J]. 应用昆虫学报,56(6):1256-1263.[Wang M,Li B, Huang J,Men X Y,Ji J,Liu Y J,Yin S Y. 2019. Comparison of Neoseiulus californicus and Neoseiulus barkeri as biological controls for Tetranychus urticae[J]. Chinese Journal of Applied Entomology,56(6):1256-1263.] doi: 10.7679/j.issn.2095-1353.2019.136. 王明明,王思琦,孟威,姜策,江幸福,付晓伟,王小奇,王兴亚. 2021. 基于SSR 分子标记的辽宁地区甜菜夜蛾遗传变异与种群遗传结构[J]. 应用昆虫学报,58(5):11431151.[Wang M M,Wang S Q,Meng W,Jiang C,Jiang X F,Fu X W,Wang X Q,Wang X Y. 2021. Genetic variation and population genetic structure of the beet armyworm, Spodoptera exigua (Lepidoptera:Noctuidae),in Liaoning, based on microsatellite marker variation[J]. Chinese Journal of Applied Entomology,58(5):1143-1151.] doi:10. 7679/j.issn.2095-1353. 2021.115. 魏丹丹,石俊霞,张夏瑄,陈世春,魏冬,王进军. 2014. 基于转录组数据的桔小实蝇微卫星位点信息分析[J]. 应用生态学报,25(6):1799-1805.[Wei D D,Shi J X,Zhang X X, Chen S C,Wei D,Wang J J. 2014. Analysis of microsatellite loci from Bactrocera dorsalis based on transcriptome dataset[J]. Chinese Journal of Applied Ecology,25(6):1799-1805.] doi: 10.13287/j.1001-9332.20140409.022. 袁远,张丽芳,吴国星,朱家颖. 2014. 云南切梢小蠹微卫星的高通量发掘[J]. 环境昆虫学报,36(2):166-170.[Yuan Y,Zhang L F,Wu G X,Zhu J Y. 2014. High-throughput discovery microsatellites in Tomicus yunnanensis (Coleoptera:Scolytinae)[J]. Journal of Environmental Entomology, 36(2):166-170.] doi: 10.3969/j.issn.1674-0858.2014.02.7. 张丹,林文忠,刘巍巍,孟威,仇贵生,曲智. 2021. 巴氏新小绥螨对苹果树苹果全爪螨的生物防治效果[J]. 中国果树, (7):62-64.[Zhang D,Lin W Z,Liu W W,Meng W,Qiu G S,Qu Z. 2021. Biocontrol effect of Neoseiulus barkeri on Panonychus ulmi in apple trees[J]. China Fruits,(7):62-64.] doi: 10.16626/j.cnki.issn1000-8047.2021.07.013. 张鹏飞,周晓榕,庞保平,谭瑶,常静,高利军. 2016. 基于转录组数据高通量发掘沙葱萤叶甲微卫星引物[J]. 应用昆虫学报,53(5):1058-1064.[Zhang P F,Zhou X R,Pang B P,Tan Y,Chang J,Gao L J. 2016. High-throughput discovery of microsatellite markers in Galeruca daurica (Coleoptera:Chrysomelidae) from a transcriptome database[J]. Chinese Journal of Applied Entomology,53(5):10581064.] doi: 10.7679/j.issn.2095-1353.2016.130. 张倩,成立新,温彩霞,常静,孟瑞霞,李海平,王振,戴桂香. 2022. 巴氏新小绥螨溴氰菊酯抗药性品系对常见药剂的交互抗性及其种群参数分析[J]. 植物保护,48(3):126130.[Zhang Q,Cheng L X,Wen C X,Chang J,Meng R X,Li H P,Wang Z,Dai G X. 2022. Cross-resistance to commonly used pesticides and analysis of population parameters in a deltamethrin-resistant strain of Neoseiulus barkeri[J]. Plant Protection,48(3):126-130.] doi:10. 16688/j.zwbh.2021495. Azandeme-Hounmalon G Y,Sikirou R,Onzo A,Fiaboe K K M,Tamo M,Kreiter S,Martin T. 2022. Re-assessing the pest status of Tetranychus evansi (Acari:Tetranychidae) on solanaceous crops and farmers control practices in Benin[J]. Journal of Agriculture and Food Research,10:100401. doi: 10.1016/j.jafr.2022.100401.
Chen J Y,Zheng L J,Ye Z P,Wang J Y,Zhang F P,Fu Y G, Zhang C H. 2023. Evaluation of predatory mite Neoseiulus barkeri against spider mites damaging rubber trees[J]. Insects,14(7):648. doi:10.3390/insects 14070648.
Deng K P,Deng R J,Fan J X,Chen E F. 2018. Transcriptome analysis and development of simple sequence repeat (SSR) markers in Zingiber striolatum Diels[J]. Physiology and Molecular Biology of Plants,24(1):125-134. doi: 10.1007/s12298-017-0485-0.
Jarne P,Lagoda P J L. 1996. Microsatellites,from molecules to populations and back[J]. Trends in Ecology and Evolution, 11(10):424-429. doi: 10.1016/0169-5347(96)10049-5.
Kalia R K,Rai M K,Kalia S,Singh R,Dhawan A K. 2011. Microsatellite markers:An overview of the recent progress in plants[J]. Euphytica,177(3):309-334. doi: 10.1007/s10681-010-0286-9.
Li C C,Li G Y,Wang Y,Peng Y. 2020. Development of SSR markers based on transcriptome sequences of the Wolf spider Pardosa pseudoannulata (Araneae:Lycosidae)[J]. Entomology News,129:6-15. doi: 10.3157/021.129.0102.
Li L T,Zhu Y B,Ma J F,Li Z Y,Dong Z P. 2013. An analysis of the Athetis lepigone transcriptome from four developmental stages[J]. PLoS One,8(9):e73911. doi: 10.1371/journal.pone.0073911.
Nigam D,Saxena S,Ramakrishna G,Singh A,Singh N K,Gaikwad K. 2017. De novo assembly and characterization of Cajanus scarabaeoides (L.) thouars transcriptome by pairedend sequencing[J]. Frontiers in Molecular Biosciences,4:00048. doi: 10.3389/fmolb.2017.00048.
Ouyang H L,Wang X Y,Zheng X L,Lu W,Qin F P,Chen C. 2021. Full-length SMRT transcriptome sequencing and SSR analysis of Bactrocera dorsalis(Hendel)[J]. Insetc, 12(10):938-955. doi: 10.3390/insects12100938.
"Simonato M,Pilati M,Magnoux E,Courtin C,Sauné L,Rousselet J,Battisti A,Auger R,Marie A,Kerdelhu C. 2019. A population genetic study of the egg parasitoid Baryscapus servadeii reveals large scale automictic parthenogenesis and almost fixed homozygosity[J]. Biological Control, 139:104097. doi: 10.1016/j.biocontrol.2019.104097.
Tian C B,Li Y Y,Wang X,Fan W H,Wang G,Liang J Y, Wang Z Y,Liu H. 2019. Effects of UV-B radiation on the survival,egg hatchability and transcript expression of antioxidant enzymes in a high-temperature adapted strain of Neoseiulus barkeri[J]. Experimental and Applied Acarology, 77(4):527-543. doi: 10.1007/s10493-019-00361-9.
Tóth G,Gáspári Z,Jurka J. 2000. Microsatellites in different eukaryotic genomes:Survey and analysis[J]. Genome Research, 10(7):967-981. doi: 10.1101/gr.10.7.967.
Xu Y,Zhou W W,Zhou Y J,Wu J X,Zhou X P. 2012. Transcriptome and comparative gene expression analysis of Sogatella furcifera (Horváth) in response to southem rice black-streaked dwarf virus[J]. PLoS One,7(4):e36238. doi: 10.1371/journal.pone.0036238.
Yang J,Huang L,Li Z R,Sun H Q,Zhao W X,Yao Y X. 2021. Development and preliminary application of novel genomewide SSR markers for genetic deversity analysis of an economically important bio-control agent Platygaster robiniae(Hymenoptera:Platygastridae)[J]. Journal of Genetics, 100(2):67-74. doi: 10.1007/s12041-021-01318-x.
-
期刊类型引用(1)
1. 陈平,刘正玲,杨亚辉,李凡,詹莜国,史爱民,谢永辉,兰平秀. 5种杀虫剂对西花蓟马的室内毒力及其对巴氏新小绥螨的安全性评价. 云南农业大学学报(自然科学). 2024(04): 23-28 . 百度学术
其他类型引用(0)
计量
- 文章访问数: 412
- HTML全文浏览量: 0
- PDF下载量: 4
- 被引次数: 1