基于种特异性COI标记的西花蓟马温室品系和羽扇豆品系一步双重快速鉴定技术

doi:10.3864/j.issn.0578-1752.2019.16.007

Abstract

Abstract:

【Background】Frankliniella occidentalis is one of the most important pests infesting agricultural and horticultural crops worldwide, also it is a national- and international-wide important quarantine pest. F. occidentalis has two races, i.e., greenhouse race (GR) and lupin race (LR). There are significant differences between the two races in host plant species, pesticide resistance, living environments, etc. However, it is difficult to identify these two races because of their high similarity in morphological characteristics.【Objective】In this study, a one-step duplex PCR procedure for rapid identification of F. occidentalis greenhouse race and lupin race was developed by using species-specific COI (SS-COI) marker based on mitochondrial DNA cytochrome c oxidase subunit I (mtDNA COI) gene, and other 14 common thrips species in the field were used as the control.【Method】The COI sequences of F. occidentalis greenhouse and lupin races, and other 14 thrips species were amplified using mtDNA COI gene universal primers. The race-specific primer combination system, TF6/GR32/LR12 (TF6, the universal forward primer; GR32, the greenhouse race-specific reverse primer; LR12, the lupin race-specific reverse primer), for one-step duplex amplification of the two races was established. The fragments amplified by the combined primers were 362 bp for the greenhouse race and 541 bp for the lupin race, respectively. The system and conditions (e.g., annealing temperature) were optimized. The species/race specificity and sensitivity/ detection threshold of the combined primers were evaluated.【Result】When the ratio of the combined primers (TF6/GR32/LR12) was 1.0/0.2/0.8 and the annealing temperature was 44℃, the amplification effect was the best. Species/race-specific tests performed with this set of combined primers indicated that all F. occidentalis greenhouse race and lupin race specimens were detected positively and no cross reaction with other 14 thrips species, including Frankliniella intonsa, Frankliniella tenuicornis, Thrips hawaiiensis, Thrips major, Thrips palmi, Thrips tabaci, Megalurothrips usitatus, Mycterothrips glycines, Sussericothrips melilotus, Fulmekiola serrate, Haplothrips aculeatus, Mesothrips jordani, Gynaikothrips ficorum, and Aeolothrips fasciatus, was identified. The system was tested on individual male and female adults, individual prepupae and pupae, individual 1st and 2nd instar larvae, and even single egg, and demonstrated to be applicable for all these stages of greenhouse and lupin races of F. occidentalis, either in single race or two races mixed together. The detection threshold for greenhouse race was 35.90 pg·μL ^-1, which was equivalent to 1/10 240 of a whole female adult. The detection threshold for lupin race was 146.95 pg·μL ^-1, which was equivalent to 1/2 560 of a whole female adult. Meanwhile, the system worked well in testing individuals from different geographic populations with different haplotypes within greenhouse race or lupin race of the thrips. 【Conclusion】The one-step duplex PCR system developed here for rapid identification of F. occidentalis greenhouse and lupin races could be useful in port quarantine and interception in national and international transportation of agricultural products, and should be significant in blocking further spreading and developing targeted prevention and control measures of F. occidentalis.

Key words: Frankliniella occidentalis greenhouse race, F. occidentalis lupin race, one-step duplex PCR, SS-COI marker, race-specific combined primer set, rapid identification

ZHANG Rong,WANG YuSheng,YANG LiMei,WAN FangHao,ZHANG GuiFen. One-Step Duplex Rapid Identification Technique of Frankliniella occidentalis Greenhouse and Lupin Races Based on Species-Specific COI Marker[J].Scientia Agricultura Sinica, 2019, 52(16): 2809-2823.

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Figures/Tables 11

Table 1

Collecting information of thrips in this study"

编号 No.	物种名称 Species	地点 Location	寄主 Host plant	时间 Date	采集人 Collector
1	西花蓟马温室品系 F. occidentalis greenhouse race	北京延庆,室内饲养 Yanqing, Beijing; indoor rearing	市售芸豆 Phaseolus vulgaris; marketed	2017-07	魏书军 WEI ShuJun
2	西花蓟马羽扇豆品系 F. occidentalis lupin race	北京延庆,室内饲养 Yanqing, Beijing; indoor rearing	市售芸豆 P. vulgaris; marketed	2017-07	魏书军 WEI ShuJun
3	花蓟马 F. intonsa	宁夏吴忠 Wuzhong, Ningxia	月季 Rosa chinensis	2016-08	田虎 TIAN Hu
4	禾花蓟马 F. tenuicornis	甘肃张掖 Zhangye, Gansu	葱 Allium fistulosum	2016-08	田虎 TIAN Hu
5	黄胸蓟马 T. hawaiiensis	山东烟台 Yantai, Shandong	未知种 Unidentified species	2016-11	田虎 TIAN Hu
6	大蓟马 T. major	青海西宁 Xining, Qinghai	月季 R. chinensis	2016-08	田虎 TIAN Hu
7	棕榈蓟马 T. palmi	浙江温州 Wenzhou, Zhejiang	西瓜 Citrullus lanatus	2018-07	薛延韬 XUE YanTao
8	烟蓟马 T. tabaci	贵州安顺 Anshun, Guizhou	茼蒿 Chrysanthemum coronarium	2018-04	杜素洁 DU SuJie
9	普通大蓟马 M. usitatus	广东河源 Heyuan, Guangdong	芸豆 P. vulgaris	2017-12	毛苗苗 MAO MiaoMiao
10	豆喙蓟马 M. glycines	山东滨州 Binzhou, Shandong	大豆 Glycine max	2018-06	薛延韬 XUE YanTao
11	草木樨近绢蓟马 S. melilotus	北京西城 Xicheng, Beijing	白三叶 Trifolium repens	2018-07	张桂芬 ZHANG GuiFen
12	蔗腹齿蓟马 F. serrata	山东青岛 Qingdao, Shandong	白茅 Imperata cylindrica	2018-07	薛延韬 XUE YanTao
13	稻简管蓟马 H. aculeatus	山东青岛 Qingdao, Shandong	芦苇 Phragmites australis	2018-07	薛延韬 XUE YanTao
14	榕端宽管蓟马 M. jordani	广东深圳 Shenzhen, Guangdong	榕树 Ficus microcarpa	2018-07	薛延韬 XUE YanTao
15	榕母管蓟马 G. ficorum	福建漳州 Zhangzhou, Fujian	榕树 F. microcarpa	2018-07	薛延韬 XUE YanTao
16	横纹蓟马 A. fasciatus	天津滨海新区 Binhai New District, Tianjin	大豆 G. max	2018-06	薛延韬 XUE YanTao

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品系 Race	引物名称 Primer name	碱基序列 Primer sequence (5′-3′)	扩增片段长度 Fragment length (bp)
温室品系 Greenhouse race	TF6/GR32	CGACTTAATAACATAAGATTTT/ GATGTATCTAAGTCTCGGTCT	362
羽扇豆品系 Lupin race	TF6/LR12	CGACTTAATAACATAAGATTTT/ GCATAGCATAGATTAGTCCC	541

采样地点 Location	种群代码 Population code	寄主植物 Host plant	采集时间 Collection date	测序个体数量 Number of sequenced
宁夏银川 Yinchuan, Ningxia	YC	美人蕉 Canna indica	2015-08	21
陕西宝鸡 Baoji, Shaanxi	BJI	月季 R. chinensis	2015-08	18
山东烟台 Yantai, Shandong	YT	蜀葵 Althaea rosea	2015-07	25
甘肃敦煌 Dunhuang, Gansu	DH	月季 R. chinensis	2015-08	8
吉林四平 Siping, Jilin	SP	美人蕉 C. indica	2014-09	2
吉林长春 Changchun, Jilin	CC	金盏菊 Calendula officinalis	2014-09	9
云南临沧 Lincang, Yunnan	LC	白三叶 T. repens	2015-04	12
甘肃兰州 Lanzhou, Gansu	LZ	辣椒 Capsicum annuum	2015-08	21
北京延庆 Yanqing, Beijing	YQ	辣椒 C. annuum	2014-07	8
河北廊坊 Langfang, Hebei	LF	辣椒 C. annuum	2015-07	3
合计Total	10			127

种群 Population	单倍型 Haplotype
	温室品系Greenhouse race			羽扇豆品系Lupin race
	GHap-1	GHap-2	GHap-3		LHap-1	LHap-2
YC	14	1			5	1
BJI	15	1			2
YT	20	3	1		1
DH	7	1
SP	1				1
CC	6				3
LC	12
LZ	19	2
YQ	7	1
LF	3
总计 Total	104	9	1		12	1

One-Step Duplex Rapid Identification Technique of Frankliniella occidentalis Greenhouse and Lupin Races Based on Species-Specific COI Marker

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