issr bemisia tabaci

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Agricultural Sciences in China2008, 7(11): 1348-1354

2008, CAAS. All rights reserved. Published by Elsevier Ltd.

November 2008

Comparative Analysis of Population Genetic Structure in Bemisia tabaci (Gennadius) Biotypes B and Q Based on ISSR Marker

CHU Dong 1, 2 , WAN Fang-hao 3, XU Bao-yun 2, WU Qing-jun 2 and ZHANG You-jun 2

1 High-Tech Research Center, Shandong Academy of Agricultural Sciences, Jinan 250100, P.R.China2 Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, Beijing 100081, P.R.China3 The State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural

Sciences, Beijing 100081, P.R.China

Abstract

Bemisia tabaci (Gennadius) biotypes B and Q are two invasive biotypes in the species complex. The comparison of the

population genetic structure of the two biotypes is of significance to show their invasive mechanism and to their control.

The intersimple sequence repeats (ISSR) marker was used to analyze the 16 B-biotype populations and 4 Q-biotype

populations worldwide with a Trialeurodes vaporariorum population in Shanxi Province, China, and a B. tabaci non-B/Q-

biotype population in Zhejiang Province, China, was used as control populations. The analysis of genetic diversity

showed that the diversity indexes of biotype Q including Neis gene diversity index, Shannon informative index, and the

percentage of polymorphic loci were higher than those of biotype B. The high genetic diversity of biotype Q might provide the genetic basis for the excellent ecological adaptation. Cluster analysis suggested that the ISSR could not be used

in the phylogenetic analysis though it could easily distinguish the biotypes of B. tabaci . The difference of the population

genetic structure between the biotype B and the biotype Q exists based on the ISSR marker. Meanwhile, the results

suggested that the molecular marker has its limitation in the phylogenetic analysis among the biotypes of B. tabaci .

Key words : invasive mechanism, Bemisia tabaci biotype B, Bemisia tabaci biotype Q, genetic structure, ISSR

INTRODUCTION

Bemisia tabaci (Gennadius) is an important agricultural pest worldwide, which can not only directly damagethe plants, but also transmit numerous plant viruses(Jones 2003). The whitefly is generally considered tobe species complex, which includes several geneticallydifferentiated populations. Some populations have beenlabeled as different biotypes, among them the biotype Band the biotype Q are the two most invasive biotypes.

B. tabaci biotype B had been an invasive pest world-wide since its outbreak in the USA during the middle-

late 1980s. B. tabaci biotype Q originated from Medi-terranean regions has successfully introduced into non-Mediterranean countries including China (Chu et al .2005), the USA, Guatemala, Mexico, and Japan (Uedaand Brow 2006), and has caused damages during therecent years. The main biotype of the whitefly hascaused severe damages in most regions of China (Zhang2000; Liu et al . 2005), and was identified as biotype Bbased on the mitochondrial cytochrome oxidase I(mtDNA COI). Meanwhile, the biotype Q has invadedinto Yunnan, Beijing, Henan (Chu et al . 2006), Zhejiang(Xu et al . 2006), and Taiwan (Hsieh et al . 2007) of China. The comparative analysis of population genetic

This paper is translated from its Chinese version in Scientia Agricultura Sinica.

CHU Dong, Ph D, E-mail: [email protected]; Correspondence ZHANG You-jun, Tel: +86-10-82109518, E-mail: [email protected]


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Comparative Analysis of Population Genetic Structure in Bemisia tabaci (Gennadius) Biotypes B and Q Based 1349


structure of the two biotypes will provide significantresults to show the genetic mechanism of invasion and

also guide in the management of these biotypes (Suarezet al . 1999; Tsutsui et al . 2001). The study will alsoshow the rapid evolutional process and its influentialfactors of alien species (Chu et al . 2004). Chu et al .(2007a) have studied the genetic structure of the twobiotypes using the random amplified polymorphic DNA(RAPD). The intersimple sequence repeat (ISSR)marker is from the RAPD, except the primer used isISSR. Compared with the RAPD primers, the ISSR

primers has the stability of DNA fingerprinting patternand the efficiency of the RAPD and is easy to operate(An et al . 2002). During the recent years, it has beenwidely used in the study of invasive alien species (Chuet al . 2007b; Gui et al . 2007; Wang et al . 2007). Theanalysis of biotype A and biotype B based on ISSR byPerring et al . (1993) showed that the genetic similarityof them is only about 10%. The study on the geneticstructure of biotype B and biotype Q based on ISSR has not been carried out until now. In the present study,the genetic structure of biotype B and biotype Q wasanalyzed using ISSR marker to further show the char-acteristics of the genetic structure of the two biotypes.The research will be helpful to recognize the geneticbasis of the invasive biotype of B. tabaci . The resultwas also compared with those based on RAPD and mt

COI markers (Chu et al . 2006, 2007a). The character-istics and limitation of the ISSR were discussed in the

biotype identification and phylogenetic analysis.

MATERIALS AND METHODS

Whiteflies

Whiteflies were collected from 22 places throughout theworld. The sites, hosts, species/biotypes, time of collection,and acronym were explained in Table 1. All samples werestored at -20C. Biotype of B. tabaci was determinedbased on mt COI. The biotype Q populations from Haidianof Beijing, and Kunming of Yunnan are invasive popula-tions (Chu et al . 2006). Biotype B from Arizona, Texas,California, and Israel, and biotype Q from Israel were usedas control populations. Dr. Judy Brown from ArizonaUniversity and Dr. Matthew Ciomperlic from the UnitedStates Department of Agriculture provided B. tabaci bio-type B from Arizona and Texas, respectively. B. tabacifrom Israel was provided by Dr. Rami Horowitz of IsraelVolcani Agricultural Research Center.

DNA extraction of different populations

The DNA extraction method proposed by Chu et al .

Table 1 Data of whitefly samples collected from different locations

Population code Whitefly species and B. tabaci biotype Sampling location Host plant Sampling date Acronym

1 B. tabaci biotype B Zhengzhou, Henan Broccoli 2003.10 HeN1-B2 B. tabaci biotype B Beijing Cucumber 2003.8 BeiJ1-B3 T . vaporariorum Yuncheng, Shanxi Cotton 2003.7 ShanX-TV4 B. tabaci biotype B Israel Cotton 2003.8 Israel1-B5 B. tabaci biotype Q Israel Cotton 2003.8 Israel2-Q6 B. tabaci biotype B Spain - 2003.8 Spain-B7 B. tabaci biotype Q Spain - 2003.8 Spain-Q8 B. tabaci biotype B Arizona, USA Hibiscus 2003.8 AZ-B9 B. tabaci biotype B California, USA Eggplant 2003.9 CL-B10 B. tabaci biotype B Australia - 2004.1 Aus-B11 B. tabaci biotype B Beijing Pepper 2003.8 BeiJ2-B12 B. tabaci biotype B Zaozhuang, Shandong Cucumber 2003.8 ShanD1-B13 B. tabaci biotype B Nanjing, Jiangsu Cotton 2003.7 JiangS-B

14 B. tabaci biotype B Hangzhou, Zhejiang - 2003.8 ZheJ-B15 B. tabaci non-B/Q biotype Hangzhou, Zhejiang - 2003.8 ZheJ-NBQ16 B. tabaci biotype Q Kunming, Yunnan Poinsettia 2003.8 YunN-Q17 B. tabaci biotype B Tulufan, Xinjiang Poinsettia 2003.10 XinJ-B18 B. tabaci biotype Q Beijing Cucumber 2003.10 BeiJ3-Q19 B. tabaci biotype B Taian, Shandong - 2003.8 ShanD2-B20 B. tabaci biotype B Zhengzhou, Henan Cotton 2003.12 HeN2-B21 B. tabaci biotype B Zhengzhou, Henan Cabbage 2003.12 HeN3-B22 B. tabaci biotype B Zhengzhou, Henan Pumpkin 2003.10 HeN4-B

- indicates unknown.


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was followed (Chu et al . 2007a). A total of 10 adultindividual whiteflies were ground in 100 L lysis buffer

(1% SDS, 10 mmol L-1

Tris-HCl, pH = 8.0, 25 mmol L-1

NaCl, 25 mmol L -1 EDTA) in the 0.2 mL Eppendorf tube.

Reaction system and conditions

The six primers used were (GACAC) 3 , (GACA) 4 ,(TCC) 5 , (AGG) 5 , (ACTG) 4 (Perring et al . 1993) and(5 -AGAGGTGGGCAGGTG-3) (Gong et al . 2001).

Polymerase chain reaction (PCR) was carried out inthe MJ-100 PCR machine, and the reaction conditionswere same as described by Perring et al . (1993). ThePCR was performed under following conditions: dena-turation at 94C for 5 min, followed by 35 cycles (94Cfor 1 min, 52C for 1 min, 72C for 2 min), and a finalextension at 72C for 5 min. The products were storedat 4C.

Data analysis

The products were separated on the 1.5% agarosegels in 0.5 TBE buffer at 80 V. The data obtained

were put into the software POPGEN32 to calculatethe genetic distance (Nei 1972), the Neis gene diver-

sity index (Nei 1973), Shannon informative index(Lewontin 1972) and percentage of polymorphic lociof different populations of B. tabaci . Meanwhile, thecluster analysis was performed in the DPS2000 (ver.3.1.0.1) (Tang and Feng 1997) using 0-1 systemiccluster (Jaccard distance/UPGMA method) based onthe statistical data.

RESULTS

Detection results

The results showed that the ISSR using six primerscould all result in the polymorphic pattern (Figs.1 and2). The result of Trialeurodes vaporariorum (code 3 inthe Figs.1 and 2) was obviously different from these of

B. tabaci .

Diversity index

The genetic diversity analysis of different populations

Fig. 1 Example of ISSR patterns generated with primer (AGG) 5 . Population codes as shown in Table 1. M, 100-bp DNA ladder.

Fig. 2 Example of ISSR patterns generated with primer (5 -AGAGGTGGGCAGGTG-3). Population codes as shown in Table 1. M, 100-bp DNA ladder.


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of B. tabaci biotypes (Table 2) showed the diversityindexes of biotype Q including Neis gene diversity

index, Shannon informative index, and percentage of polymorphic loci (0.1764, 0.2544, 41.10, respectively)were higher than those of biotype B (0.0991, 0.1577,38.36, respectively).

Genetic distance and cluster

The data in Table 3 showed the genetic distance be-tween the T . vaporariorum and B. tabaci ranged from

0.5528 to 0.8565. The genetic distance of total bio-type B ranged from 0.0420 to 0.2474 and total bio-type Q from 0.1961 to 0.3586. The genetic distancebetween the biotype Q and biotype B ranged from0.5063 to 0.8565. The distances between the Zhejiangnon-B/Q biotype and biotype Q ranged from 0.4403

to 0.4838 and Zhejiang non-B/Q biotype and biotypeB from 0.4403 to 0.6016. The ISSR could distin-

guish different biotypes of B. tabaci based on the clus-ter analysis (Fig.3).

DISCUSSION

Genetic diversity of biotypes B and Q based onISSR analysis

The result on the genetic structure of biotype B andbiotype Q analyzed using ISSR (Table 2) showed di-versity indexes of biotype Q including Neis gene di-versity index, Shannon informative index, and percent-age of polymorphic loci were higher than those of bio-type B. This result is similar with the analysis based on

Table 2 Genetic diversity of Bemisia tabaci biotype-B and biotype-Q populations and all Bemisia tabaci populations

Populations 1) Neis gene diversi ty index Shannon informat ive index Percentage of polymorphic lociBiotype B (16) 0.0991 0.1577 38.36Biotype Q (4) 0.1764 0.2544 41.10Total B. tabaci biotypes 0.2398 0.3808 89.04

1) The number in the parenthesis indicates the number of populations analyzed.

RAPD (Chu et al . 2007a; Moya et al . 2001). It isgenerally considered that the genetic diversity is closelyrelated to the adaptability to the environment and po-tential evolution. Both biotype Q and biotype B areinvasive biotypes of B. tabaci , which have introducedinto many countries whereas there is a certain differ-ence between the two biotypes. Some of the publisheddata showed that the biotype Q is better than the bio-type B in some biological characteristics, for example,biotype Q has better biological advantages on some planthosts (Muniz 2000; Muniz and Nombela 2001; Nombelaet al . 2001), has similar or stronger ability to transmitviruses (Berdiales et al . 1999; Parrella et al . 2004), andhas higher and more steady resistance to some chemi-

cal pesticides than biotype B (Nauen et al . 2002; Elbertand Nauen 2000; Dennehy et al . 2005). The high ge-netic diversity of biotype Q might provide the geneticbasis for biological variance and ecological adaptation.

Although the genetic diversity of alien species mightbe influenced by all kinds of factors during the invasion,can the influence result in the biological changes of

Fig. 3 Dendrogram for different Bemisia tabaci biotypes withTrialeurodes vaporariorum as outgroup acronyms as shown inTable 1.


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AcknowledgementsThis work was funded by the National Program on

Basic Research Projects of China (973 Program,2002CB111400), National Natural Science Foundationof China (30500331, 30771410), the Natural ScienceFoundation of Beijing Municipal (6062024), ExcellentYoung Scientist Foundation of Shandong Province(2007BS06013), Innovation Foundation of ShandongAcademy of Agricultural Sciences (Q2006B05;2007YCX030), and the National Key Technologies R&DProgram of China during the 11th Five-Year Plan pe-

riod (2006BAD08A18). We are grateful to Dr. JudyBrown of Arizona University, Dr. Matthew Ciomperlicof United States Department of Agriculture, Dr. RamiHorowitz of Israel Volcani Agricultural Research Cen-ter for providing whiteflies.

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