Genetic differentiation among geographic populations of Gonatocerus ashmeadi the predominant egg parasitoid of the glassy-winged sharpshooter Homalodisca coagulata

摘要

The aim of genetically comparing different populations of the same species of natural enemies is to identify the strain that is most adapted to the environment where it will be released. In the present study, Inter-Simple Sequence Repeat-Polymerase Chain Reaction (ISSR–PCR) was utilized to estimate the population genetic structure of Gonatocerus ashmeadi (Girault) (Hymenoptera: Mymaridae), the predominant egg parasitoid of Homalodisca coagulata (Say) (Homoptera:Cicadellidae), the glassy-winged sharpshooter. Six populations from throughout the U.S. and a population from Argentina identified as near G. ashmeadi were analyzed. Four populations (California; San Antonio, Texas; Weslaco, Texas [WTX-2]; and Florida) were field collected and two (Louisiana and Weslaco, Texas [WTX-1]) were reared. Three ISSR–PCR reactions were pooled to generate 41 polymorphic markers among the six U.S. populations. Nei's expected heterozygosity values (h), including the reared population from Louisiana, were high (9.01–14.3%) for all populations, except for a reared population from WTX-1 (2.9%). The total genetic diversity value (Ht) for the field populations was high (23%). Interestingly, the Florida population that was collected from one egg mass (siblings) generated the greatest number of polymorphic markers (20) and was observed with the highest gene diversity value (14.3%). All populations, except WTX-2 generated population-specific markers. Comparison of genetic differentiation estimates, which evaluate the degree of genetic subdivision, demonstrated good agreement between GST and θ values, 0.38 and 0.50, respectively for field populations, and 0.44 and 0.50, respectively for all populations. Genetic divergence (D) indicated that the WTX-1 population was the most differentiated. Average D results from the Argentina population support the taxonomic data that it is a different species. The present results estimate the population genetic structure of G. ashmeadi, demonstrating genetic divergence and restricted gene flow (Nm = 0.83) among populations. These results are of interest to the Pierce's disease/glassy-winged sharpshooter biological control program because the key to successful biological control may not be in another species, but instead in different geographic races or biotypes.

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ISSR–PCR

Inter-Simple Sequence Repeat-Polymerase Chain Reaction

class="kwd-title">Keywords: DNA fingerprinting, genetic differentiation, grapevines, molecular markers, natural enemies, Pierce's disease, polymorphic loci, population genetics class="head no_bottom_margin" id="s1title" style="text-transform: uppercase;">IntroductionGonatocerus ashmeadi (Girault) (Hymenoptera: Mymaridae) is a primary egg parasitoid of Homalodisca coagulata (Say) (Homoptera: Cicadellidae), the glassy-winged sharpshooter (). A survey conducted by in California, Florida, and Louisiana concluded that this mymarid wasp was the predominant or the most common na tural enemy of H. coagulata. A biological control program is currently in progress in California against H. coagulata because this xylem feeding leafhopper is a serious economic pest that vectors a strain of Xylella fastidiosa, a bacterium that causes Pierce's disease in grapevines (Vitis vinifera and V. labrusca) (). H. coagulata are native to the southern United States, ranging from Florida to Texas and northern Mexico (; ; ; ). Within the last 10 years, H. coagulata have established in southern California where they pose a serious threat to the wine and table grape industry ().Studies of allele or marker frequencies in naturally occurring parasitoid populations are important, not only for identifying genetic variation of potential benefit in the selection and screening of biological control organisms, but also for the detection of genetic markers indicative of specific biological traits or geographic origins. In addition, the recognition of intraspecific variation can be as crucial for the success of biological control programs as is sound species determination. Populations of parasitoids from distinct geographical regions may differ in relevant biological characteristics of importance to biological control (; ; ). An aim of genetically comparing different populations of the same species of natural enemies is to identify the strain that is most adapted to the environment where it will be released (). In other words, the key to successful biological control may not be in another species, but instead in different geographic races or biotypes of one species (). Laboratory populations of parasitoids are maintained for three main purposes: to provide material for research into their basic biology and behavior, to meet quarantine requirements, and to mass-rear insects for release in biological control programs (; ; ). Reliable methods are needed for distinguishing various exotic strains of these biological control agents from those indigenous to the U.S., including parasitoids from different states within the U.S. Release of unidentified and uncharacterized strains can make it difficult to document their establishment and dispersal. Therefore, genetic typing of strains prior to their release in the field is highly desirable (href="#i1536-2442-005-02-0001-narang2" rid="i1536-2442-005-02-0001-narang2" class=" bibr popnode">Narang et al. 1993b). Molecular methods are also needed to monitor the quality of laboratory cultures, to detect development of major deviations in genetic composition from that existing in field populations, and for the detection of cross-contamination between cultures of sibling species (href="#i1536-2442-005-02-0001-powell1" rid="i1536-2442-005-02-0001-powell1" class=" bibr popnode">Powell and Walton 1989; href="#i1536-2442-005-02-0001-menken1" rid="i1536-2442-005-02-0001-menken1" class=" bibr popnode">Menken and Ulenberg 1987; href="#i1536-2442-005-02-0001-narang2" rid="i1536-2442-005-02-0001-narang2" class=" bibr popnode">Narang et al. 1993b; href="#i1536-2442-005-02-0001-hopper1" rid="i1536-2442-005-02-0001-hopper1" class=" bibr popnode">Hopper et al. 1993; href="#i1536-2442-005-02-0001-unruh3" rid="i1536-2442-005-02-0001-unruh3" class=" bibr popnode">Unruh and Woolley 1999).A sensitive approach for obtaining polymorphic DNA markers is based on the use of simple sequence repeats (SSR) or microsatellites. Microsatellites are ubiquitous in eukaryotic genomes and can be found in both protein-coding and noncoding regions (href="#i1536-2442-005-02-0001-toth1" rid="i1536-2442-005-02-0001-toth1" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_109490431">Tóth et al. 2000). Microsatellites are widely used as genetic markers because they are co-dominant, multiallelic, easily scored, and highly polymorphic, although drawbacks for their use are the time and cost required to characterize them (href="#i1536-2442-005-02-0001-karp1" rid="i1536-2442-005-02-0001-karp1" class=" bibr popnode">Karp and Edwards 1997). However, a DNA fingerprinting procedure, Inter-Simple Sequence Repeat-Polymerase Chain Reaction (ISSR–PCR) (href="#i1536-2442-005-02-0001-zietkiewicz1" rid="i1536-2442-005-02-0001-zietkiewicz1" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_109490436">Zietkiewicz et al. 1994), including adding a primer pair to the reaction (pp-ISSR–PCR) (href="#i1536-2442-005-02-0001-prevost1" rid="i1536-2442-005-02-0001-prevost1" class=" bibr popnode">Prevost and Wilkinson 1999; href="#i1536-2442-005-02-0001-cekic1" rid="i1536-2442-005-02-0001-cekic1" class=" bibr popnode">Cekic et al. 2001), permits detection of DNA variation in microsatellites without the need to isolate and sequence specific DNA fragments. The approach for this technique involves amplification with oligonucleotide primers corresponding directly to random SSR sites. This involves the use of 5′-anchored or compound ISSR primers where the anchor serves to fix the annealing of the primer to a single position of the target site, thus resulting in a low level of slippage during amplification. Many classes of microsatellite repeat motifs have been identified, though the class most abundant in eukaryotic genomes is the CA-repeat. The presence of these repeat motifs in high copy number and their dispersion throughout the genome of all eukaryotes tested has been demonstrated by earlier studies (href="#i1536-2442-005-02-0001-hamada1" rid="i1536-2442-005-02-0001-hamada1" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_333682803">Hamada et al. 1982; href="#i1536-2442-005-02-0001-tautz1" rid="i1536-2442-005-02-0001-tautz1" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_109490443">Tautz and Renz 1984; href="#i1536-2442-005-02-0001-toth1" rid="i1536-2442-005-02-0001-toth1" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_109490435">Tóth et al. 2000). Therefore, because of their high density, a few oligonucleotides complementary to these CA-repeat motifs can be used to target a significant portion of the genome and reveal highly polymorphic banding patterns (href="#i1536-2442-005-02-0001-zietkiewicz1" rid="i1536-2442-005-02-0001-zietkiewicz1" class=" bibr popnode tag_hotlink tag_tooltip" id="__tag_109490437">Zietkiewicz et al. 1994).To gain an understanding of the population genetics of G. ashmeadi, molecular genetic markers were developed by the ISSR–PCR DNA fingerprinting procedure. Recently, we developed DNA markers for H. coagulata for the purpose of estimating genetic variation in natural populations (href="#i1536-2442-005-02-0001-deleon1" rid="i1536-2442-005-02-0001-deleon1" class=" bibr popnode">de León and Jones 2004). We screened and compared four DNA fingerprinting procedures and determined that procedures incorporating SSR or microsatellites were the most sensitive with H. coagulata template. To this end, the specific objectives of the present study were to 1) estimate genetic variation or gene diversity within and among populations, 2) estimate the population genetic structure, 3) determine whether ISSR–PCR was sensitive enough to identify diagnostic markers in geographic populations, and 4) confirm the species identification of a population of egg parasitoids from Argentina identified as near G. ashmeadi. We demonstrated the ability of the ISSR–PCR procedure with compound and 5′-anchored ISSR primers, including combining them, to generate polymorphic markers and estimate geographic variation in G. ashmeadi populations.