Cross-species amplification of microsatellite loci in aphids: assessment and application

Microsatellites have revolutionized aphid population biology (Wilson et al. 2003). They have been widely applied in population genetic studies both at the scale of a country (e.g. Sunnucks et al. 1996; Simon et al. 1999; Wilson et al. 1999, 2002; Llewellyn et al. 2003; Papura et al. 2003) and more recently to study metapopulation structure within fields (e.g. Haack et al. 2000; Massonnet et al. 2002b). In aphids, microsatellites are currently the only codominant genetic markers that are sufficiently polymorphic to identify clones and clonal lineages (e.g. Wilson et al. 2003), the occurrence of sexual reproduction (e.g. Sunnucks et al. 1997; Papura et al. 2003), and evolution by mutation in asexual lineages (e.g. Wilson et al. 1999, 2003). Microsatellites and their flanking sequences, in conjunction with mitochondrial markers, have also been useful in elucidating the mechanisms by which obligately parthenogenetic aphid lineages could arise (Delmotte et al. 2001, 2002, 2003).

Microsatellites have now been isolated from a wide range of aphid species. These include: Sitobion miscanthi (Sunnucks et al. 1996; Wilson et al. 1997; Simon et al. 1999), Sitobion avenae (Simon et al. 1999), Aphis gossypii (Vanlerberghe-Masutti et al. 1999), Pemphigus bursarius (Miller et al. 2000), Pemphigus spyrothecae (Johnson et al. 2000), Myzus persicae (Sloane et al. 2001), Rhopalosiphum padi (Simon et al. 2001), Macrosiphoniella tanacetaria (Massonnet et al. 2001), Metopeurum fuscoviride (Massonnet et al. 2002a), and Dysaphis plantaginea (Harvey et al. 2003). Additionally, we provide previously unpublished primer sequences for another 21 aphid microsatellite loci, 14 of which were isolated from Sitobion miscanthi, three from Sitobion avenae and four from Myzus persicae (Table 1).

Despite the relative ease of cloning microsatellite loci from aphids (cf. e.g. avian species; Zane et al. 2002), microsatellite development still requires a skilled molecular biologist, and a considerable investment of time and resources on the part of the researcher. Thus, using loci already developed in a related species may provide a cost-effective alternative to microsatellite isolation and development in a species of interest. Cross-species amplification is only effective if primer sequences are conserved between species. Generally the number of loci amplifying tends to decrease with increasing divergence between species (Moore et al. 1991; Peakall et al. 1998), although extreme conservation of loci has also been reported (Schlötterer et al. 1991; FitzSimmons et al. 1995; Rico et al. 1996; Scott et al. 2003). For this reason we have investigated, in a range of nontarget species, the potential for cross-species amplification of 48 aphid microsatellite loci, isolated from five target species (Supplementary material Table S1). Three of these target species belong to the tribe Macrosiphini: Myzus persicae (10 loci), Sitobion avenae (four loci) and Sitobion miscanthi (18 loci) and two to the tribe Aphidini: Aphis gossypii (eight loci) and Rhopalosiphum padi (eight loci). The materials and methods used for cross-species amplification tests are detailed in the Appendix S1.

We found that amplification success in species belonging to the same genus as the target species was high, in the order of 80% (e.g. Sitobion, Table 2), whereas amplification success in noncongeners of the same tribe was lower (e.g. for non-Sitobion of the Macrosiphini, 59% of tested loci amplified, Table 2). Surprisingly, of the small total number of loci tested outside the subfamily from which they were isolated, 35% amplified successfully (Tables 2 and 3). These results overall suggest that time and money invested in investigating the cross-species amplification of microsatellite loci in nontarget species may be well spent. However, our more extensive cross-species amplification data (Supplementary material Table S2) indicate that allelic diversity can be very low in nontarget species compared with target species (see especially data for Macrosiphoniella tanacetaria, Metopeurum fuscoviride and Uroleucon tanaceti). In cases where levels of allelic diversity are extremely low, the usefulness of these loci is greatly limited and workers should consider isolating microsatellite loci in their species of interest. Further caution needs to be used in utilizing microsatellite loci in nontarget species by investigating the occurrence of null alleles, as detected for example in S. fragariae samples amplified with microsatellite primers designed from S. miscanthi sequences (Sunnucks et al. 1997).

Sitobion miscanthi loci, S10, S17b, S49 and S43ii are X-linked in S. miscanthi (Wilson 2000) and locus Sm11 is X-linked in Sitobion near fragariae (Wilson et al. 1997). We have confirmed the X-linkage of loci S10, S17b and S49 in Sitobion near fragariae, loci Sm11, S10, S17b and S49 in S. avenae and locus Sm11 and S17b in R. padi. Loci Sm11, S10 and S17b are X-linked in A. pisum (Caillaud et al. 2002). Finally, locus S17b is also X-linked in M. persicae (Sloane et al. 2001). It is worth noting that whilst the X-linkage of these loci has not been confirmed in species other than those listed above, many of these X-linked loci have been shown to cross-amplify in many species (Table 2). These include: Sm11, which has amplified in 22/33 species including the taxonomically distant species, Neuquenaphis staryi; S10, which has amplified in 5/6 species in which it has been tested; S17b, which has amplified in 15/16 species; and S49, which has amplified in 6/13 species. Overall, X-linked loci amplified in 71% of tests compared with 51% amplification of all loci tested (austosomal, X-linked and those of unknown location). The X-chromosomes are the largest chromosome pair in most aphid species, accounting for over 25% of the genome (e.g. Wilson et al. 1997). These data suggest that X-linked loci may be more conserved in aphids than autosomal loci. Finally, because X-linkage of loci is easily determined in cyclically parthenogenetic aphids, we encourage researchers to investigate the chromosomal location of their markers using the simple diagnostic test described in Wilson et al. (1997).

Supplementary material

The following material is available from http://www.blackwellpublishing.com/products/journals/suppmat/MEN/MEN584/MEN584sm.htm

Appendix S1. Supplementary information.

Table S1. Source of microsatellite primer sequences for each of the loci used in the cross-species amplification study.

Table S2. Detailed cross-species amplification data for fourteen non-target species.