Molecular Ecology Notes (2004) 4, 306 –308 doi: 10.1111/j.1471-8286.2004.00652.x © 2004 Blackwell Publishing Ltd Blackwell Publishing, Ltd. PRIMER NOTE Polymorphic microsatellites loci in the black Aphid, Aphis fabae Scopoli, 1763 (Hemiptera, Aphididae) A. COEUR D’ACIER,* M. SEMBÈNE,† P. AUDIOT* and J. Y. RASPLUS* *CBGP-INRA, Campus International de Baillarguet, CS 30016, 34988 Montferrier-sur-Lez Cedex, France, †Département de Biologie Animale, Faculté des Sciences et Techniques, B.P. 5005 Dakar, Sénégal. Abstract Aphis fabae is a widespread pest in Europe. This species includes four morphological cryptic subspecies which are mostly identified by their partially distinct secondary host range. To determine the extent of gene flow and isolation between these four taxa, we isolated and characterized eight microsatellite loci from A. fabae fabae and tested them on the three other subspecies. Polymorphism ranged from five to 35 alleles on the 138 individuals collected. When analysing each taxon separately, significant deviations from Hardy-Weinberg (HW) equilibrum were observed. This could have different explanations, including the presence of null alleles, or the Wahlund effect or the mating system. Keywords: Aphids, A. fabae cirsiiacanthoids, A. fabae mordvilkoi, A. fabae solanella, genetic differentiation, host plant Received 30 October 2003; revision received 20 December 2003; accepted 25 February 2004 Aphis fabae Scopoli is a European widespread pest. The species is of particular importance because of its direct feeding dam- age to Vicia faba and its role as a virus vector in sugar beet. In Europe A. fabae probably represents a complex of at least four heteroecious holocyclic taxa, which are presently considered as subspecies (namely A. f. fabae , A. f. cirsiiacan- thoidis Scop., 1763 A f. mordvilkoi Börner & Janisch, 1922 and A. f. solanella Theobald, 1914). These co-occur over much of their natural geographical range. They all use the same primary host, Evonymus europaeus L., but are extremely polyphagous for their secondary host as they can use more than 350 host plant species. Each subspecies can also feed on preflowering stages of host-plants which are not used by any other subspecies. These plants are consequently called ‘subspecies specific secondary hosts’, and are used to identify Aphis fabae taxa (Raymond et al . 2001). These subspecies are partially reproductively isolated. Hybrids have been observed in experimental conditions but their occurence in the field has never been detected (Müller 1982). In order to clarify the putative gene flow between these taxa, we isolated and characterized microsatellite loci from A. fabae fabae and tested them on other subspecies develop- ing on natural ‘subspecies specific secondary hosts’. We performed microsatellite screening following Estoup & Cornuet (1994). Genomic DNA was isolated using a phenol-chloroform protocol (Doyle & Doyle 1997) from approximately 400 A. f. fabae individuals, belonging to the same colony collected on Beta vulgaris maritima at Veulette/ Mer (France) the 06/20/2001. Total genomic DNA was digested using Rsa I restriction enzyme. A 300–900 bp fraction of the digested DNA was selected, purified and ligated to Rsa linkers. The enrichment procedure followed the protocol from Kijas et al . (1994) based on streptavidin-coated magnetic particles (Magnesphere, Promega), with slight modifications. 5 ′ -biotinylated (CT) 10 (GT) 10 (AAG) 10 (AAT) 10 (TGTA) 10 oligonucleotides were used as probes. We amplified the enriched single stranded DNA using one of the Rsa linkers as primer to obtain more enriched product and to recover double stranded DNA. PCR prod- ucts were purified and ligated into pGEM-T Easy vector (Promega), and the plasmid transformed into Escherichia coli supercompetent cells (XL1 blue, Stratagene). In total, for all motifs 3632 recombinant clones were transferred on Hybond-N nylon membranes (Amersham). Colonies were hybridized at 50 ° C with the mixture of oligonucleotide probes (CT) 10 (TG) 10 (AGG) 10 (AAT) 10 (ATGT) 10 labelled with the DIG oligonucleotid tailing kit (Boehringer). We detected 272 positive clones. We prepared plasmid DNA Correspondence: A. Coeur d’acier. Fax: 33(0)4 99 62 33 43; E-mail: coeur@ensam.inra.fr