Journal Name: Potato Research

Article title: Investigation and development of potato parentage analysis methods using multiplexed SSR fingerprinting

Journal name: Potato Research

Authors name: M. Spanoghe1,*, T. Marique1,2, J. Rivière1, D. Lanterbecq1 and M. Gadenne1,2

Affiliation:1 Haute Ecole Provinciale de Hainaut-CONDORCET, Laboratory of Biotechnology – AppliedGenetics Unit, 17 Chemin du Champ de Mars, 7000 Mons, Wallonia, Belgium2Centre pour l’Agronomie et l’Agro-Industrie de la Province de Hainaut (CARAH), Labotory of Food Technology, 11 rue Paul Pastur, 7800 Ath, Wallonia, Belgium

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Supplementary material

Here, the explanation andthe description of the intensive preliminary testing phase, resulting in the combination of the17 primer pairs in the three multiplexed-PCRs used in the study.

The multiplexed SSR constitutes a variant of polymerase chain reaction (PCR), which allows amplifying several locus simultaneously in a single reaction and thus considerably decreases the time of analyses(Chamberlain et al. 1988). While numerous papers have been published about primers for potatoes, either through potato database searches (Veilleux et al. 1995; Feingold et al. 2005), enriched genomic libraries (Milbourne et al. 1998; Ashkenazi et al. 2001) or expressed sequence tags (Ghislain et al. 2004) only one study in our knowledge did set up functional multiplexed SSR sets allowing potatoes cultivar fingerprinting,which succeed to co-amplify all loci with a maximum of three markers per set(Reid et al. 2011). Such a tool is in need to be chosen as compared to the simplex SSRmethods of potato varieties fingerprinting, as it allows considerable time and effort saving within laboratory. In addition, by increasing the number of markers per multiplexed set and consequently the informativeness, it is possibleto cope withthe increasing expectations ofcertification programs, traceability programs, parentage analysis programs (i.e. kinship testing method, parentage inference analysis) and in some case breeding programs, as it generates more genetic data that be used according to the specificsofeach of these respective program. In particular, the kinship testing method, which is based on astrict examination of allelicphenotypesbetween the tested individuals, requires alarge number ofgenotypicdata to perform a reliable analysis. For instance, paternity testingin humans (Chakraborty and Stivers 1996) is performed using one multiplexed SSR set included at least 12 autosomal SSR markers (Junge et al. 2006), which are in normal paternity cases adequate to clarify the case and to reach a paternity probability of more than 99.9%.Kinship testing method using microsatellite markers was also performed on plant. For example, Grattapaglia et al. (2004) used a set of 14 markers to carry out parentage tests of 256progeny individuals of Eucalyptus.

In this study, 26 primer pairs were firstly taken from the literature (Veilleux et al. 1995; Provan et al. 1996; Milbourne et al. 1998; Ghislain et al. 2004; Feingold et al. 2005; Ghislain et al. 2009) in order to achieve reliable parentage analysis on potato using SSR markers. They wereselected based on their annealing temperature ranging from 53°C to 57°C; this range included the highest number of available primers. They were also chosen based on the Polymorphic Information Content (PIC) or Diversity Index (DI) and on the length of the obtained products.According to the Henegariu protocol (1997), many assays were carried out to set up the optimal combinations of the primers in the multiplex sets in order to obtain an equal amplification between all loci and to detect undesirable primer-primer interactions. During all steps described by this protocol, two repetitions of each assaywere conducted over different runs on the on a 4-capillary ABI 3100-avant Genetic Analyzer (Applied Biosystems).

In a first time, five cultivars were arbitrary chosen to perform an initial PCR program and to amplify all lociindividually. This step allowed us to adjust cycling conditions of the PCR program and to identify the primers pairs producing either artifacts or unspecific peaks for each tested 26 simplex (3 primer pairs generating unspecific peaks were discarded). The lengths of PCR products were determined using GeneMapper Software 4.0 (Applied Biosystems) and a threshold for scoring was first automatically performed by the same Software (automatic threshold).Some peaks werein few cases nocalled due to the presence of stutter peaks or when peaks were below the threshold. For these reasons, all genetic profiles were then checked two times (by two peoples) in order to avoid outputting wrong allelic phenotypes. We followed the Gill et al. (1997)procedure to recognize good peak within stutter and pull-up peaks in order to carry out the interpretative process.

In a second time, all the possible combinations (in regard to the fluochrome label and the length of the obtained products) of these pre-selected primers were performed using respectively duplex, triplex, 4-plex, 5-plex and hexaplex. The development ofthe multiplex-PCR set were set upusing the same 5 cultivars and the reactions were carried out with equimolar amounts of pre-selected primers and the same PCR conditions than those used during the initial program.This step allowed the detection of all combinations of primer-primer interactions (not revealed by alignment sequence test), due to either a lack of amplification or an unspecific peak. The primers causing such interactions were then removed from the analysis.

From the initial primers selected, 17 pairs were chosen to set up the three multiplexed SSR markers set (Table 1). All of these succeeded to generate the expected amplification products with an acceptable level of polymorphism. The combination of selected primers in the three sets of multiplexed-PCR did not show undesirable primer-primer interactions or any unexpected amplified fragments.In one multiplexed set, some selected primers co-amplified with a higher resolution for short products. To overcome this non uniform amplification, the amount of “weak loci” primers was increased, the amount of “intense loci” primers was decreased and the extension time wasalso increased of a few seconds. Finally, two sets of six loci and one set of five loci were co-amplified at a same annealing temperature and were used for the subsequent analysis (Table 1).

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