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 – Applied Genetics Unit, 17 Chemin du
Champ de Mars, 7000 Mons, Wallonia, Belgium
2
Centre 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
Email address : *martin.spanoghe@condorcet.be
Supplementary material
Here, the explanation and the 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 SSR methods 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 possible to cope with the increasing expectations of certification 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 specifics of each of these
respective program. In particular, the kinship testing method, which is based on a strict examination of allelic
phenotypes between the tested individuals, requires a large number of genotypic data to perform a reliable
analysis. For instance, paternity testing in 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 256 progeny 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 were selected 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 assay were 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
loci individually. 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 were in few cases no called 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, 5plex and hexaplex. The development of the multiplex-PCR set were set up using 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 was also 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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