5. Comparisons across studies of SSR-based genotyping: Reference marker sets and reference plant varieties

In most cases, it is impossible to make valid comparisons across studies on the same species since different sets of SSR loci are used in different laboratories [162]. For some species, the choice of microsatellites begins to be fairly uniform (Table 4). For instance, almost all of the studies aimed to genotype Olea europaea L. cultivars make use of SSR markers belonging to four main datasets developed by Sefc et al. [163], Carriero et al. [164], Cipriani et al. [165], and de La Rosa et al. [166]. Based on these studies, two informal universal sets of SSR markers were proposed for genotyping Olea europaea L. cultivars by Doveri et al. [52] and Baldoni et al. [53]. Cipriani et al. [74] suggested a list of 38 markers with excellent quality of peaks, high power of discrimination, and uniform genome distribution (1–3 markers/chromosome) for genotyping Vitis vinifera L. cultivars. Li et al. [144] assembled a reference kit of SSR markers for genetic analysis in Triticum spp. that comprises 46 microsatellites. Moriya et al. [108] developed a set of SSR markers for genotyping Malus � domestica Borkh. cultivars, which includes 15 microsatellites. Not only independent research works, but also some international programs and projects attempted to pursue this goal. The European Cooperative Programme for Plant Genetic Resources (ECPGR) has recommended a new set of 12 SSR marker loci distributed in different linkage groups of the Malus � domestica Borkh. genome, organized in three multiplexes and designed for a four-dye system [147]. Comparable considerations have been presented within two projects focused on the grapevine genetic resources conservation and characterization (EU-project GENRES CT96 No 81, [139]) and on the Traceability of Origin and Authenticity of Olive Oil (Oliv-Track, [167]). It is worth noting that, to the best of our knowledge, for Solanum Lycopersicum L., no SSR set of reference has been proposed yet.

Unfortunately, by establishing a reference set of microsatellite markers to use in each analysis for a given species, it is not sufficient to ensure the comparability among different studies and the reproducibility among different laboratories. Some tests have been carried out in order to investigate the reproducibility of SSR data produced by different laboratories under varying local conditions. Four different laboratories performed independent marker analyses on a common set of 21 DNA samples of Olea europaea L. cultivars and with the same set of SSR markers, using different DNA polymerase enzymes, PCR cycling conditions, amplicon separation, and visualization methods [53]. The results are not encouraging. Many cases of allele drop out and discrepancies in allele length, up to five nucleotides for identical microsatellite loci, were recorded. This finding is probably attributable to a combination of different equipments, different sequencers, and different internal ladders, which may have affected the relative mobility estimates leading to noncomparable electropherograms. Similar results have been achieved from ten laboratories distributed in seven countries that analyzed the same 46 Vitis vinifera, L. cultivars at the same 6 SSR loci [72].

One of the main discoveries is that the specific microsatellite sequence dramatically influences the efficiency of analysis. Marmiroli et al. [168] showed that the repeatability of results among different laboratories was good enough for some microsatellites but rather low for others, confirming that the choice of SSR loci and of their primers is crucial for an efficient analysis.

6. Conclusions

the urgent need to establish a common procedure.

maximizes the genetic information.

of discrimination (PD, Figure 1).

The genetic characterization of plant varieties by means of multilocus genotyping through SSR markers in the main crop species is still not based on standardized protocols making the acquisition of reproducible and transferable datasets difficult. What emerges from the analysis of the literature is a lack of wider consensus among the authors regarding the strategy to design and to adopt for genotyping plant varieties with SSR markers. This finding highlights

Critical Aspects on the Use of Microsatellite Markers for Assessing Genetic Identity of Crop Plant Varieties…

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Some conclusions of general validity can be drawn on the basis of the articles here reviewed. First of all, it is quite difficult to define exactly the ideal number of microsatellite loci to assay. Usually, the number of SSR markers depends on the type and goal of the analysis. If the purpose is merely to distinguish among two or more cultivars (i.e., individual genotypes), it is possible to adopt an "as simple as possible strategy." For example, a novel approach called the cultivar identification diagram (CID) strategy has been recently developed. This method was designed so that, at each step, a polymorphic marker generated from each PCR analysis directly allows the separation of cultivar samples [109]. In this specific study, eight is considered the minimum number of SSR markers necessary to distinguish 60 cultivars in Malus domestica Borkh.. Supposedly, the number of SSR markers could depend on the number of cultivars to distinguish, on their relationship and on the polymorphic degree of each marker locus. In this regard, we suggest AMaCAID [170] and UPIC [171], two very interesting tools that able the investigation of the minimum number of markers required to distinguish a specific number of accessions and, thus, the identification of the best marker combination that

When the purpose is to genetically characterize a cultivar in order to fulfill the requirements of a varietal register that could include hundreds or thousands of different varieties, the selection of SSR markers should be oriented to an exhaustive representation of the genome as whole. This is the reason why different authors consider one or two microsatellite for each linkage group for the minimum number required to reconstruct a reliable and selectable genotype for a given plant accession. For instance, Cipriani et al. [74] implemented an efficient method for Vitis vinifera L. fingerprinting using a set of 38 microsatellite marker loci scattered throughout the genome. In particular, two SSR loci were carefully chosen, on average, for each linkage group, selecting the best ones in terms of polymorphism information content (PIC) and power

It is worth noting that despite some international programs and projects attempted to establish reference SSR set, there is still a lack of wider consensus. For instance, in 2003, the partners of the EU-project Genres CT96 No81 [139] agreed on the utilization of six highly polymorphic SSR-markers for the identification of Vitis vinifera L. cultivars, but, since then, several studies continue to be performing using a higher number of markers [74, 76, 78, 84, 86]. As reported by Cipriani et al. [74], grape varieties selected in Western Europe, which account for most of the worldwide production of wine, likely have extensive coancestry that is a common origin from the hybridization of a few ancestors. Because of this, using too few markers for fingerprinting could hamper the discrimination of sibling varieties. For this

Despite all the precautions and the establishment of a reference set of SSR markers, some residual variation in laboratory equipment and procedures cannot be completely avoided, and representative reference material with many different alleles should be adopted by all laboratories involved in a genotyping program for a given species [162]. For this purpose, 21 out of 90 studies included reference cultivars, promoting new ones or exploiting cultivars already used as reference in previous works. Independent researches and international institutions are trying to find an agreement filling lists of reference accessions in order to prevent that each group uses its own reference cultivars and to standardize all works performed on these species. For example, the ECPGR has chosen eight Malus domestica Borkh. cultivars as reference set for this species [147]. Baldoni et al. [53] and Doveri et al. [52] proposed two different lists of reference cultivars for Olea europaea L. (Table 4).

Even if this approach is fully applicable also to the crop derivatives here taken into account (olive oil and wine), there are some additional aspects that must be considered when talking about processed products. First, sometimes, it is very difficult to make SSR marker analyses on food products and beverages because of the low DNA quantity and the lack of DNA integrity. For example, Baleiras-Couto and Eiras-Dias [45] reported their difficulties to investigate wines after about eight months of fermentation, as well as Recupero et al. [115] highlighted technical problems during the isolation of genomic DNA from Nebbiolo wine. Nevertheless, both of them managed to characterize must. For olive oil, Martins-Lopes et al. [119] as well as Vietina et al. [122], took advantage from extraction methods able to give good yield of genomic DNA and PCR amplificability. It is therefore evident how an optimized DNA extraction method is also a crucial step to carry out a reliable study on the applicability of molecular markers for identifying the varietal origin or assessing the varietal composition of crop plant derivatives.

It is not trivial considering the match between genetic profiles of crop plants and their derivatives. In this regard, there are some contrasting points of view. In the review of Agrimonti et al. [169], it is reported that several authors (e.g., [46, 118, 120]) have noticed a satisfying conformity between olive oil and leaf profiles with SSR markers. On the contrary, Doveri et al. [117] have proposed a cautionary note about the use of SSR markers, stressing the nonperfect concordance between the molecular genetic profiles of the olive oil and the original leaf sample. Furthermore, it is necessary to underline the extreme difficulty in characterizing multivarietal derivatives through SSR analysis. Most of the Italian PDO wines and olive oils are produced blending two or more cultivars in percentages strictly defined in the production regulation. In these cases, each SSR locus is represented by the combination of the marker alleles of each variety. For examples, Baleiras-Couto and Eiras-Dias [45], after having analyzed with six SSR markers in different divarietal musts at different percentages, reported results that confirm the complexity and difficulty of assessing multiple genotypes.
