6. Conclusions

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

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]

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

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.

proposed two different lists of reference cultivars for Olea europaea L. (Table 4).

efficient analysis.

144 Rediscovery of Landraces as a Resource for the Future

composition of crop plant derivatives.

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 the urgent need to establish a common procedure.

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 maximizes the genetic information.

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 of discrimination (PD, Figure 1).

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

deriving from noncoding ones, because they are associated with transcribed regions of the genome and thus reflecting the genetic diversity within genes or adjacent to genes [149]. Moreover, the association with trait loci with Mendelian inheritance is particularly requested

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

http://dx.doi.org/10.5772/intechopen.70756

147

About the localization of target microsatellites in the cellular genomes, nuclear SSR (nSSR) markers seem to be more polymorphic than plastidial and mitochondrial ones (cpSSR and mtSSR markers) and because of their co-dominance, the former are the only markers useful for assessing the genetic value of breeding stocks, even if the abundance and the haploid nature of the latter ones make them particularly suitable for phylogenetic and genetic diver-

As far as the microsatellite repeat is concerned, the most recommended motifs are dinucleotide and trinucleotide repeats, whereas mononucleotide repeats need caution because of technical drawbacks, which can be experienced in the allele discrimination. SSR markers with tetranucleotide or more repeats display a polymorphism inversely proportional to the complexity of the motif. The so-called perfect SSR markers are preferred because of their ease of scorability. It is also worth emphasizing that the choice of SSR markers is also dependent on the occurrence of null alleles for a given locus and the informativeness in terms of allele diversity indexes. First of all, any rate of null alleles can underestimate heterozygosity and affect the reliability of the analysis. Second, the calculation of some informative indexes cannot be underrated: it represents a crucial step of the planning of any analysis. What emerges from the 90 studies here reviewed is a lack of wider consensus among the authors regarding the best informative index to calculate and this makes the comparison difficult also among studies performed on the same cultivars and with the same markers. The power of discrimination (PD), the confusion probability (C), the polymorphism information content (PIC), the probability of identity (PI), the total probability of identity (PIt), and the probability of null allele (r) are all parameters able to describe exhaustively the efficiency of the set of SSR markers used in

In conclusion, there is the urgent need to establish a common procedure for SSR genotyping with a universal set of marker loci to be analyzed in each species. In parallel, the reference varieties must be defined in each species in order to maximize not only the reproducibility but also the portability of marker data, being aware that the residual variation in laboratory

Laboratory of Genomics and Plant Breeding, DAFNAE – University of Padua, Campus of

procedures and equipment cannot be completely avoided.

\*Address all correspondence to: gianni.barcaccia@unipd.it

in case of needs for marker-assisted selection (MAS).

sity studies.

a given species.

Author details

Agripolis, Legnaro, Italy

Fabio Palumbo and Gianni Barcaccia\*

Figure 1. Schematic representation of the 19 basic linkage groups of Vitis vinifera L. with indication of the 38 mapped marker loci chosen on the basis of their discriminant informativeness. In addition to the marker name, each locus reports the individual power of discrimination value (PD) and the polymorphism information content (PIC). Figure modified from Cipriani et al. [74].

reason, they recommend using at least 19 markers (among the 38 markers employed in their work). In general, for the selection of the panel of SSR markers, the following criteria should be followed. Based on previous works, the SSR marker loci with the highest number of marker alleles and the highest PIC and PD scores should have the priority. In addition, the position of the SSR markers across the genome, as mapped in different linkage groups and associated with adjacent chromosome blocks, is crucial in order to get a representative multilocus marker genotype. In fact, microsatellites retrieved from noncoding regions (genomic SSR markers) meet this requirement more precisely than those derived from expressed regions (EST-SSR markers). Nevertheless, the application of EST-SSR markers cannot be excluded when phylogenetic relationships have to be investigated. It is well known that SSR markers belonging to coding regions may be functionally more informative than those deriving from noncoding ones, because they are associated with transcribed regions of the genome and thus reflecting the genetic diversity within genes or adjacent to genes [149]. Moreover, the association with trait loci with Mendelian inheritance is particularly requested in case of needs for marker-assisted selection (MAS).

About the localization of target microsatellites in the cellular genomes, nuclear SSR (nSSR) markers seem to be more polymorphic than plastidial and mitochondrial ones (cpSSR and mtSSR markers) and because of their co-dominance, the former are the only markers useful for assessing the genetic value of breeding stocks, even if the abundance and the haploid nature of the latter ones make them particularly suitable for phylogenetic and genetic diversity studies.

As far as the microsatellite repeat is concerned, the most recommended motifs are dinucleotide and trinucleotide repeats, whereas mononucleotide repeats need caution because of technical drawbacks, which can be experienced in the allele discrimination. SSR markers with tetranucleotide or more repeats display a polymorphism inversely proportional to the complexity of the motif. The so-called perfect SSR markers are preferred because of their ease of scorability. It is also worth emphasizing that the choice of SSR markers is also dependent on the occurrence of null alleles for a given locus and the informativeness in terms of allele diversity indexes. First of all, any rate of null alleles can underestimate heterozygosity and affect the reliability of the analysis. Second, the calculation of some informative indexes cannot be underrated: it represents a crucial step of the planning of any analysis. What emerges from the 90 studies here reviewed is a lack of wider consensus among the authors regarding the best informative index to calculate and this makes the comparison difficult also among studies performed on the same cultivars and with the same markers. The power of discrimination (PD), the confusion probability (C), the polymorphism information content (PIC), the probability of identity (PI), the total probability of identity (PIt), and the probability of null allele (r) are all parameters able to describe exhaustively the efficiency of the set of SSR markers used in a given species.

In conclusion, there is the urgent need to establish a common procedure for SSR genotyping with a universal set of marker loci to be analyzed in each species. In parallel, the reference varieties must be defined in each species in order to maximize not only the reproducibility but also the portability of marker data, being aware that the residual variation in laboratory procedures and equipment cannot be completely avoided.
