4. The choice of the best microsatellite motifs and the problem of the null alleles

Microsatellite repeat units typically vary from one to six bases. Shortest motifs (mono- or dinucleotide repeats) usually have a high number of alleles [74], and they allow packing more loci on a given separation system, resulting in larger multiplexes. However, this kind of SSR motifs can be difficult to assay accurately. It is very common to observe a stuttering in terms of multiple bands or peaks, a phenomenon commonly caused by slippage of the DNA polymerase, but the main problem arises when there is a difference of one or two base-pairs between marker alleles: in case of homozygous loci, the electrophoretic analysis results in one main band or peak, but with heterozygous loci very often one of the two marker alleles is masked by the stutter. SSR markers containing trinucleotide or higher order repeats usually eliminate this technical problem because target sequences appear to be significantly less prone to slippage [52]. Nevertheless, microsatellite loci with long motifs are known to be less polymorphic and, in some cases, due to lack of stutter bands or peaks, which is not always possible to distinguish SSR amplicons from other aspecific PCR products and it may lead to an overestimation of the level of polymorphism of these loci [159].

<sup>r</sup> <sup>¼</sup> He � Ho 1 þ He

Critical Aspects on the Use of Microsatellite Markers for Assessing Genetic Identity of Crop 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 knowl-

being He the expected heterozygosity and Ho the observed heterozygosity.

marker sets and reference plant varieties

Vitis vinifera, L. cultivars at the same 6 SSR loci [72].

5. Comparisons across studies of SSR-based genotyping: Reference

edge, 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

(10)

143

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

Among the 90 studies we surveyed, only 25 of them specify the length of the SSR motifs employed and very few justifies the choice. Cipriani et al. [80] performed two distinct molecular analyses on the same set of cultivars, using the genetic profiles obtained from the two sets of microsatellites, the dinucleotide repeats from one side, and the tri-, tetra-, and pentanucleotide repeats from the other, with the aim of comparing their performance in the discrimination of the genotypes analyzed. Both microsatellite data sets produced identical consensus tree topology, but the authors underlined that dinucleotide SSR markers scored a higher number of alleles per locus, and consequently, a potentially higher power for identifying and distinguishing closely related genotypes. On the other hand, themicrosatellite dataset based on tri-, tetra-, and pentanucleotide SSR markers proved to have the advantage of ease in scorability, while maintaining a very high power of discrimination for successful genotyping of the Vitis vinifera L. cultivars.

Microsatellites have also been classified according to the type of repeat sequence as perfect or imperfect, according to the occurrence of simple or uneven repeats, respectively [160]. The preference should be given to perfect motifs because using imperfect ones, there is no more equivalency between fragment length and amplicon sequence, and hence several sequences can correspond to a given length variant [39]. This is the reason why only four studies employed imperfect SSRs among the 25 ones specifying the motifs.

The occurrence of null alleles is something to avoid when using SSR markers for genotyping plant materials. A microsatellite null allele is any marker allele at a genomic locus that consistently fails to amplify by the polymerase chain reaction, resulting in the lack of detectable amplicons. Lack of amplified fragments could preclude the detection of heterozygous loci, which would be computed as homozygotes. In the same way, null alleles at homozygous loci are characterized by a complete lack of amplification with the consequent production of missing data. On the whole, null alleles may interfere with the genetic identification of cultivars, by wrongly reducing the genetic diversity among accessions [149]. In the 90 studies surveyed, only 38 of them estimated the probability of null alleles, mainly using the formula of Brookfield [161]: Critical Aspects on the Use of Microsatellite Markers for Assessing Genetic Identity of Crop Plant Varieties… http://dx.doi.org/10.5772/intechopen.70756 143

$$\mathbf{r} = \frac{\mathbf{H}\_{\text{e}} - \mathbf{H}\_{\text{o}}}{1 + \mathbf{H}\_{\text{e}}} \tag{10}$$

being He the expected heterozygosity and Ho the observed heterozygosity.

4. The choice of the best microsatellite motifs and the problem

Microsatellite repeat units typically vary from one to six bases. Shortest motifs (mono- or dinucleotide repeats) usually have a high number of alleles [74], and they allow packing more loci on a given separation system, resulting in larger multiplexes. However, this kind of SSR motifs can be difficult to assay accurately. It is very common to observe a stuttering in terms of multiple bands or peaks, a phenomenon commonly caused by slippage of the DNA polymerase, but the main problem arises when there is a difference of one or two base-pairs between marker alleles: in case of homozygous loci, the electrophoretic analysis results in one main band or peak, but with heterozygous loci very often one of the two marker alleles is masked by the stutter. SSR markers containing trinucleotide or higher order repeats usually eliminate this technical problem because target sequences appear to be significantly less prone to slippage [52]. Nevertheless, microsatellite loci with long motifs are known to be less polymorphic and, in some cases, due to lack of stutter bands or peaks, which is not always possible to distinguish SSR amplicons from other aspecific PCR products and it may lead to an overestimation of the

Among the 90 studies we surveyed, only 25 of them specify the length of the SSR motifs employed and very few justifies the choice. Cipriani et al. [80] performed two distinct molecular analyses on the same set of cultivars, using the genetic profiles obtained from the two sets of microsatellites, the dinucleotide repeats from one side, and the tri-, tetra-, and pentanucleotide repeats from the other, with the aim of comparing their performance in the discrimination of the genotypes analyzed. Both microsatellite data sets produced identical consensus tree topology, but the authors underlined that dinucleotide SSR markers scored a higher number of alleles per locus, and consequently, a potentially higher power for identifying and distinguishing closely related genotypes. On the other hand, themicrosatellite dataset based on tri-, tetra-, and pentanucleotide SSR markers proved to have the advantage of ease in scorability, while maintaining a very

high power of discrimination for successful genotyping of the Vitis vinifera L. cultivars.

employed imperfect SSRs among the 25 ones specifying the motifs.

Microsatellites have also been classified according to the type of repeat sequence as perfect or imperfect, according to the occurrence of simple or uneven repeats, respectively [160]. The preference should be given to perfect motifs because using imperfect ones, there is no more equivalency between fragment length and amplicon sequence, and hence several sequences can correspond to a given length variant [39]. This is the reason why only four studies

The occurrence of null alleles is something to avoid when using SSR markers for genotyping plant materials. A microsatellite null allele is any marker allele at a genomic locus that consistently fails to amplify by the polymerase chain reaction, resulting in the lack of detectable amplicons. Lack of amplified fragments could preclude the detection of heterozygous loci, which would be computed as homozygotes. In the same way, null alleles at homozygous loci are characterized by a complete lack of amplification with the consequent production of missing data. On the whole, null alleles may interfere with the genetic identification of cultivars, by wrongly reducing the genetic diversity among accessions [149]. In the 90 studies surveyed, only 38 of them estimated the probability of null alleles, mainly using the formula of Brookfield [161]:

of the null alleles

142 Rediscovery of Landraces as a Resource for the Future

level of polymorphism of these loci [159].
