2. Applications of SSR markers for the genetic characterization of crop plant varieties

Some of the most economically important crops in Italy have been chosen for this study, and the search has been focused on their varietal characterization through SSR analysis. In particular, olive (Olea europaea L.), grape (Vitis vinifera L.), and apple (Malus domestica Borkh.) were reviewed among the fruit trees, whereas wheat (Triticum spp.) and tomato (Solanum lycopersicum L.) were selected as representative of cereals and vegetables, respectively. A large number of commercial cultivars are available for each of these species, and the annual Italian GPV for these crops is about 18 billion Euro [2]. Moreover, scientific articles dealing with the genetic identification in wines and olive oils were also evaluated because these two derivatives contribute to the annual Italian GPV for another 15 billion Euro [2].

Although passport data, morphological, and agronomical descriptors have been collected, data are not informative enough to assess the numerous cases of misidentification, mislabeling, homonymies, and synonymies as well as voluntary or accidental frauds [47]. With regard to this, several research groups characterized and identified cultivars using SSR markers (Table 3).


Table 3. Crops and derivatives reviewed.

traits and that has been extensively used for the characterization of plant varieties [22–24] and

Microsatellites (or simple sequence repeats (SSRs)) are PCR-based molecular markers valued for their abundant and uniform genome coverage, high levels of polymorphism information content as a consequence of their marked mutation rates, and other valuable qualities such codominant inheritance of DNA amplicons/alleles and request of little amount of DNA for the amplifications [28]. A unique pair of primers defines each SSR marker locus; as a consequence, the molecular information exchange among laboratories is easy and allows individuals to be

SSR markers have been shown repeatedly as being one of the most powerful marker methodologies for genetic studies in many crop species. In fact, since they are multiallelic chromosomespecific and well distributed in the genome, microsatellite markers have already been used for mapping genes with Mendelian inheritance [30], for identifying quantitative trait loci (QTLs, [31]) and for molecular marker-assisted selection [32]. In many species, microsatellite markers have also been used for ascertaining the genetic purity of seed lots [33], as well as to assess the capability to protect the intellectual property of plant varieties [34]. These markers are also largely used for assessing the genetic diversity and relationships among populations and lines,

The advantages of SSRs over single-nucleotide polymorphisms (SNPs), another co-dominant marker system increasingly exploited in breeding programs, include relative ease of transfer between closely related species [35, 36] and high allelic diversity [37, 38]. On the contrary, SSRs when compared to SNPs have some limits: the development phase is quite long and expensive for multilocus assays and the throughput is relatively low because of drawbacks for automation and output data management. Recently, progresses in the development of multilocus assays have been made in several directions, suggesting that SSR markers still remain as relevant molecular tools at least for specific applications and genetic studies [39]. In fact, PCRbased SSR genotyping has rapidly evolved in plants, and methods for the simultaneous amplification of multiple marker loci coupled to semi-automated detection systems have been developed [40]. The identification and selection of SSR markers have become cheaper and faster due to the emergence of next-generation sequencing technology means. Moreover, the possibility to multiplexing specific combinations of microsatellite markers has become much easier and the availability of capillary electrophoresis equipment relying on automated laserinduced fluorescence DNA technology has facilitated the adoption and exploitation of this

Genotypic characterization through SSR loci analysis represents a molecular tool applicable to all species and able to support the phenotypic observation in order to characterize and describe a cultivated variety as well as to define its uniformity, distinctiveness, and stability (DUS testing). At the same time, SSR markers are largely used for the genetic identification of

The main goal of this work is to provide an updated and detailed description of the applications of SSR markers for varietal characterization and identification, reviewing the state of the art of genotyping in the most economically relevant Italian crop plants and food products: Olea

varieties and the authentication and traceability of their foodstuffs [44–46].

the certification of food products [25–27].

134 Rediscovery of Landraces as a Resource for the Future

uniquely genotyped in a reproducible way [29].

methodology in applied breeding programs [41–43].

and for identifying crop varieties.

Article searches were performed using the three most popular sources of scientific information: Scopus, Web of Science, and Google Scholar, while PubMed was excluded from the queried datasets because it focuses mainly on medicine and biomedical sciences and also because Google scholar already includes its index [126]. A total of 90 articles based on SSR genotyping analysis were selected from the international literature in the last 15 years, covering all the plant species/ food products taken as reference list. Only articles dating from 2000 to now were reviewed assuming that researches published earlier would have lost their steering effects on the activities of plant DNA genotyping, given that the development of new and large marker datasets, and technologically advanced and automated protocols has been very fast in the last 15 years.

entire genome, not only the number of homologous chromosomes but also their size (i.e., total amount of DNA) should be considered when choosing the optimal panel of microsatellite loci to be investigated. Finally, in tomato (Solanum lycopersicum L., 2n = 2 = 24), the average

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

Only few studies [65, 74, 96, 106] evaluated the position within linkage groups of the microsatellites selected: the choice often falls on SSR markers with unknown or not specified position or mapped on few chromosomes, thus resulting in a poor representation of the entire genome. In this regard, the results from Cipriani et al. [74] and van Treuren et al. [106] represent a good model for the choice of molecular markers to investigate the genetic diversity in germplasm collections and to solve synonymy/homonymy cases as well as paternity and kinship issues. The former group selected microsatellite sequences from scaffolds anchored to the 19 linkage groups of Vitis vinifera L. with the aim of analyzing 38 well-distributed SSR

> SSR employed (mean st. dev)

11 5 21 [53], 17

No. of reference cultivars

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

[52]

15 11 49 [139] 6 [139], 38

18 3 n.a. 46 [144]

12 6 7 [147] 12 [147], 15

14 7 n.a. n.a.

No. of reference SSRs

137

11 [53], 8 [52]

[74]

[108]

number of SSR markers employed for genotyping varieties is 14 7 (Table 4).

Ploidy SSR available (SSR database)

[134]

[135]

[136]

microsatellite database)

66,823 (Tomato genomic resources database)

21,100 (Tomato: Kazusa Marker Database) [137]

collection) [138]

443 (Italian Vitis Database) [140] 6 (The European Vitis Database) [141]

microsatellite consortium) [143]

2n = 6 = 42 21 6

database) [146]

Table 4. Information on the five species analyzed in this book chapter, including genome size, ploidy, available SSR database and number of microsatellite regions included, average number of SSR employed in the articles reviewed,

2449 (Genome database for Rosaceae) [128]

1.42–2.28 [133] 2n = 2 = 46 12 (OLEA Database)

0.48 [129] 2n = 2 = 38 56 (Grape microsatellite

2n = 4 = 28 588 (Wheat

0.75 [145] 2n = 2x = 34 664 (HiDRAS SSR

0.90–0.95 [132] 2n = 2 = 24 146,602 (Tomato

Species Genome size (Gb)

Olea europaea

Solanum Lycopersicum L.

Vitis vinifera L.

Malus domestica Borkh.

Triticum spp. 12.3–13.00

(T. durum Desf) [142]

number of cultivars, and microsatellite used as reference.

16.50–17.00 (T. aestivum L.) [142]

L.
