1. The Italian agriculture scenery and the utility of SSR markers to develop a reference method for genotyping plant varieties

The Food and Agriculture Organization (FAO) indices of agricultural production describe the relative level of the aggregate volume of agricultural production for each year in comparison with the base period 2004–2006 [1]. According to the most recent data available in The Food and Agriculture Organization Corporate Statistical Database, the gross value of the total Italian agricultural production was equal to \$ 41.9 billion, about € 32.7 billion [2]. It is worth

© The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons © 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and eproduction in any medium, provided the original work is properly cited.

noting that 20 products contribute to over 50% of gross production value (GPV), as shown in Table 1.

and wine (Table 2). It is worth noting that the wine GPV (Table 2) is four times higher than the grape GPV and slightly less than half of the total GPV shown in Table 1, a demonstration that

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One of the main problems that needs to be addressed is the lack of a uniform, complete, and updated register of cultivars. For the cultivars of some, species like cereals or vegetables are already available as official registers provided by the Ministry of Agricultural, Food and Forestry Policies (MIPAAF, National register of agricultural varieties and National register of horticultural varieties). Concerning fruit trees, on the contrary, there is not a register yet, although Article 7 of the Italian Legislative Decree no. 124/2010 has established a "National Register of fruit trees varieties" [11]. For this reason, the inventory of cultivars of some fruit species is still ongoing and there is a total lack of official data for some of them (see for instance orange, lemon, and mandarin, Table 1). Moreover, for species of particular interest, there exist

In the past, cultivars have been extensively characterized by morphological traits, including plant, leaf, fruit, and seed characteristics. Since objectivity is crucial to perform an accurate morphological typing, it is constraining to use exclusively morphological descriptors for plant cultivars, especially because most of the morphological traits are influenced by environmental factors. Several cases of misidentification, owing to classifications carried out only employing morphological traits, are reported in the scientific literature for a wide range of vegetal crops [12–14] and fruit trees [15–18]. Moreover, the uneven distribution, simultaneous cultivation of local varieties, ambiguous names, continuous interchange of plant materials among varieties and/or farmers of different regions and countries, possibility of the cultivation of varietal clones, and uncertainty of varietal certification in nurseries have complicated the identification of genotypes [19–21]. At the same time, cultivar and clone identity is also very important for protecting plant breeders' rights not only for commercial seeds but also for processed materials and food derivatives, especially for the final consumers' safeguard. Another important aspect to highlight is the need to ensure that each specific variety grown by farmers and its food product bought by consumers is the one declared on the label. This is especially true if the product is sold in a processed or transformed form (thus difficult to recognize phenotypically) and/or if the product is subjected to a form of certification (PDO or PGI). In a modern market, it is crucial being able to identify agricultural products and foodstuffs by means of reliable

The method of DNA genotyping based on microsatellite markers represents an efficient, reliable, and suitable technique that is able to complement the information provided by morphological

Value [2] of agriculture production USD (10<sup>6</sup>

Wine 11603.83 403 Oil, olive, virgin 2126.78 41 Total 13,730.61 444

) PDO and PGI [10]

shows producing food derivatives could be more profitable than selling raw products.

registers apart (see for example, Olea europaea L. and Vitis vinifera L.).

traceability systems, including genetic molecular markers.

Table 2. GPV, PDO, and PGI products for wine and olive oil in Italy.

On average, each species is characterized by dozens or hundreds of cultivars and, as defined in Article 2 of the International Code of Nomenclature for Cultivated Plants, a "cultivar is an assemblage of plants that has been selected for a particular character or combination of characters, that is distinct, uniform, and stable in those characters, and that when propagated by appropriate means, retains those characters" [3]. If some cultivars are virtually ubiquitous, some others are associated with specific geographical contexts and often provide the basis for the establishment of protected designation of origin (PDO) and protected geographical indication (PGI) products (Table 1).

It is not a coincidence that Italy, with its 268 brand products, including 106 PGI, 160 PDO, and 2 traditional specialty guaranteed (TSG) labels, is the European leader in terms of certified productions and that 20% of them arise from the 20 crops listed in Table 1. As a whole, the Italian certified products reach around 500 units, including two important derivatives such as olive oil


Table 1. GPV, registered cultivars, PDO, and PGI products for the 20 most economically important crops in Italy.

and wine (Table 2). It is worth noting that the wine GPV (Table 2) is four times higher than the grape GPV and slightly less than half of the total GPV shown in Table 1, a demonstration that shows producing food derivatives could be more profitable than selling raw products.

noting that 20 products contribute to over 50% of gross production value (GPV), as shown in

On average, each species is characterized by dozens or hundreds of cultivars and, as defined in Article 2 of the International Code of Nomenclature for Cultivated Plants, a "cultivar is an assemblage of plants that has been selected for a particular character or combination of characters, that is distinct, uniform, and stable in those characters, and that when propagated by appropriate means, retains those characters" [3]. If some cultivars are virtually ubiquitous, some others are associated with specific geographical contexts and often provide the basis for the establishment of protected designation of origin (PDO) and protected geographical indica-

It is not a coincidence that Italy, with its 268 brand products, including 106 PGI, 160 PDO, and 2 traditional specialty guaranteed (TSG) labels, is the European leader in terms of certified productions and that 20% of them arise from the 20 crops listed in Table 1. As a whole, the Italian certified products reach around 500 units, including two important derivatives such as olive oil

Olives (table and oil) 5064.24 644 [5] 3 Tomatoes 4753.00 445 [6] 3 Grapes (table and wine) 2770.60 638 [7] 3 Wheat (durum, common, spelt) 2558.23 489 [8] 0 Maize 2363.83 1739 [8] 0 Apples 1129.69 75 [9] 5 Oranges 990.80 n.a. 3 Potatoes 751.58 56 [8] 3 Rice, paddy 750.28 194 [8] 3 Peaches and nectarines 570.69 311 [9] 4 Pumpkins 532.35 8 [6] 0 Pears 521.17 32 [9] 2 Mandarins, Clementines 431.27 n.a. 2 Artichokes 386.11 14 [6] 4 Carrots and Turnips 355.33 8 [6] 2 Cauliflowers and Broccoli 314.07 41 [6] 0 Beans 311.63 39 [6] 6 Lemons 267.16 n.a. 6 Onions 264.10 71 [6] 2 Hazelnuts (with shell) 247.36 25 [9] 3 Total 25,333.49 4829 54

Table 1. GPV, registered cultivars, PDO, and PGI products for the 20 most economically important crops in Italy.

Registered cultivars PDO and PGI [4]

Table 1.

tion (PGI) products (Table 1).

132 Rediscovery of Landraces as a Resource for the Future

Crop plants Value [2] of agriculture production USD (10<sup>6</sup> )

One of the main problems that needs to be addressed is the lack of a uniform, complete, and updated register of cultivars. For the cultivars of some, species like cereals or vegetables are already available as official registers provided by the Ministry of Agricultural, Food and Forestry Policies (MIPAAF, National register of agricultural varieties and National register of horticultural varieties). Concerning fruit trees, on the contrary, there is not a register yet, although Article 7 of the Italian Legislative Decree no. 124/2010 has established a "National Register of fruit trees varieties" [11]. For this reason, the inventory of cultivars of some fruit species is still ongoing and there is a total lack of official data for some of them (see for instance orange, lemon, and mandarin, Table 1). Moreover, for species of particular interest, there exist registers apart (see for example, Olea europaea L. and Vitis vinifera L.).

In the past, cultivars have been extensively characterized by morphological traits, including plant, leaf, fruit, and seed characteristics. Since objectivity is crucial to perform an accurate morphological typing, it is constraining to use exclusively morphological descriptors for plant cultivars, especially because most of the morphological traits are influenced by environmental factors. Several cases of misidentification, owing to classifications carried out only employing morphological traits, are reported in the scientific literature for a wide range of vegetal crops [12–14] and fruit trees [15–18]. Moreover, the uneven distribution, simultaneous cultivation of local varieties, ambiguous names, continuous interchange of plant materials among varieties and/or farmers of different regions and countries, possibility of the cultivation of varietal clones, and uncertainty of varietal certification in nurseries have complicated the identification of genotypes [19–21]. At the same time, cultivar and clone identity is also very important for protecting plant breeders' rights not only for commercial seeds but also for processed materials and food derivatives, especially for the final consumers' safeguard. Another important aspect to highlight is the need to ensure that each specific variety grown by farmers and its food product bought by consumers is the one declared on the label. This is especially true if the product is sold in a processed or transformed form (thus difficult to recognize phenotypically) and/or if the product is subjected to a form of certification (PDO or PGI). In a modern market, it is crucial being able to identify agricultural products and foodstuffs by means of reliable traceability systems, including genetic molecular markers.

The method of DNA genotyping based on microsatellite markers represents an efficient, reliable, and suitable technique that is able to complement the information provided by morphological


Table 2. GPV, PDO, and PGI products for wine and olive oil in Italy.

traits and that has been extensively used for the characterization of plant varieties [22–24] and the certification of food products [25–27].

europaea L., Solanum lycopersicum L., Vitis vinifera L., Triticum spp., and Malus domestica Borkh., wine and olive oil. In this respect, the chapter aims to assess the real achievements of different genotyping analyses, to evaluate the strengths and limitations according to applied research studies, and to emphasize the striking lack of data related to the applications of SSR technology. Through the careful investigation and evaluation of a large number of scientific papers, our review highlights some critical aspects on the use of microsatellite markers and formulates recommendations for standardizing the strategies and methods for ascertaining the genetic identity of plant varieties and for achieving the genetic traceability of their food derivatives. Here, we focus on three main aspects: (i) how to choose and use SSR markers, (ii) which parameters/indices calculate for the genetic characterization of plant materials, and (iii) assess a standardized way to make SSR data from different works on the same species comparable.

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

contribute to the annual Italian GPV for another 15 billion Euro [2].

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

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).

Crops References Olive (Olea europaea L.) [23, 25, 48–63] Tomato (Solanum lycopersicum L.) [27, 44, 64–70] Grape (Vitis vinifera L.) [15, 19, 24, 71–89] Wheat (Triticum spp.) [22, 26, 90–98] Apple (Malus domestica Borkh.) [99–111]

Wine [45, 112–116] Olive oil [46, 58, 117–125]

plant varieties

Derivatives

Table 3. Crops and derivatives reviewed.

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 uniquely genotyped in a reproducible way [29].

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, and for identifying crop varieties.

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 methodology in applied breeding programs [41–43].

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

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 europaea L., Solanum lycopersicum L., Vitis vinifera L., Triticum spp., and Malus domestica Borkh., wine and olive oil. In this respect, the chapter aims to assess the real achievements of different genotyping analyses, to evaluate the strengths and limitations according to applied research studies, and to emphasize the striking lack of data related to the applications of SSR technology. Through the careful investigation and evaluation of a large number of scientific papers, our review highlights some critical aspects on the use of microsatellite markers and formulates recommendations for standardizing the strategies and methods for ascertaining the genetic identity of plant varieties and for achieving the genetic traceability of their food derivatives. Here, we focus on three main aspects: (i) how to choose and use SSR markers, (ii) which parameters/indices calculate for the genetic characterization of plant materials, and (iii) assess a standardized way to make SSR data from different works on the same species comparable.
