**5. DNA markers**

fruit, while holding production costs as low as possible. Therefore, many of the breeding goals focus on characteristics that reduce production costs or ensure reliable production of high yields with high-quality fruits. The genetics of a quantitative trait is hard to study, since the effect of each gene is small and often influenced by environment or by the interaction with other genes (epistasis). Many important tomato traits as described above are genetically controlled by a combined action of QTLs with favorable alleles often present in the wild species [18–20]. To introgress the wild favorable allele into cultivated tomato, marker-assisted selection plays an important role and the map positions and markers linked to the QTLs provide a basis for breeders to design optimal breeding strategies. To map QTLs in tomato, interspecific populations have been extensively used. However, in an interspecific cross, multiple segregating QTLs at the whole genome level often tend to mask the effects of one another [21, 22].

An alternative approach to improving selection efficiency in tomato is to discover genetic markers that are associated through linkage or pleiotropy with genes that control the trait(s) of interest. Genetic markers are biological features that can be transmitted from one generation to another. They can be used as experimental probes or tags to track an individual, a tissue, a cell, a nucleus, a chromosome or a gene. The value of genetic markers as indirect selection criteria has been known to breeders since early 1900s. Genetic markers can be classified into two categories namely classical markers and DNA markers [23, 24]. Classical markers comprise morphological markers, cytological markers and biochemical markers. DNA markers such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), rapid amplified polymorphic DNA (RAPD), simple sequence repeats (SSR), single nucleotide polymorphism (SNP), etc. have been developed. These DNA markers have developed into many systems based on different polymorphism detecting techniques or methods including northern and southern blotting of nucleic acid hybridization, polymerase chain

Breeders have used morphological markers to select for superior phenotypes for many decades. During the history of plant breeding, markers mainly used included visible traits such as flower color, leaf shape, seed shape, fruit shape, flesh color, stem length, etc. These morphological markers can easily be identified and therefore usually used in the construction of linkage maps. Some of these markers are also linked with other agronomic traits and thus can be used as indirect selection criteria in breeding. However, morphological markers available are limited, and many of these markers are not associated with important economic traits like yield and quality. In addition, some even have undesirable effects on the development and growth of the plant. In tomato, there are over 1300 morphological, physiological (e.g., male sterility, fruit ripening, and fruit abscission), and disease-resistance genes [26] of which

**3. Development of genetic markers**

96 Recent Advances in Tomato Breeding and Production

reaction (PCR), and DNA sequencing [25].

only less than 400 have been mapped [27].

**4. Classical markers**

In overcoming limitations associated with classical markers, development of DNA markers have proven to be of great significance in enhancing genetics and breeding of crop varieties [30]. A DNA marker is a fragment of DNA showing mutations/variations, which can be used to detect polymorphism between different genotypes in a population. These fragments are usually associated with a specific location within the genome and may be detected using modern molecular tools. In the past, different types of molecular markers have been developed and utilized. This includes both dominant and codominant markers. Dominant markers are markers that are unable to differentiate between homozygotes and heterozygotes, while codominant markers can differentiate between homozygotes and heterozygotes. A lot of molecular markers have been developed for tomato. Notable among them are RFLP markers; however, this marker is time and labor intensive and requires the use of large amount of DNA. As a result, RFLP markers have been replaced with PCR-based markers that are easy to handle (http://solgenomics.net). Other marker techniques that have been developed for tomato include random amplified polymorphic DNA (RAPD) and amplified fragment length polymorphism (AFLP) [30]. Large amount of sequence information have been released for tomato species and subsequently, SSR markers developed. These SSR markers are widely used because they are easy to handle and able to detect multiple alleles. Currently, over 20,000 SSR markers have been developed from expressed sequence tag (EST) and BAC-end sequences and used as genetic and genomic tools in tomato species [31]. Single nucleotide polymorphism (SNP), which is now the marker technology of choice, has also been discovered by a resequencing strategy, and several SNP genotyping methodologies have been developed for application in tomato research. As a result, this high-throughput SNP analysis can be performed effectively in a large number of samples by array-based assays as genotyping platforms and applied to the construction of high-density genetic linkage maps and performance of genome-wide association studies [32]. The diversity arrays technology (DArT) platform, which is one of the array-based methods, has also been used to develop polymorphic markers across introgression line (ILs) population of tomatoes [33].

### **6. Genetic maps in tomato**

The first linkage map of tomato was reported in 1968. This linkage map was constructed based on both morphological and physiological markers [34]. The map was later improved and was assigned to the 12 linkage groups in tomato [35]. This facilitated the development of other maps including the tomato isozyme linkage map that was published in 1980. Then in 1986, another map consisting of RFLP and isozyme loci was also generated. Since then, several interspecific genetic linkage maps have been generated with RFLPs incorporating cleaved amplified polymorphic sequences (CAPS), SSR and SNP markers. Varying number of markers ranging from 93 to 4491 have been used for constructing linkage maps with a coverage of about 50% of the genome. Other intraspecific maps were later constructed using SSR and SNP markers. Identification and construction of these markers and maps, respectively, will be helpful in identifying useful genes or QTLs that can be introgressed into desirable genetic backgrounds for marker-assisted breeding [36]. This may not only hasten the breeding process, but will also allow pyramiding of desirable genes and QTLs from different genetic backgrounds, which will serve as an effective complementary approach to substantial crop improvement.

PCR-based markers reported; with powdery mildew, several QTLs [44] have been identified, but there are no PCR-based markers closely linked to these QTLs identified; and with Septoria leaf spot, there has been no report of genetic mapping studies for resistance breeding. MAS has, however, been successful for resistance breeding in tomato for Fusarium wilt, late blight, leaf mold and Verticillium wilt. Molecular markers associated with Fusarium wilt resistance *I*, *I-1*, *I-2* and *I-3* [45] conferring resistance to four different races of the pathogen were identified, and PCR-based markers developed for all with the exception of *I-1* and used effectively for MAS; markers associated for late blight resistance *Ph*-*1*, *Ph*-*2* and *Ph*-*3* [46] has also been developed and used for tomato breeding; several PCR-based markers linked to the *Cf* gene for leaf mold

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QTLs and molecular markers associated with resistance have also been identified in tomato for the various bacterial diseases; however, it is only markers that are tightly linked to RFLPs and PCR-based markers for gene *Pto* in bacterial speck [49] that have been used for resistance breeding via MAS. With the other bacterial diseases including the bacteria canker, bacterial spot and bacterial wilt, QTLs or RFLP markers have been identified and reported but are not commercially used for MAS. With bacterial canker, two QTLs [50] have been developed and could be useful for MAS. RFLP markers associated with *Rx*-*1* and *Rx*-*2* and Rx-*3* for bacterial spot have been reported [51], but *Rx*-*1, Rx*-*2* and Rx-*3* are independently associated with hypersensitive response in the greenhouse and are not polymorphic in most breeding populations and hence not useful for MAS breeding, while *Rx*-*3* is associated with both hypersensitive response and field resistance. CAPs markers have been developed for the gene *Rx*-*3* and used for MAS breeding. Several QTLs have also been identified for breeding for bacteria wilt resistance in tomato; however, two dominant markers associated with the gene *TRST*-*1* [52] have been suggested to be useful.

Although there has been reports on the identification of the resistant gene *Cmr* for the cucumber mosaic virus [53], *pot*-*1* gene for Potyviruses [54] and two QTLs associated with the tomato mottle virus, there are no reports of use of these markers in tomato breeding.

have been reported to be used for MAS [55]. Several genes have also been reported to be resistant to the tomato spotted wilt virus; however, PCR-based markers for only resistant gene *Sw*-*5* have been reported to be developed and utilized by most tomato breeding programs [56]. With the tomato yellow leaf curl virus, PCR-based markers have been identified for and developed for *Ty*-*1*, *Ty*-*2*, *Ty*-*3* and *Ty*-*4*-resistant loci [57]; hence, these markers are not very consistent and hence the challenge in using them for MAS. In the early 1980s, linkage association between the gene *Mi* [58] controlling nematode (*Meloidogyne incognita*)

locus and PCR-based markers associated with the *Mi* gene [60] have been routinely used for the selection of root knot nematode resistance in tomato. The *Mi* gene has also been reported to be resistant to two biotypes of the whitefly *Bemisia tabaci*. Several studies have tried to identify genes or QTLs for insect resistance in tomato; however, there are fewer reports on the identification of these genes/QTLs [61]. This may be attributed to difficulties in phenotypic screening for insect resistance, linkage drag and ease of using pesticides for insect control. However, with the increasing crusade on integrated pest management and restrictions on the use of pesticides, new discoveries in marker development, it is expected

locus was reported [59]. RFLP markers associated with the *Aps-11*


With the tomato mosaic virus, PCR-based markers for *Tm*-*1*, *Tm*-*2*, and *Tm*-*22*

resistance and *Aps-11*

[47] and Verticillium wilt [48] has also been reported and widely used for MAS.
