II. Identification of QTLs and

Advanced Studies in Biology, Vol. 7, 2015, no. 4, 149 - 168 HIKARI Ltd, www.m-hikari.com http://dx.doi.org/10.12988/asb.2015.41163

Characterization of Early Blight Resistance in a Recombinant Inbred Line Population of Tomato: II. Identification of QTLs and Their Co-localization with Candidate Resistance Genes Hamid Ashrafi Department of Plant Science The Pennsylvania State University, University Park, PA 16802, USA Present address: Department of Plant Sciences, Seed Biotechnology Center, University of California, 1 Shields Ave., Davis, CA 95616 USA Majid R. Foolad* Department of Plant Science The Pennsylvania State University, University Park, PA 16802, USA *Corresponding author Copyright © 2014 Hamid Ashrafi and Majid R. Foolad. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract Early blight (EB), caused by fungi Alternaria solani and A. tomatophila, is a major foliar disease of the tomato in many growing regions. Sources of resistance have been identified within the related wild species of tomato, including S. habrochaites, S. peruvianum and S. pimpinellifolium. Breeding for EB resistance via traditional protocols has been difficult due to the complexity of resistance and influence of several plant physiological and morphological characteristics on tomato response to EB. Identification of genetic markers associated with EB resistance and application of marker-assisted selection (MAS) would facilitate development of elite tomato breeding lines and hybrid cultivars with EB resistance. The goals of this study were to identify quantitative trait loci (QTLs) conferring resistance to EB

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in a resistant accession (LA 2093) of the tomato wild species S. pimpinellifolium, and discover putative candidate resistance genes and expressed sequence tags (ESTs) that co-localize with the QTLs. Previously, a recombinant inbred line (RIL) population of tomato was developed from a cross between LA 2093 and S. lycopersicum breeding line NCEBR-1 and a genetic linkage map with 294 molecular markers spanning the 12 tomato chromosomes constructed. In the present study, the RIL population was grown and evaluated for EB resistance under field conditions in four successive years and generations (F7, F8, F9 and F10). Early blight disease severity, measured as the final % defoliation as well as the area under disease progress curve (AUDPC), was subjected to QTL analysis using simple interval mapping (SIM) and composite interval mapping (CIM). The SIM and CIM analyses resulted in identification of the same QTLs, though CIM detected QTLs with substantially more accuracy. Across the four generations, 5 major QTLs (LOD ≥ 2.4, P ≤ 0.001) were identified for EB resistance on tomato chromosomes 2 (2 QTLs), 5, 6 and 9. Three QTLs on chromosomes 2 and 6 were contributed from LA 2093 with individual phenotypic effects ranging from 8% to 16%, and two QTLs on chromosomes 5 and 9 were contributed from NCEBR-1 with individual phenotypic effects ranging from 7% to 18%. The QTLs on chromosomes 5 and 6 exhibited largest effects (10% to 18%) among all QTLs and were identified in three of the four generations. These two QTLs should be most useful for MAS and improvement of tomato EB resistance using LA 2093 and NCEBR-1 as resistant resources. The identified QTLs showed co-localization with several resistance genes and candidate ESTs, including Mi-1, ethylene response factor-5, lipoxygenase B, wound-induced protein-1, and phosphoenolpyruvate carboxylase kinase-2. With the genome sequence of tomato available, further investigation of these QTLs may lead to the identification of candidate genes underlying EB resistance in tomato. Keywords: Alternaria solani; candidate resistance genes; disease resistance; QTL mapping; quantitative resistance; resistance breeding; Solanum lycopersicum; Solanum pimpinellifolium

1. Introduction Early blight, caused by fungi Alternaria tomatophila E. G. Simmons and A. solani Sorauer (hereafter referred to as A. solani), is one of the most devastating foliar diseases of tomato (Solanum lycopersicum L.) in the area with heavy dew, rainfall, and high relative humidity [11, 17, 18]. Currently there are few commercial cultivars of tomato with sufficient resistance to EB. Common approaches to EB disease control include sanitation, long crop rotation, and routine application of fungicides. Sources of genetic resistance to tomato EB have been identified mainly in the related wild species of tomato, including S. habrochaites S. Knapp & D.M. Spooner, S. peruvianum L. and S. pimpinellifolium L. (see review article [17]). Several studies have examined the inheritance of EB resistance in tomato [5, 16, 19, 26,

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44, 45]

and a few breeding lines and hybrid cultivars of tomato with moderate level of resistance to EB have been developed via traditional breeding [23-25, 27-29]. However, there are major challenges when using conventional breeding protocols to develop tomatoes with EB resistance and yet with other desirable horticultural characteristics. The plant-related challenges include polygenic nature of resistance [69] and association of resistance with several characteristics, including physiological maturity, fruit load and plant type [5, 6, 23, 45]. For example, late maturing and/or low yielding plants appear more resistant, while they may not possess genetic resistance [20]. Similarly, indeterminate plants may outgrow the disease and emerge as resistant, while they may not have genes for resistance. Therefore, selection for EB resistance without considering other associated traits may result in low yielding, late maturing genotypes with undesirable plant type. It is imperative to formulate methodologies which allow developing genotypes with EB resistance and yet with other desirable horticultural characteristics. An approach to better understanding the genetic control of complex traits and improving selection efficiency is to discover genetic markers that are associated with genes or quantitative trait loci (QTLs) controlling the trait of interest and use them as indirect selection criteria. Molecular markers technology can facilitate determination of the number, chromosomal location, and individual and interactive effects of genes or QTLs that control a complex trait. Following their identification, useful genes or QTLs can be introgressed into desirable genetic backgrounds by marker-assisted selection (MAS). Further, because EB resistance may be controlled by different mechanisms and that different resistance genes may be involved in different genetic backgrounds, identification, characterization and pyramiding of resistance genes from different resources via MAS may be an effective approach to substantial improvement in EB resistance in tomato. Previously, QTLs for EB resistance were identified in tomato wild species S. habrochaites [20, 68] and S. peruvianum [10]. However, as these species are distantly related to the cultivated tomato and often have numerous undesirable characteristics, it is often difficult to transfer desirable traits from these species to the cultivated tomato without significant linkage drag or extensive pre-breeding activities [8]. In contrast, genes or QTLs identified in closely-related wild species of tomato, including S. pimpinellifolium, may be more attractive for practical breeding purposes. Among the wild species of tomato, S. pimpinellifolium is the most closely related and the only species for which natural introgression with the cultivated tomato has been demonstrated [53]. Accessions within S. pimpinellifolium produce red fruits and hybridize in both directions with the cultivated tomato and the interspecific progeny do not exhibit many undesirable characteristics [54], facilitating gene transfer from this species. For these reasons, accessions of S. pimpinellifolium have been used frequently in breeding programs for improving the cultivated tomato for numerous important traits, including disease and insect resistance, fruit quality and abiotic stress tolerance [7, 12, 13, 15, 30, 32, 42, 54, 60]. In a recent screening of hundreds of accessions within S. pimpinellifolium, we identified one accession (LA 2093) with numerous desirable horticultural charac-

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teristics, including EB resistance, high fruit quality, and abiotic stress tolerance (MR Foolad, unpublished data). To facilitate characterization of the genetic basis of desirable horticultural characteristics in LA 2093 via the use of molecular markers and QTL mapping approaches, we developed a recombinant inbred line (RIL) population from a cross between LA 2093 and a cultivated tomato and constructed a genetic linkage map of the population [3]. The RIL population was previously used for characterization of the genetic basis of several fruit quality characteristics, including fruit lycopene content [2, 33]. In the present study, we evaluated the RIL population for EB resistance in four successive generations (F7 to F10) and identified QTLs contributing to EB resistance. The use of a RIL population for genetic mapping or QTL analysis has several advantages over the use of early filial or backcross populations, including more accurate estimation of map position (due to reduced linkage disequilibrium), trait evaluation in multiple years or locations, and possible use of dominant markers [3, 9, 51, 58]. A practical approach to identifying genetic factors underlying expression of a trait of interest is to examine putative candidate genes. A gene is considered a candidate either if its encoded protein represents a logical possibility for being involved in the trait of interest or if the gene is physically located within a region of the genome known to be containing the gene or QTL for the trait. The latter situation is frequently encountered in positional cloning projects, where a resistance gene is identified based on its position within the genome. The candidate gene approach has successfully led to the identification of many new genes in human as well as in several plant and animal species [65]. In plants, candidate gene approach has been particularly useful for the identification of new pest and disease resistance genes because many genes involved in resistance pathways have been already characterized [52]. Limited studies have been conducted to identify candidate resistance genes underlying EB resistance in tomato, and only few genes have been reported [14, 35, 36, 59]. On the other hand, a few studies have investigated the use of resistance gene analogues (RGAs) as an alternative approach to better dissect the nature of EB resistance in tomato [20, 47]. In the latter studies, chromosomal location of seven RGAs coincided with the location of EB resistance QTLs, and many of the RGAs were clustered together in chromosomal regions that were known to contain tomato R genes. In the present study, a QTL mapping approach using ESTs for disease resistance/defense response genes was employed to identify potential candidate resistance genes, which might be co-localized with QTLs for EB resistance. A similar approach in rice resulted in the identification of a few candidate genes potentially responsible for resistance to rice blast [64]. The induction of three candidate genes (i.e., ESTs linked to a QTL) was investigated after rice plants were challenged with rice blast fungus, Magnaporthe grisea. Northern blot analysis revealed that two of the genes were over-expressed in infected rice plants. This study demonstrated the power of QTL mapping in association with candidate gene approach to identify new resistance genes in a plant. The purpose of the present study was to identify QTLs for EB resistance in a RIL population of

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tomato and compare the genomic locations of QTLs with genomic locations of known resistance genes and candidate resistance expressed sequence tags (ESTs) to determine potential co-localizations.

2. Materials and Methods Genetic materials Previously we developed a recombinant inbred line (RIL) population of tomato from a cross between S. pimpinellifolium accession LA 2093 (staminate parent) and S. lycopersicum breeding line NCEBR-1 [3]. LA 2093 is a self-compatible inbred accession, which was previously identified as a genetic source for several desirable horticultural and agronomic characteristics, including EB resistance (MR Foolad, unpubl. data). NCEBR-1 was previously released as a fresh-market tomato breeding line with multiple desirable horticultural traits, including EB resistance [23]. Original seeds of LA 2093 was obtained from the C.M. Rick Tomato Genetics Resources Center (TGRC; http://tgrc.ucdavis.edu/), and seed of NCEBR-1 was obtained from R.G. Gardner (North Carolina State University, Fletcher, NC). A total of 172 RILs from each of the F7, F8, F9 and F10 generations were used in this study for EB disease evaluation and identification of EB-resistance QTLs. Early blight disease evaluation In each of the F7, F8, F9 and F10 generations, the 172 RILs were grown under field conditions in 2004, 2005, 2006 and 2007, respectively, at the Pennsylvania State University Research Farm, Russell E. Larson Agricultural Research and Education Center, Rock Springs, PA. In each year, the RILs, parental lines and F1 progeny were grown in two replications of 12 plants each. Each year, seeds were first grown under greenhouse conditions around mid April and 6-8-week old seedlings were transplanted into a field and grown to maturity. No artificial inoculation was made, as there was natural occurrence of EB under field conditions in Rock Springs every year. Evaluation of the plants for EB resistance was conducted as described in the companion paper (Foolad and Ashrafi, 2014). The final % defoliation and AUDPC (Area Under Disease Progress Curve) were used as disease severity indices for QTL mapping, as described below. Genetic map construction A genetic linkage map of the RIL population was previously developed using DNAs from 172 F7 plants and 294 polymorphic markers, including standard restriction fragment length polymorphisms (RFLPs), expressed sequence tag (EST) sequences (used as RFLP probes), cleaved amplified polymorphic sequences (CAPS), and simple sequence repeats (SSRs) [3]. The genetic map spanned 1091 cM of the tomato genome with an average marker distance of 3.7 cM. A majority of the EST sequences were chosen mainly based on the putative role of their unigenes in disease resistance and defense-related response. The linkage map was used for mapping QTLs underlying EB resistance and identification of candidate

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resistance genes or ESTs co-localizing with the identified QTLs, which could potentially be associated with EB resistance in this population. QTL mapping Final % defoliation as well as AUDPC data were used as disease severity indices for identifying QTLs underlying EB resistance. For QTL analysis, while in F7 generation disease severity indices of a select plant within each RI line were used, in each of the F8-F10 generations disease severity indices of each RI line as averaged over the two replications were used. Windows QTL Cartographer v2.5 software [63] was used for QTL analysis. Simple interval mapping (SIM) and composite interval mapping (CIM) using the default parameters (model 6) were employed. A stepwise regression was used to perform the CIM analysis to enter or remove background markers from the model. SIM analysis was also carried out using QGENE mapping program [46] and the results were compared to that of Windows QTL cartographer software. A 1000 time permutation was performed in order to obtain the LOD threshold necessary to declare a significant QTL. Windows QTL cartographer generated a relatively higher values (LOD > ~3.0) for the thresholds than QGENE (LOD > ~2.4). Because the LOD threshold values obtained by Windows cartographer were very stringent, it was highly likely that it would lead to increased frequency of Type-II error (not detecting valid QTLs). Therefore a reduced LOD score of 2.4 (P = 0.001) was chosen to assure sufficient stringency and yet with reduced chance of Type-I error (false positive).

3. Results Responses of the parental lines, F1 progeny, and RIL population to EB The S. pimpinellifolium parent (accession LA 2093) exhibited significantly more EB resistance than the S. lycopersicum parent (breeding line NCEBR-2) over the four years of disease evaluation, based on both final % defoliation and AUDPC (Table 1). However, as expected, NCEBR-2 exhibited some level of resistance to EB in all four years of disease evaluation. The resistant level in the F1 progeny was similar to that of LA 2093 (Table 1). Table 1. Early Blight (EB) disease severity indices, based on final percentage defoliation (% Defoliation) and area under disease progress curve (AUDPC), for parental lines, F1 progeny, and four generations of the Solanum lycopersicum × S. pimpinellifolium recombinant inbred line (RIL) population. Adapted from Foolad and Ashrafi 2014.

Genotypes P1 (NCEBR1)

% Defoliation

Number of plants

Mean ± SE

Range

96

53.7 ± 14.0

10.0 - 75.0

AUDPC Mean ± SE Range 1349 ± 334 254 - 2505

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Table 1. (Continued): Early Blight (EB) disease severity indices, based on final percentage defoliation (% Defoliation) and area under disease progress curve (AUDPC), for parental lines, F1 progeny, and four generations of the Solanum lycopersicum × S. pimpinellifolium recombinant inbred line (RIL) population. Adapted from Foolad and Ashrafi 2014.

P2 (PSLP125) F1

96 96

28.3 ± 2.1 29.2 ± 3.0

25.0 - 32.5 10.5 - 30.0

507 ± 37 564 ± 66 1357 ± 608 957 ± 641 1283 ± 540 964 ± 524

F7-RILs F8-RILs

172 4128

69.4 ± 23.0 42.7 ± 24.9

15.0 100.0 6.0 - 100.0

F9-RILs F10-RILs

4128 4128

68.6 ± 26.8 46.8 ± 22.7

10.0 -100.0 6.5 -98.0

368 - 650 440 - 875

201 - 2720 71 - 3085 208 - 2596 132 - 2769

In all four years of disease evaluation, the RIL population exhibited large variation in response to EB, from very resistant (≤ 15% defoliation) to highly susceptible (e.g. 100% defoliation) (Table 1). There were generally higher levels of EB disease in F7 (2004) and F9 (2006) generations compared to those in F8 (2005) and F10 (2007) generations. This was most likely due to higher levels of rainfall and humidity in the field during 2004 and 2006 growing seasons, compared to those in 2005 and 2007 seasons. Although the distribution of disease response in the RIL population was continuous in every year of disease evaluation, it was not normally distributed (presented and discussed in the companion paper Foolad and Ashrafi, 2014).

QTLs identified for early blight resistance The results of SIM and CIM analyses were similar, and thus only those of the CIM analyses are presented here. Further, QTL results were similar based on both final % defoliation and AUDPC, therefore only results of AUDPC are presented.

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Over the course of the four years (4 generations) of disease evaluation and QTL mapping, a total of 5 major QTLs for EB resistance were identified. These QTLs were located on chromosomes 2 (two QTLs), 5, 6 and 9 (Table 2; Fig. 1). Of these, the positive alleles for 3 QTLs (those on chromosomes 2 and 6) were contributed from the wild parent,

Table 2. QTLs identified for early blight (EB) resistance by composite interval mapping in a recombinant inbred line (RIL) population of tomato developed from a Solanum lycopersicum (NCEBR-1) × S. pimpinellifolium (LA 2093) cross.

Chrom. 2

Position Interval (cM) 46.9-64.1

LOD 3.4

Additive Effect -197.4

EB5.1 EB9.1

Marker Interval cTOF19J9-TG-463 cLEY-18H8-cTOA24J24 TG343-cLED4N20

5 9

71.4-77.4 60.8-69.8

3.9 3.0

267.8 179.7

R2 8% 11% 7%

F8 generation

EB2.1 EB2.2 EB5.1 EB6.1

cLEC73K6b-cLEC73I19a CT103-TG463 cLEY-18H8-cTOC20J21 TG274-cLEN10H12

2 2 5 6

1.1-4.57 54.0-67.9 69.4-81.5 14.8-29.0

3.6 2.5 5.6 4.9

-178.8 -195.1 283.2 -225.3

8% 3% 18% 13%

F9 generation

EB5.1 EB6.1 EB9.1

cLEY-18H8-cTOC2J24 TG274-TG590 TG348-cTOE10J18

5 6 9

71.4-77.1 14.8-17.3 52.8-54.6

7.1 4.6 5.1

249.8 -182.2 205.6

17% 16% 14%

F10 generation

EB2.1 EB6.1

cLEC73K6b-CT205

2 6

1.1-12.2 14.8-29.0

3.0 3.7

-184.9 -224.2

8% 10%

F7 generation

QTL Name EB2.2

TG274-cLEN10H12

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Figure 1. Identified QTLs for early blight resistance based on AUDPC data in four generations of a RIL population developed from a Solanum lycopersicum (NCEBR1) × S. pimpinellifolium cross.

LA 2093, and 2 QTLs (on chromosomes 5 and 9) from the cultivated parent, NCEBR-2. Of the 5 major QTLs, those located on chromosomes 5 and 6 exhibited the largest effects, were most stable and each was detected in three of the four generations. The individual effect of the QTL on chromosome 5 (EB5.1) ranged from 11% (in F7) to 18% (in F8), with an average of 15.3% of the total phenotypic variation in disease severity (Table 2). The individual effect of the QTL on chromosome 6 (EB6.1) ranged from 10% (in F10) to 16% (in F9), with an average of 13% of the total phenotypic variation in disease severity (Table 2). The two QTLs on chromosome 2 (EB2.1 and EB2.2) and the QTL on chromosome 9 (EB9.1) were each detected in 2 of the 4 years of disease evaluation and comparatively had smaller effects than the QTLs on chromosomes 5 and 6 (Table 2). The average individual effect of each of EB2.1, EB2.2 and EB9.1 QTLs was 8.0%, 5.5% and 11.5%, respectively, of the total phenotypic variation in EB severity (Table 2).

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There were multiple co-localizations of QTLs with resistance genes and/ or candidate resistance ESTs, as discussed below.

4. Discussion Response of the RIL population to EB In all four years (generations) of evaluations, the disease response of the RIL population was continuous, from very resistant to highly susceptible, suggesting that the nature of EB resistance in this population is quantitative and possibly polygenic, in agreement with several previous genetic studies of EB resistance in different populations of tomato [19, 20, 39-41, 44, 61]. Further, the identification of multiple QTLs for EB resistance in the present study, as well as in previous studies [10, 20, 68] , confirms the notion of polygenic nature of EB resistance in tomato. These results, along with the fact that several other factors, including physiological, morphological, maturity and yield characteristics, affect plant response to EB, further confirm challenges in breeding for EB resistance in tomato [4, 17, 18, 22, 26, 40, 44] . Identification of QTLs underlying EB resistance in tomato may facilitate breeding for this complex trait. QTLs identified Of the five major QTLs identified in this study, three were contributed from the wild parent (LA 2093) and two from the cultivated parent (NCEBR-2). This was not unexpected, as both parents were previously reported to have EB resistance [1, 23] . Three of the QTLs identified in the present study (EB2.2, EB5.1 and EB6.1) were also previously reported in other tomato populations, whereas the two QTLs EB2.1 and EB9.1 are new. Of the two QTLs identified on chromosome 2 (contributed from LA 2093), EB2.1 was detected in F8 and F10 generations, mapped to a marker-saturated region on the short arm of the chromosome (Fig. 1), and was not previously reported. In comparison, EB2.2 was detected in F7 and F8 generations, mapped to the long arm of the chromosome in an area with fewer markers (Fig. 1), and located in the vicinity of a QTL for EB resistance previously reported in a S. peruvianum accession [10]. Further, Foolad et al. [20] reported a QTL for EB resistance on the lower region of the long arm of chromosome 2, which was contributed from a S. habrochaites accession. It is likely that the two previouslyreported QTLs for EB resistance on the long arm of chromosome 2 are the same as EB2.2 detected in the present study, and the shifting in position may be due to variation in marker density in the various populations used for QTL mapping (Fig. 1; [10, 20]). The third QTL contributed from the wild parent (LA 2093) was identified on tomato chromosome 6 (EB6.1), spanning ~20 cM region (Table 2; Fig. 1). This QTL, which was identified in F8, F9 and F10 generations, had very large individual effects, ranging from 10% to 16% of the total phenotypic variation for this trait in different RIL generations. This is considered a major QTL, and should be useful for MAS and transferring of EB resistance from LA 2093 to the cultivated tomato.

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A QTL in the same chromosomal region was previously reported in a S. lycopersicum  S. habrochaites population [68] as well as in a S. lycopersicum  S. peruvianum population [10]. Because the effect of this QTL was inherited from the wild resistant parents in all of these three studies, it is possible that S. pimpinellifolium, S. habrochaites and S. peruvianum share the same QTL region for EB resistance. Of the two QTLs contributed from the cultivated parent (NCEBR-1), one was located on chromosome 5 (EB5.1), identified in F7, F8 and F9 generations with individual effects of 11% – 17%, and the second was located on chromosome 9 (EB9.1), identified in F7 and F9 generations with individual effects of 7% – 14% of the total phenotypic variation for EB resistance (Table 2; Fig. 1). EB5.1 had the larges individual effects among all the QTLs identified in the present study. In the same chromosomal position, Foolad et al. [20] and Chaerani el al. [10] had reported a QTL for EB resistance in two different interspecific populations of tomato. While in the latter two studies the QTL on chromosome 5 was contributed from a S. habrochaites and a S. peruvianum accession, respectively, in the present study the positive allele of this QTL was contributed from the cultivated parent, NCEBR-1. However, as the EB resistance of NCEBR-1 was incorporated from a wild S. habrochaites accession [23], it is possible that this QTL is inherited from the same ancestral origin. The presence of this QTL might explain the observation of transgressive segregants observed in the RIL population with high EB resistance. The QTL EB9.1 from the cultivated parent (NCEBR-1) was not previously identified in any study and is considered new. However, as the EB resistance in NCEBR-1 was incorporated from a wild accession (PI 126445) of S. habrochaites [23, 26] , it is highly likely that the original source of this QTL is the wild species S. habrochaites. This QTL also has moderately large effect (7% – 14% of total phenotypic variation) and should be important for development of tomato breeding lines with EB resistance. An overall comparison of the EB resistance QTLs identified in the present study based on a S. lycopersicum  S. pimpinellifolium cross with those previously identified based on either a S. lycopersicum  S. habrochaites [20, 68] or a S. lycopersicum  S. peruvianum cross [10] indicates that while some QTLs appear to map to the same genomic locations, other QTLs are different from population to population (see review papers [17, 18]). Presence of the same or similar resistance QTLs across populations validates the authenticity of the QTLs for further genetic studies or exploitation in breeding programs for improving EB resistance in tomato. However, identification of different QTLs in different interspecific populations suggests the presence of different genes for EB resistance in different genetic backgrounds, and thus the potential for pyramiding resistance from multiple sources and producing stronger and more durable resistances to tomato EB. Candidate genes underlying QTLs for EB resistance The genetic map of the RIL population was developed based on the use of 294 molecular markers, including 132 candidate resistance/defense-response genes (ESTs) (see Ashrafi et al. 2009 for the description of the ESTs). In the present study,

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we investigated co-localization of QTLs for EB resistance with known resistance genes, candidate resistance ESTs, and quantitative resistance loci (QRLs) previously reported for resistance to different diseases in tomato. A few such colocalizations were observed. For example, in the very distal end of the short arm of chromosome 2, at least 7 ESTs were mapped in a 1.6 cM interval, near the location of EB2.1 (Fig. 1). These ESTs, coded for jasmonic acid, xyloglucan-specific fungal endoglucanase inhibitor protein and several kinases. Further, EB2.1 is co-localized with Tm-1 gene, which confers resistance to tomato mosaic virus [47, 50]. The presence of Tm-1 gene and a cluster of candidate resistance ESTs nearby EB2.1 suggest richness of this region in resistance/ defense-response genes. A cluster of resistant gene analogues (RGAs) was also observed in the same region of chromosome 2 in an F2 population of the same cross (NCEBR-1  LA 2093), in which a QTL was also identified for EB resistance at the same location as EB2.1 (Foolad et al. unpublished data; [17]). Further research is needed to determine whether any or a combination of these ESTs or resistance genes are involved with EB resistance in tomato. At both sides of the QTL EB2.2 interval (within ~10 cM), three resistance ESTs were mapped, though the QTL peak was at neighboring markers TG463 and CG21 and not exactly at the ESTs’ location (Fig. 1). EST marker cLEY1K9 (pathogeninducible alpha-dioxygenase) mapped to the right side of the QTL EB2.2 and EST markers cLEC72P14 (acidic 27 kDa endochitinase precursor) and cLET10E15 (acidic 26 kDa endochitinase precursor) mapped to the left of EB2.2. The role of acidic and basic chitinases were previously investigated in EB resistance [35]. However, to our knowledge, the role of pathogen-inducible alpha-dioxygenase in EB resistance has not been determined. The two strongest QTLs identified in the present study for EB resistance, those on chromosomes 5 (EB5.1) and 6 (EB6.1), are co-localized with several resistancerelated ESTs or resistance gene families. The QTL on chromosome 5 spans ~15 cM interval in which region important ESTs such as disease resistance gene homolog Mi-copy1 (cTOC2J14), Ribosomal protein L6 (cTOA24J24), 60S ribosomal protein L6 (cTOC20J21), ethylene response factor-5 and metallothionein-like protein type-2 have been located. Co-localization of the peak of QTL EB5.1 with Mi gene further suggests that resistance to EB and root-knot nematodes (Meloidogyne spp.) may share a common pathway. Because the Mi-1 gene encodes a protein sharing structural features with the nucleotide-binding site leucine-rich repeat (NBS-LRR) type of plant resistance genes [62], it is likely that it has other functions and contribute to resistance to EB. The fact that Mi-1 gene confers resistance to three unrelated plant pests, root-knot nematodes [62], potato aphid, Macrosiphum euphorbiae [55], and whitefly, Bemisia tabaci [48, 49], raises the possibility that it may also confer resistance to fungal diseases such as EB. Challenging the lines carrying the Mi-1 gene with A. solani may test this hypothesis. The QTL EB5.1 is also co-localized with rx gene family for resistance to bacterial spot [66] and pto and prf genes for resistance to bacterial speck [43, 47, 57]. The involvement of these genes with EB resistance is unknown.

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The QTL on chromsosme 6, EB6.1, is co-localized with several known resistance genes, including the cf gene family for resistance to leaf mould, QRLs for resistance to bacterial canker, and Ty-1 for resistance to yellow leaf curl virus [47, 67] . Further, the Log Likelihood Ratio (LR) plot of EB6.1 starts at marker cLEG32E10 for lipoxygenase B (LOX B) and ends at marker (cLEN10H12) for alcohol dehydrogenase-2 (ADH-2) (Fig. 1). In this chromosomal region two other ESTs were mapped, including cLEC76A13 for wound-induced protein-1 and cLEG49O24 for Phosphoenolpyruvate carboxylase kinase-2. Although these are not the only ESTs in this chromosomal region and no conclusion can be made regarding their potential roles in EB resistance, knowledge of their function may help speculating any potential relationship. Lipoxygenase (LOX) activity has been noted during pathogen-induced defense responses [34] and mechanical wounding and insect attack [31]. It has been suggested that LOX is involved in the development of an active resistance mechanism known as the hypersensitive response (HR), a form of programmed cell death [56]. Mechanical wounding of potato leaves, stems, roots and tubers lead to a rapid increase of wound induced protein wun1 mRNA [37]. Because the peak of the QTL is at the EST marker for wun1 it is possible that this gene is involved in EB resistance. Transforming susceptible tomato plants with this gene may verify whether it enhances the level of tomato resistance to A. solani. Alternatively, one could study up-regulation of this gene in tomato plants challenged with A. solani. Alcohol dehydrogenase (ADH) is up-regulated in potato plants challenged with Phytophthora infestans and its role in fatty acid degrading pathways of diseased potato is well characterized [38]. Phosphoenolpyruvate carboxylase (PEPC), is a key enzyme of primary metabolism of higher plants and is regulated by reversible phosphorylation, which is catalyzed by PEPC kinase (PPCK) [21]. PEPC is normally up-regulated upon pathogen infection or wounding [38] but its role in metabolic pathway of disease resistance has not yet been characterized. Despite the apparent co-localizations of EB resistance QTLs with disease resistance genes, QRLs or resistance ESTs, at this point no conclusion can be made as to active roles of such genes in EB resistance. Such co-localizations could simply be due to random association. Further studies are needed to determine the potential involvement of the noted genes or ESTs in EB resistance. Acknowledgments. We thank Professors Mark Guiltinan, David Huff, Seogchan Khang and David Braun for reviewing an earlier version of this manuscript and making constructive comments and suggestions. We also thank all Penn State staff and students who helped with the disease screening experiments, Dr. Randolph Gardner of the NC State University for providing seed of breeding line NCEBR-1, and TGRC for providing the original seed of the wild accession LA 2093. This research was supported in part by Agricultural Research Funds administered by the Pennsylvania Department of Agriculture, the Pennsylvania Vegetable Marketing and Research Program, and the College of Agricultural Sciences at the Pennsylvania State University.

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Received: November 15, 2014; Published: February 13, 2015