LINKAGE MAPPING OF CANDIDATE GENES FOR INDUCE RESISTANCE AND GROWTH PROMOTION BY Trichoderma koningiopsis (Th003) IN TOMATO Solanum lycopersicum
Mapeo de genes candidatos relacionados con inducción de resistencia sistemica y promoción de crecimiento por Trichoderma koningiopsis (Th003) en tomate Solanum lycopersicum
JAIME SIMBAQUEBA1, M.Sc., ALBA MARINA COTES1, Ph. D.; LUZ STELLA BARRERO1, Ph. D. 1 Plant Molecular Genetics Lab, Center for Biotechnology and Bioindustry (CBB), Corporación Colombiana de Investigación Agropecuaria (CORPOICA). Km 14, vía Mosquera, Cundinamarca, Colombia. Corresponding author: Km 14, vía Mosquera, Cundinamarca, Colombia. Phone: 57 1 422 73 00, ext. 1428 lbarrero@corpoica.org.co
Presentado 8 de julio de 2010, aceptado 9 de marzo de 2011, correcciones 23 de marzo de 2011.
ABSTRACT
Induced systemic resistance (ISR) is a mechanism by which plants enhance defenses against any stress condition. ISR and growth promotion are enhanced when tomato (Solanum lycopersicum) is inoculated with several strains of Trichoderma ssp. This study aims to genetically map tomato candidate genes involved in ISR and growth promotion induced by the Colombian native isolate Trichoderma koningiopsis Th003. Forty-nine candidate genes previously identified on tomato plants treated with Th003 and T. hamatum T382 strains were evaluated for polymorphisms and 16 of them were integrated on the highly saturated genetic linkage map named “TOMATO EXPEN 2000”. The location of six unigenes was similar to the location of resistance gene analogs (RGAs), defense related ESTs and resistance QTLs previously reported, suggesting new possible candidates for these quantitative trait loci (QTL) regions. The candidate gene-markers may be used for future ISR or growth promotion assisted selection in tomato.
Key words: Trichoderma koningiopsis-Solanum lycopersicum-induced systemic resistance growth promotion- candidate genes -genetic linkage map.
RESUMEN
La resistencia sistémica inducida (ISR) es un mecanismo mediante el cual las plantas aumentan sus defensas frente a cualquier condición de estrés. El objetivo de este trabajo fue localizar en el mapa genético de tomate, genes candidatos involucrados en ISR y promoción de crecimiento inducidos por la cepa colombiana nativa Th003 de Trichoderma koningiopsis. Se realizó una búsqueda de polimorfismos en cuarenta y nueve genes candidatos previamente identificados en plantas de tomate inoculadas con Th003 y la cepa T382 de T. hamatum. Diez y seis de estos genes candidatos fueron integrados en el mapa genético de tomate altamente saturado, llamado “TOMATO EXPEN 2000”. La ubicación de seis unigenes fue similar a la localización de genes análogos de resistencia (RGAs), ESTs relacionados con defensa y QTLs de resistencia previamente identificados, sugiriendo posibles nuevos candidatos para estas regiones de QTLs. Los genes candidatos o marcadores pueden ser usados en futuros programas de selección asistida relacionados con ISR o promoción de crecimiento en tomate.
Palabras clave: Trichoderma koningiopsis, Solanum lycopersicum, Inducción de resistencia sistémica, promoción de crecimiento, genes candidatos, mapeo genético
INTRODUCTION
Tomato, Solanum lycopersicum, is the most important vegetable crop in the world and constitutes approximately 30% of vegetable consumption with developing countries contributing about 65% of world production; however, phytosanitary constraints are major limiting factors for this production (Peralta and Spooner, 2007). Biological control is an alternative for sustainable disease control and crop production in agriculture. In this context, plant induced systemic resistance (ISR) has aroused great interest. ISR is a mechanism where the defensive capability is enhanced by a specific elicitor by which the innate plant defenses are activated against subsequent biotic targets. It is effective against a broad range of pathogens and is mostly caused by necrotrophic pathogens, herbivorous insects, beneficial microorganisms as growth promoter rhizobacteria (PGPRs) and some strains of Trichoderma spp. This mechanism involves production of the plant jasmonic acid (JA) and ethylene (ET) (Van Loon et al., 1998; Hammerschmidt 1999; Harman 2004; Segarra et al., 2009).
Trichoderma ssp. are cosmopolitan soil fungi, widely used to control plant pathogens and pests and promote plant growth (Yedidia et al., 2000; Howell 2003; Harman et al., 2004). For example, in maize, differentially expressed proteins induced by T. harzianum T22 suggested that T22 stimulates both increased growth, which is mediated by an increase in photosynthetic and respiratory rates, and systemic induced resistance (Shoresh and Harman, 2008). Other examples involving pathosystems demonstrate the biocontrol activity of Thrichoderma. In cucumber, T. asperellum T203 strain induced resistance against Pseudomonas syringae pv. lachrymans (Shoresh et al., 2005). In Arabidopsis, T. asperellum T34 has a great inducer effect against P. syringae, Hyaloperonospora parasítica (biotrophic) and Plectosphaerella cucumerina (necrotrophic); (Segarra et al., 2009).
In tomato, studies concerned with the control activity of a native T. koningiopsis isolate namely Th003 from Colombia have shown that in addition to its antagonistic activity, this strain induced resistance against Fusarium oxysporum, promoted growth and enhanced seed germination rate (Moreno et al., 2009). Using the TOM1-ESTs microarray available at the Solanaceae Genomics Network (SGN); (Mueller et al., 2005) coupled with real-time PCR, (Moreno et al., 2009) also showed differential expression of 45 genes (41 in roots and 4 in leaves) in Th003 treated vs. non treated tomato plants. The genes belonged to the functional categories of transport, signaling, cell wall degradation and hormone responses (auxin and ethylene). Some genes were also differentially expressed by T. hamatum T382, which induced resistance against Xanthomonas euvesicatoria in tomato (Alfano et al., 2007).
The present study aims to contribute the understanding of ISR and growth promotion triggered by Th003 and T382, through the integration of differentially expressed genes from (Moreno et al., 2009), in the “TOMATO EXPEN 2000” linkage map. This map is based on 80 F2 individuals from the cross S. lycopersicum LA925 and S. pennellii LA716 and contains about 2506 RFLPs, COS, SSRs, CAPS and other markers; furthermore, this map is anchored to the BAC physical map, used for the tomato genome sequence (Mueller et al., 2005). The location of these candidate genes on the tomato map provides a key starting point for validation through subsequent QTL analyses for ISR or growth promotion, the discovery of novel genes affecting the phenotype not identified by microarray analysis, and their subsequent use in breeding programs related to biological control.
MATERIALS AND METHODS
PLANT MATERIAL AND DNA EXTRACTION
The 80 F2 plant population along with the parents S. lycopersicum and S. pennellii (referred as to Sl and Sp, respectively) of the TOMATO EXPEN 2000 map (Mueller et al., 2005), were kindly donated by professor Steven D. Tanksley, Cornell University, USA, and were propagated in vitro. DNA extractions from in vitro foliar tissues were carried out according to Fulton et al.,2002.
GENE PRIMER DESIGN
Forty-five gene sequences were obtained from Moreno et al., 2009 and four from Alfano et al., 2007 (Table 1). Primers derived from candidate genes were designed based on the SGN unigene sequences (EST assembles), and each sequence was compared usingBLASTn against the SGN unigene and the SGN BAC sequence database (Mueller et al., 2005). Unigenes not found in tomato BACs were compared against Arabidopsis thaliana mRNA and genome databases (NCBI, http://www.ncbi.nlm.nih.gov) usingtBLASTx. Each unigene vs. its corresponding tomato BAC or Arabidopsis homolog were aligned using Dialign 2.2.1 (Lassmann and Sonnhammer 2002). Primers were designed from a region spanning coding positions and a complete intron using Primer3 v 0.2 (Rozen and Skaletsky 2000).
PCR AMPLIFICATION
Candidate genes were PCR amplified using the designed primers (Table 1) initially in parents, and those amplifying, were screened in the F2 population. Briefly, each amplification was carried out in a 10 µl final reaction containing 1X PCR buffer, 2.5 mM MgCl 2 , 0.2 µM dNTPs, 0.2 µM each primer, 0.05 U/µl Taq polymerase and 5 ng/µl DNA. All PCR reactions were carried out on an I-cycler (BIORAD) thermocycler programmed for 4 minutes at 94ºC (initial denaturation), 30 seconds at 94ºC (denaturation), 40 seconds at 56ºC (annealing), 2 minutes at 72ºC (extension) for 34 cycles, and then, 10 minutes at 72ºC (final extension).
POLYMORPHISMS IN PCR PRODUCTS
PCR products were run on 2% agarose gel electrophoresis, and visualized with the GeneSnap Windows XP software (SYNGENE). Products showing different sizes in bp (i.e. polymorphic by insertions/deletions, InDels>20pb) in parents, were amplified directly in the F2 population for mapping analysis. PCR products not showing gel size differences in both parents were converted to CAPS markers by using two approaches. The first one was based on in silico analyses of parental sequences, which were cleaned, edited, and aligned to search for polymorphisms (SNPs or small InDels) using PHRED/PHRAP/CONSED (Gordon et al., 2001) and to predict restriction enzymes recognizing the polymorphic region using CAPS designer (Mueller et al., 2005). The second one was performed by using nine randomly chosen restriction enzymes (EcoRI, EcoRV, HindIII, HaeIII, RsaI, MspI, BglII, DraIy PstI) (Table 1). PCR digested products were visualized on 2% agarose gel.
LOCATION OF CANDIDATE GENES ON THE TOMATO EXPEN 2000 MAP
Linkage analysis was carried out using the MapDisto software for windows (Lorieux, 2007), which uses similar algorithms implemented in MapMaker (Lander et al., 1987). Two-hundred four framework markers were selected from SGN (Mueller et al., 2005), 200 based on location at LOD ≥3 and average distance of 5 cM between markers along the 12 chromosomes of the tomato map and four markers selected from chromosome 11 with LOD≤2 since they served for comparisons with the tomato map developed by (Ashrafi et al., 2009). Candidate genes were located with threshold parameters of LOD≥4, a maximum distance of 30cM, and the Kosambi mapping function (Kosambi, 1944).
RESULTS
MARKER DEVELOPMENT OF CANDIDATE GENES
From 49 primers designed from candidate genes, 33 produced successful PCR amplification products in both parents (66%); of these, two were derived from Alfano et al., 2007, six from Moreno et al., 2009, and 25 from this study. Seven of the 33 (21%) were polymorphic by size and 26 (79%) were monomorphic (Table 1). The CAPS approach based on in silico prediction was useful for three genes (Fig. 1A) in which alignment of good quality sequence (PHRED score >15) had a minimum of 100 base pairs overlap in both parents. The Random CAPS approach was done with nine restriction enzymes on 23 candidate genes of low quality sequence (PHRED score <15), obtaining polymorphisms on eight genes in both parents (Fig. 1B; Table 1). Therefore, marker conversion was performed for 18 of the 49 candidate genes analyzed in this study.
INTEGRATION OF MARKER GENES ON THE TOMATO EXPEN 2000 MAP
To determine the location of the 18 marker genes on the tomato map, each one was PCR amplified in the F2 population and either visualized directly on a 2% agarose gel or digested with the corresponding CAPS enzyme (Table 1). The map was constructed with 204 framework markers, 16 candidate gene markers and either an allele or a duplication of the gene locus Ara (Fig. 2; Table 1). One unigene (U586438) out of the 18 was unlinked. The candidate genes integrated on the map were located on chromosomes 1 (U573196, U579779), 2 (U569177), 3 (Ab), 4 (U580089, Sol, Ext and Asr3), 6 (Exp and Pr), 7 (U586360), 11 (U584666 and Tr8) and 12 (Ara1, Ara2, Sre1b and Pl2). The generated map covered a length of 1083 cM, which is similar to previous tomato maps made from the same species (Grandillo and Tanksley, 1996; Frary et al., 2004; Sharma et al., 2008; Ashrafi et al., 2009).
DISCUSSION
Comparisons with similar marker data obtained by Zhang et al., 2002, Sharma et al., 2008 and Ashrafi et al., 2009, were performed since the authors report a thorough localization of candidate resistance/defense genes and QTLs in tomato. Zhang et al., 2002, constructed a map based on Restriction Fragment Length Polymosphisms (RFLPs) and resistance gene analog (RGAs) markers, Sharma et al., 2008, constructed a map based on RFLPs, ESTs similar to known resistance, defense, signaling, transcription factors and RGAs mainly obtained from Zhang et al., 2002. Whereas, Ashrafi et al., 2009, using an advanced F7 population derived from the same population used by Sharma et al., 2008, constructed a map based on RFLPs, SSRs, CAPS markers and ESTs chosen based on their putative unigene roles in disease resistance and defense-related response.
Furthermore, data obtained in the present map were also compared with the location of other disease and pest resistance QTLs in tomato (Kaloshian et al., 1998; Ammiraju et al., 2003; Brower and St.Clair 2004; Scott et al., 2004 and Davis et al., 2009). Eleven unigenes mapped to chromosomes 1 (U573196, U579779), 4 (Sol, Ext and Asr3), 6 (Pr), 7 (U586360) and 12 (Pl2, Sre1b, Ara1 and Ara2) in regions were no previous resistance markers or QTLs were reported. However, six unigenes were positioned to chromosomal regions where other resistance QTLs, ESTs or RGAs have been placed in tomato. The chromosomal position of these six unigenes as well as other six not necessarily co-localizing with previous QTLs, RGAs or ESTs related to defense but otherwise interesting because their putative role in resistance or growth is described as follows. Unigene U569177 was located on chromosome 2, whereas, Ab (U578305), was placed on chromosome 3. The former putatively functions in intracellular transport and the latter is related to metabolic function (Table 1). Both co-localized within a 1 to 2 cM EST region reported by Sharma et al., 2008 and Ashrafi et al., 2009, with putative metabolic functions related to ripening.
Unigenes U580089, Sol (U573896), Ext (U568616) and Asr3 (U577352) were located on chromosome 4, these unigenes were separated from each other by 2 to 4.5 cM (Fig. 2). U580089 and Sol are predicted to be involved in metabolic processes (Mueller et al., 2005; Table 1). Ext belongs to the extensin gene family, whose members are related to structural cell wall function and play important roles in plant defense (Baumberger et al., 2003; Merkouropoulos and Shirsat, 2003; Wei and Shirast, 2006). Asr3 belongs to the Asr gene family of transcription factors expressed in response to abcisic acid (ABA) under drought stress conditions (Rossi et al., 1996; Frankel et al., 2003). The position of Asr3 agrees with previous studies where three Asr genes were placed at about a 19 cM of CD55, which is located at about 1 cM of the T1050 marker close to theAsr3 mapped in this study (Rossi et al., 1996; Tanskley et al., 1992). Furthermore, the region where U580089 was placed expands an EST related with water/salt stress response described by Ashrafi et al., 2009 (Fig. 2).
Exp (U581296) and Pr (U577360) candidates were located on chromosome 6 (Fig. 2). Exp belongs to the expansin gene super family involved in growth, expansion of cell wall and is downregulated by growth hormones (auxins, gibberelic acid and ethylene; Catalá.et al., 2000; Cosgrove 2000). Pr is related to ISR and functions in response to ethylene. An EST framework marker (cLES1K3) which maps at 1,4 cM of Pr mapped in this study is also a member of the unigene construct (SGN-U577360) of Pr (Mueller et al., 2005), which could be the result of a Pr loci duplication.
Kaloshian et al., 1998; Ammiraju et al., 2003; Sharma et al., 2008 and Ashrafi et al., 2009, located several Meloidoyine spp. (Mi) resistance QTLs (Mi-1, Mi-3 and Mi-9) close to Exp (Fig. 2). Plant originated Exp expression have been associated with a successful parasitic nematode-plant interaction in tomato (Gal et al., 2006; Fudali et al., 2008); therefore, this co-localization suggest Exp as a candidate for Mi-tomato interaction.
U584666 and Tr8 (U585601) were located on chromosome 11. U584666 is related to intracellular transport through vacuoles and Tr8 functions in cellular metabolism as proteinase inhibitor, related to insects attack (Graham et al., 1985). The position of Tr8 agrees with previous reports where it was located near TG400, also used in the present study as a frame marker (Taylor et al., 1993; Tanskley et al., 1992; Fig. 2). In addition, a Fusarium spp. (I) and a Phytophtora infestans (Ib11b) resistance QTL, as well as a Botrytis cinerea susceptible QTL (Fbc11) were located between CT182 and TG400 markers, which span the U584666 and Tr8 region (Scott. et al., 2004; Brower and St.Clair, 2004; Davis et al., 2009; Fig. 2). Moreover, Sharma et al., 2008, located four RGAs and a resistance QTL (I) to Fusarium spp. in the same region where U584666 was placed. Ara (U576250) was located on chromosome 12.
Two PCR amplified fragments named Ara1 and Ara2, were located at 0 cM from each other (>Fig. 2). In addition, a third PCR amplification product (Ara3) was observed only in Ara1 and Ara2 F2 heterozygote individuals (Fig. 1C); this observation might suggest either gene duplication events or different alleles of the same locus. A sequence alignment would be important to clarify this. These observations might agree with the fact that Ara belongs to one of the largest gene family of plants MYB transcription factors (Yanhui et al., 2006).
Based on this positional information and the results obtained by Moreno et al., 2009, we suggest that some genes analyzed in this study may not only be involved in T. koningiopsis Th003 and T. hamatum T382 response, but also, may constitute candidates for pathogen resistance QTLs (for example, Exp for Meloidoyine spp. resistance; U584666 and Tr8 for Fusarium spp., Phytophtora infestans and Botrytis cinerea resistance or susceptibility). However, these hypotheses must be verified by further studies (e.g. QTL mapping, association to resistance, mutant complementation, gene silencing, among others). Further research on this topic should also consider information derived from the recently released tomato genome sequence draft (Mueller et al., 2005). This sequence is derived from BAC clones anchored to the genetic map of tomato used in the present study. This is a powerful tool to identify several candidates through the whole tomato genome.
Genetic linkage mapping analysis of the differentially expressed candidate genes for Th003 and T382 response in tomato coupled with QTL analyses for specific growth and resistance phenotypes should be assessed. Preliminary measurements of phenotypic differences in response to Th003 in parents Sl and Sp of the TOMATO EXPEN 2000 population show differences in root volume of Th003 treated vs. non-treated plants, suggesting that is possible to evaluate growth promotion or ISR in response to Th003 to further QTL analysis in this population. This will foster future assisted selection for induced resistance or growth promotion triggered by native biocontrol agents in tomato breeding programs.
ACKNOWLEDGMENTS
The authors want to acknowledge Dr. Steven D. Tanksley for providing a copy of the TOMATO EXPEN 2000 population. To Ligia Suescún for assistance in the in vitro establishment of this population. To Oscar Bedoya for assistance in primer design. To Carlos Moreno for advice related to Th003. To Drs. Carolina González, Camilo López and Leonardo Mariño for critical reading of the manuscript. Funding was provided by CORPOICA and COLCIENCIAS.
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