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Molecular Selection Of Tomato And Pepper Breeding Lines Possessing Resistance Alleles Against Tobamoviruses

Molecular Selection Of Tomato And Pepper Breeding Lines Possessing Resistance Alleles Against... DOI: 10.1515/agri-2015-0008 SHORT COMMUNICATION MOLECULAR SELECTION OF TOMATO AND PEPPER BREEDING LINES POSSESSING RESISTANCE ALLELES AGAINST TOBAMOVIRUSES MARTINA HUDCOVICOVÁ 1*, ERIKA KORBELOVÁ 2, SVETLANA SLIKOVÁ 1, LENKA KLCOVÁ 1, DANIEL MIHÁLIK 1, JÁN KRAIC 1,3 National Agricultural and Food Center ­ Research Institute of Plant Production Piesany 2 Zelseed spol. s.r.o., Horná Potô 3 University of SS. Cyril and Methodius in Trnava HUDCOVICOVÁ, M. KORBELOVÁ, E. SLIKOVÁ, S. KLCOVÁ, L. MIHÁLIK, D. KRAIC, J.: Molecular selection of tomato and pepper breeding lines possessing resistance alleles against tobamoviruses. Agriculture (Ponohospodárstvo), vol. 61, 2015, no. 1, pp. 33­37. Molecular selection among newly created tomato and pepper lines was applied for identification of lines possessing DNA markers linked with the resistant loci against tobamoviruses ToMV, TMV, PaMMV, and PMMoV. Only four tomato lines among 184 had DNA marker linked with the resistant allele Tm-2 conferring homozygosity at this locus. Resistance of these four lines was tested and confirmed also by virological testing by inoculation with TMV strain 0. Simultaneously tested lines heterozygous at this locus expressed full or unbalanced resistance. Fifty-eight out of 62 tested pepper lines had only marker linked to susceptible allele of the locus L. Three lines derived from tobamovirus resistant pepper cultivars Brill and Brilliant expressed marker linked to resistant allele L 3 , and only one line derived from resistant cultivar Hurricane possessed both markers. Four selected pepper lines declared resistance also after artificial inoculation with the TMV P 0 pathotype. Molecular selection, both in tomato and pepper breeding lines, may be useful in breeding programs directed to tobamovirus resistance. Key words: tomato, pepper, molecular marker, tobamovirus, Tm-2, tm-2, L 3 gene Selection of advanced lines within the newly created breeding materials is a crucial operation in the plant breeding programs. Individuals possessing better resistance against different biotic and abiotic stresses can be efficiently selected by molecular markers linked with relevant loci. Also transfer of resistance genes from donor genotypes into elite cultivars is more effective using the marker assisted selection (MAS). This is particularly useful for incorporation of disease resistance genes that are dif- ficult to detect in the same genotype and accumulate by gene pyramiding (Ordon et al. 1999). Molecular markers have been already widely used in genetic mapping and MAS related to resistance against different diseases in tomato and pepper (Foolad & Sharma 2005; Yang et al. 2012). Tobamoviruses such as pepper mild mottle virus (PMMoV), paprika mild mottle virus (PaMMV), tobacco mosaic virus (TMV), and tomato mosaic virus (ToMV) are easily transmitted between plants mechanically or in seeds. Mgr. Martina Hudcovicová, PhD. (*Corresponding author), Ing. Svetlana Sliková PhD., Mgr. Lenka Klcová, Mgr. Daniel Mihálik PhD., Doc. RNDr. Ján Kraic, PhD., National Agricultural and Food Center ­ Research Institute of Plant Production Piesany, Bratislavská cesta 122, 921 68 Piesany, Slovak Republic. E-mail: hudcovicova@vurv.sk Ing. Erika Korbelová, Zelseed spol. s.r.o., Horná Potô 1269, 930 36 Horná Potô, Slovak Republic Doc. RNDr. Ján Kraic, PhD., University of SS. Cyril and Methodius in Trnava, Faculty of Natural Sciences, Nám. J. Herdu 2, 917 00 Trnava, Slovak Republic These viruses cause serious damages with systemic mosaic symptoms associated with yield and quality losses. The development of improved resistant genotypes is challenging especially if resistance genes originate from wild relatives. Phenotypic selection of resistant lines after pathogen inoculation is time-consuming, affected by environmental factors and plant developmental stage, moreover depends on availability of virus pathotypes. Therefore DNA markers in genetic linkage with resistance genes are very useful tools for effective breeding. Response of commercial tomato (Lycopersicon esculentum L.) cultivars to ToMV infections is influenced by presence or absence of resistance genes Tm-1, Tm-2, and Tm-22 (Pelham 1966; Hall 1980) introgressed from the wild tomato species Lycopersiocon hirsutum f. hirsutum (Tm-1) and Lycopersiocon peruvianum L. (Tm-2, Tm-22), respectively. The gene Tm-1 provides resistance to ToMV strains 0 and 2 (Ohmori et al. 1996; Levesque et al. 1990). Genes Tm-2 and Tm-22 are considered to be allelic and the allele Tm-2 conferred resistance to ToMV strains 0 and 1, whereas allele Tm-22 resistance to ToMV strains 0, 1 and 2 (Hall 1989; Lanfermeijer et al. 2003). Three alleles Tm-2, Tm-22, and tm-2 (sensitive) have been cloned and sequenced (GenBank accessions AF536199, AF536200, AF536201) and cleaved amplified polymorphic sequences (CAPS) markers were developed to distinguish these alleles in tomato (Lanfermeijer et al. 2005). Shi et al. (2011) identified four allele-specific PCR-based markers for Tm-2, Tm-22, and two for the susceptible allele tm-2 as well as three allele-derived CAPS markers for distinguishing between them. Other three specific SNP markers developed for the locus Tm-2 provide advanced tools for tomato breeders to detect Tm-2 and Tm-22 resistance alleles. Genetic resistance against Tobamovirus spp. in pepper (genus Capsicum) provides the L locus. Five alleles at this locus L were revealed: L0 ­ related to susceptibility; L1 resistance to P0 pathotypes of TMV, L2 resistance to P0 and P1 pathotypes of PaMMV that overcome L1 resistance; L3 resistance to P0, P1, and P1,2 pathotypes of PMMoV that overcome L2 resistance; and L4 resistance to P0, P1, P1,2, and P1,2,3 pathotypes of PMMoV that overcome L3 resistance (Tomita et al. 2011). Also other alleles as L1a, L1c, and L2b with different temperature sensitivity were identified (Tomita et al. 2011). Recently pathotypes P1,2,3,4 of the PMMoV braking the L4 resistance were identified (Antignus et al. 2008; Genda et al. 2007). Alleles L1, L2, L3, and L4 were identified in Capsicum annuum L. (e.g. cv. Bruinsma Wonder and Verbeterde Glas), Capsicum frutescens L. (cv. Tabasco), Capsicum chinense Jacq. (PI159236), and Capsicum chacoense Hunz. (PI260429), respectively. Different molecular markers linked to the L locus were developed, e.g. RAPDs (Sugita et al. 2004), AFLPs (Tomita et al. 2008), SCARs converted from RAPD and AFLP markers (Kim et al. 2008; Matsunaga et al. 2003), and SNPs (Yang et al. 2009; Yang et al. 2011). The goal of this study was to test the applicability of the molecular selection by DNA markers linked to alleles of resistance against tobamoviruses ToMV, TMV, PaMMV, and PMMoV within tomato and pepper breeding lines. Altogether 184 tomato and 62 pepper lines developed within the breeding programs of the company Zelseed Ltd. (Horná Potô, Slovakia) were screened with the aim to select lines possessing the desired resistance alleles. Total plant DNA was isolated from young leaves by the DNeasy Plant Mini kit (Qiagen). Polymerase chain reactions (PCR) Figure 1. Analysis of the amplified 393 bp DNA fragment linked to the susceptible allele tm-2 in tomato (M size marker, 1 negative control, 2 ­ cv. Monalbo, 3 ­ cv. Moperou, 4-21 ­ breeding lines) were carried out using specific primers. Analysed tomato and pepper lines were tested simultaneously by artificial inoculation with relevant virus isolates ToMV pathotype P0 and TMV pathotype P0, both obtained from the Netherlands Inspection Service for Horticulture (Naktiunbouw, Roelofarendsveen, The Netherlands). Symptoms manifested on the pepper and tomato plants were evaluated according to official guidelines of the European Union Community Plant Variety Office (CPVO) Protocol for Distinctness Uniformity and Stability Tests for Capsicum annuum L. (Technical Protocol 076) and Protocol for Distinctness Uniformity and Stability Tests for Lycopersicon esculentum Mill. (Technical Protocol 044/3). Two tomato cultivars Moperou and Monalbo were used as the control genotypes carrying resistant allele Tm-2 and susceptible allele tm-2, respectively. Presence or absence of the susceptible allele tm-2 in all tomato lines was determined by dominant allele-specific PCR markers amplified using two primer pairs Tm2S-f1/Tm2S-r1 and Tm2S-f2/ Tm2S-r2, designed based on the nucleotide sequences of the Genbank accession AF536199 (Shi et al. 2011). These primers amplified DNA fragment in genotypes containing the allele tm-2 in homozygous or heterozygous status. Presence or absence of the resistant allele Tm-2 in 184 tomato lines were determined by the dominant allele-specific PCR marker using primer pair Tm2R-f1c/Tm2R-r3 designed from the sequences of the GenBank accessions AF536200 and AF536201 (Shi et al. 2011). Parameters of the PCRs for all three primer pairs were set according to Shi et al. (2011). Amplified products were separated by gel electrophoresis in 1.5% agarose gels and stained with ethidium bromide. These dominant allele-specific markers are not able to distinguish homozygous and heterozygous genotypes at the locus Tm-2 individually, but combination of these markers can do it. Similar to Shi et al. (2011), three types of fragments were amplified. The 393 bp fragment relates to the susceptible allele tm-2 (amplified by primers Tm2S-f1/Tm2S-r1, Figure 1), 284 bp fragment for the susceptible allele tm-2 (using primers Tm2S-f2/Tm2S-r2), and 444 bp fragment for the resistant allele Tm-2 (using primers Tm2R-f1c/ Tm2R-r3, Figure 2). Altogether 133 tomato lines out of 184 analysed possessed only fragment for the susceptible allele tm-2 (homozygotes) and 33 lines both fragments for susceptible and resistant alleles (heterozygotes). The remaining 4 lines expressed only fragment related to the resistant allele Tm-2 (homozygotes) and they were also tested by artificial inoculation with TMV strain 0. All of them showed full resistance (9 points), while heterozygotes showed full or unbalanced resistance. These lines were selected as the most promising for further exploitation in tomato virus resistance breeding program. The pepper cultivars Hurricane and Century were used as positive controls carrying resistance genes L3 and L4, respectively. Others two Piperade and Yolo Wonder were used as negative controls. Presence or absence of the L3 gene in 62 pepper lines was determined using the codominant SCAR markers PMFR11269 and PMFR11283 derived from RAPD markers E18272 and E18286 (Sugita et al. 2004). Also PCR reactions with primer pair PMF1 and PMR1 were according to Sugita et al. (2004). Amplified products were separated in 6% polyacrylamide gel and stained by the silver staining method (Bassam et al. 1991). Similar to Sugita et al. (2004) two types Figure 2. Analysis of the amplified 444 bp DNA fragment linked to the resistant allele Tm-2 in tomato (M size marker, 1 negative control, 2 ­ cv. Monalbo, 3 ­ cv. Moperou, 4-21 ­ breeding lines) of fragments were amplified ­ the 283 bp fragment (PMFR11283) corresponding to the susceptible allele and 269 bp fragment (PMFR11269) to the resistant allele (Figure 3). Fifty-eight from 62 analysed pepper lines expressed only fragment for the susceptible allele. Three genotypes expressed only the fragment for the resistant allele. All were derived from the Hungarian cultivars Brill and Brillant, both declared as resistant to tobamoviruses. Only one line possessed both fragments. It was derived from the Hungarian cultivar Hurricane declared as resistant to tobamoviruses. Above mentioned four lines were tested by artificial inoculation with the TMV pathotype P0 and all confirmed resistance. Molecular selection process thus enabled to select for a number of resistant materials suitable for subsequent crossing with elite pepper cultivars. However, Sugita et al. (2004) found out that the fragment PMFR11269 was not detected in some genotypes regardless of the presence of the L3gene. The reason can be either the presence of different sources of the L3gene or recombination between PMFR11269 and the L3gene. Presence or absence of the L4 gene in all screened pepper lines were determined by the dominant SCAR marker L4SC340 derived from the AFLP marker L4-b closely linked to the locus L4 (Kim et al. 2008). PCR reactions with primers L4SC340F and L4SC340R were similar to Kim et al. (2008), nevertheless optimized by the mix FailSafe PCR PreMix Selection kit (Epicentre). The amplified products were separated by electrophoresis on a 6% polyacrylamide gel using the silver staining method (Bassam et al. 1991). Unfortunately this marker did not show any polymorphism in our group of pepper lines. The reason can be similar as described Kim et al. (2008), that in pepper differentials for the Tobamovirus pathotype this marker showed band for plants harbouring loci L+, L2, L3 and L4 and did not show band only for the L1 locus. Thus the applicability of this marker depends on the genetic background of tested breeding material. Obtained results showed that DNA markers used for detection of resistance genes against tobamoviruses in pepper and tomato can be successfully used in breeding programs. They enabled to identify genotypes which could be used as donors of the genes conferring effective resistance against tobamoviruses. These donors of resistance genes could be transferred into elite pepper or tomato cultivars and DNA markers can be used for screening within hundreds of off-springs in selection process. Acknowledgement: This work is supported by OP Research and Development: Transfer of effec- Figure 3. Analysis of two DNA fragments amplified by the primer pair PMF1 and PMR1 in pepper 283 bp fragment linked to the susceptible allele and 269 bp fragment linked to the resistant allele (M 1 ­ marker 1, M 2 ­ marker 2, 1-11 ­ pepper lines, 12 ­ cv. Hurricane, 13 ­ cv. Yolo Wonder, 14 ­ cv. Piperade) tive procedures for selection and identification of plants in breeding (ITMS 26220220142) from European Regional Development Fund. OHMORI, T. ­ MURATA, M. MOTOYOSHI, F. 1996. Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato. In Theoretical and Applied Genetics, vol. 92, no. 2, pp. 151156. DOI: 10.1007/BF00223369. ORDON, F. SCHIEMANN, A. PELLIO, B. DAUCK, V. BAUER, E. STRENG, S. FRIEDT, W. GRANER, A. 1999. Application of molecular markers in breeding for resistance to the barley yellow mosaic virus complex. In Journal of Plant Diseases and Protection, vol. 106, no. 3, pp. 256264. DOI: ISSN/ ISBN: 0340-8159. PELHAM, J. 1966. Resistance in tomato to tobacco mosaic virus. In Euphytica, vol. 15, no.2, pp. 258267. DOI: 10.1007/BF00022331. SHI, A. VIERLING, R. GRAZZINI, R. CHEN, P. CATON, H. PANTHEE, D. 2011. Molecular markers for Tm-2 alleles of tomato mosaic virus resistance in tomato. In American Journal of Plant Sciences, vol. 2, no. 2, pp. 180189. DOI: 10.4236/ajps.2011.22020. SUGITA, T. ­ YAMAGUCHI, K. ­ SUGIMURA, Y. ­ NAGATA, R. ­ YUJI, K. ­ KINISHITA, T. ­ TODOROKI, A. 2004. Development of SCAR markers linked to L 3 gene in Capsicum. In Breeding Science, vol. 54, no. 2, pp. 111­115. DOI: 10.1270/jsbbs.54.111. TOMITA, R. MURAI, J. MIURA, Y. ISHIHARA, H. LIU, S. KUBOTERA, Y. HONDA, A. HATTA, R. KURODA, T. HAMADA, H. SAKAMOTO, M. MUNEMURA, I. NUNOMURA, O. ISHIKAWA, K. GENDA, Y. KAWASAKI, S. SUZUKI, K. MEKSEM, K. KOBAYASHI, K. 2008. Fine mapping and DNA fiber FISH analysis locates the tobamovirus resistance gene L 3 of Capsicum chinense in a 400-kb region of R-like genes cluster embedded in highly repetitive sequences. In Theoretical and Applied Genetics, vol. 117, no. 7, pp. 11071118. DOI: 10.1007/s00122-008-0848-6. TOMITA, R. ­ SEKINE, K.T. ­ MIZUMOTO, H. ­ SAKAMOTO, M. ­ MURAI, J. ­ KIBA, A. ­ HIKICHI, Y. ­ SUZUKI, K. ­ KOBAYASHI, K. 2011. Genetic basis for the hierarchical interaction between Tobamovirus spp. and L resistance gene alleles from different pepper species. In Molecular Plant-Microbe Interactions, vol. 24, no. 1, pp. 108­117. DOI: 10.1094/ MPMI-06-10-0127. YANG, H.B. ­ LIU, W.Y. ­ KANG, W.H. ­ JAHN, M. ­ KANG, B.C. 2009. Development of SNP markers linked to the L locus in Capsicum spp. by a comparative genetic analysis. In Molecular Breeding, vol. 24, no. 4, pp. 433­446. DOI: 10.1007/s11032-009-93049. YANG, H.B. ­ LIU, W.Y. ­ KANG, W.H. ­ KIM, J.H. ­ CHO, H.J. ­ YOO, J.H. ­ KANG, B.C. 2012. Development and validation of L allele-specific markers in Capsicum. In Molecular Breeding, vol. 30, no. 2, pp. 819­829. DOI 10.1007/s11032-011-9666-7. Received: March 3, 2015 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Agriculture de Gruyter

Molecular Selection Of Tomato And Pepper Breeding Lines Possessing Resistance Alleles Against Tobamoviruses

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DOI: 10.1515/agri-2015-0008 SHORT COMMUNICATION MOLECULAR SELECTION OF TOMATO AND PEPPER BREEDING LINES POSSESSING RESISTANCE ALLELES AGAINST TOBAMOVIRUSES MARTINA HUDCOVICOVÁ 1*, ERIKA KORBELOVÁ 2, SVETLANA SLIKOVÁ 1, LENKA KLCOVÁ 1, DANIEL MIHÁLIK 1, JÁN KRAIC 1,3 National Agricultural and Food Center ­ Research Institute of Plant Production Piesany 2 Zelseed spol. s.r.o., Horná Potô 3 University of SS. Cyril and Methodius in Trnava HUDCOVICOVÁ, M. KORBELOVÁ, E. SLIKOVÁ, S. KLCOVÁ, L. MIHÁLIK, D. KRAIC, J.: Molecular selection of tomato and pepper breeding lines possessing resistance alleles against tobamoviruses. Agriculture (Ponohospodárstvo), vol. 61, 2015, no. 1, pp. 33­37. Molecular selection among newly created tomato and pepper lines was applied for identification of lines possessing DNA markers linked with the resistant loci against tobamoviruses ToMV, TMV, PaMMV, and PMMoV. Only four tomato lines among 184 had DNA marker linked with the resistant allele Tm-2 conferring homozygosity at this locus. Resistance of these four lines was tested and confirmed also by virological testing by inoculation with TMV strain 0. Simultaneously tested lines heterozygous at this locus expressed full or unbalanced resistance. Fifty-eight out of 62 tested pepper lines had only marker linked to susceptible allele of the locus L. Three lines derived from tobamovirus resistant pepper cultivars Brill and Brilliant expressed marker linked to resistant allele L 3 , and only one line derived from resistant cultivar Hurricane possessed both markers. Four selected pepper lines declared resistance also after artificial inoculation with the TMV P 0 pathotype. Molecular selection, both in tomato and pepper breeding lines, may be useful in breeding programs directed to tobamovirus resistance. Key words: tomato, pepper, molecular marker, tobamovirus, Tm-2, tm-2, L 3 gene Selection of advanced lines within the newly created breeding materials is a crucial operation in the plant breeding programs. Individuals possessing better resistance against different biotic and abiotic stresses can be efficiently selected by molecular markers linked with relevant loci. Also transfer of resistance genes from donor genotypes into elite cultivars is more effective using the marker assisted selection (MAS). This is particularly useful for incorporation of disease resistance genes that are dif- ficult to detect in the same genotype and accumulate by gene pyramiding (Ordon et al. 1999). Molecular markers have been already widely used in genetic mapping and MAS related to resistance against different diseases in tomato and pepper (Foolad & Sharma 2005; Yang et al. 2012). Tobamoviruses such as pepper mild mottle virus (PMMoV), paprika mild mottle virus (PaMMV), tobacco mosaic virus (TMV), and tomato mosaic virus (ToMV) are easily transmitted between plants mechanically or in seeds. Mgr. Martina Hudcovicová, PhD. (*Corresponding author), Ing. Svetlana Sliková PhD., Mgr. Lenka Klcová, Mgr. Daniel Mihálik PhD., Doc. RNDr. Ján Kraic, PhD., National Agricultural and Food Center ­ Research Institute of Plant Production Piesany, Bratislavská cesta 122, 921 68 Piesany, Slovak Republic. E-mail: hudcovicova@vurv.sk Ing. Erika Korbelová, Zelseed spol. s.r.o., Horná Potô 1269, 930 36 Horná Potô, Slovak Republic Doc. RNDr. Ján Kraic, PhD., University of SS. Cyril and Methodius in Trnava, Faculty of Natural Sciences, Nám. J. Herdu 2, 917 00 Trnava, Slovak Republic These viruses cause serious damages with systemic mosaic symptoms associated with yield and quality losses. The development of improved resistant genotypes is challenging especially if resistance genes originate from wild relatives. Phenotypic selection of resistant lines after pathogen inoculation is time-consuming, affected by environmental factors and plant developmental stage, moreover depends on availability of virus pathotypes. Therefore DNA markers in genetic linkage with resistance genes are very useful tools for effective breeding. Response of commercial tomato (Lycopersicon esculentum L.) cultivars to ToMV infections is influenced by presence or absence of resistance genes Tm-1, Tm-2, and Tm-22 (Pelham 1966; Hall 1980) introgressed from the wild tomato species Lycopersiocon hirsutum f. hirsutum (Tm-1) and Lycopersiocon peruvianum L. (Tm-2, Tm-22), respectively. The gene Tm-1 provides resistance to ToMV strains 0 and 2 (Ohmori et al. 1996; Levesque et al. 1990). Genes Tm-2 and Tm-22 are considered to be allelic and the allele Tm-2 conferred resistance to ToMV strains 0 and 1, whereas allele Tm-22 resistance to ToMV strains 0, 1 and 2 (Hall 1989; Lanfermeijer et al. 2003). Three alleles Tm-2, Tm-22, and tm-2 (sensitive) have been cloned and sequenced (GenBank accessions AF536199, AF536200, AF536201) and cleaved amplified polymorphic sequences (CAPS) markers were developed to distinguish these alleles in tomato (Lanfermeijer et al. 2005). Shi et al. (2011) identified four allele-specific PCR-based markers for Tm-2, Tm-22, and two for the susceptible allele tm-2 as well as three allele-derived CAPS markers for distinguishing between them. Other three specific SNP markers developed for the locus Tm-2 provide advanced tools for tomato breeders to detect Tm-2 and Tm-22 resistance alleles. Genetic resistance against Tobamovirus spp. in pepper (genus Capsicum) provides the L locus. Five alleles at this locus L were revealed: L0 ­ related to susceptibility; L1 resistance to P0 pathotypes of TMV, L2 resistance to P0 and P1 pathotypes of PaMMV that overcome L1 resistance; L3 resistance to P0, P1, and P1,2 pathotypes of PMMoV that overcome L2 resistance; and L4 resistance to P0, P1, P1,2, and P1,2,3 pathotypes of PMMoV that overcome L3 resistance (Tomita et al. 2011). Also other alleles as L1a, L1c, and L2b with different temperature sensitivity were identified (Tomita et al. 2011). Recently pathotypes P1,2,3,4 of the PMMoV braking the L4 resistance were identified (Antignus et al. 2008; Genda et al. 2007). Alleles L1, L2, L3, and L4 were identified in Capsicum annuum L. (e.g. cv. Bruinsma Wonder and Verbeterde Glas), Capsicum frutescens L. (cv. Tabasco), Capsicum chinense Jacq. (PI159236), and Capsicum chacoense Hunz. (PI260429), respectively. Different molecular markers linked to the L locus were developed, e.g. RAPDs (Sugita et al. 2004), AFLPs (Tomita et al. 2008), SCARs converted from RAPD and AFLP markers (Kim et al. 2008; Matsunaga et al. 2003), and SNPs (Yang et al. 2009; Yang et al. 2011). The goal of this study was to test the applicability of the molecular selection by DNA markers linked to alleles of resistance against tobamoviruses ToMV, TMV, PaMMV, and PMMoV within tomato and pepper breeding lines. Altogether 184 tomato and 62 pepper lines developed within the breeding programs of the company Zelseed Ltd. (Horná Potô, Slovakia) were screened with the aim to select lines possessing the desired resistance alleles. Total plant DNA was isolated from young leaves by the DNeasy Plant Mini kit (Qiagen). Polymerase chain reactions (PCR) Figure 1. Analysis of the amplified 393 bp DNA fragment linked to the susceptible allele tm-2 in tomato (M size marker, 1 negative control, 2 ­ cv. Monalbo, 3 ­ cv. Moperou, 4-21 ­ breeding lines) were carried out using specific primers. Analysed tomato and pepper lines were tested simultaneously by artificial inoculation with relevant virus isolates ToMV pathotype P0 and TMV pathotype P0, both obtained from the Netherlands Inspection Service for Horticulture (Naktiunbouw, Roelofarendsveen, The Netherlands). Symptoms manifested on the pepper and tomato plants were evaluated according to official guidelines of the European Union Community Plant Variety Office (CPVO) Protocol for Distinctness Uniformity and Stability Tests for Capsicum annuum L. (Technical Protocol 076) and Protocol for Distinctness Uniformity and Stability Tests for Lycopersicon esculentum Mill. (Technical Protocol 044/3). Two tomato cultivars Moperou and Monalbo were used as the control genotypes carrying resistant allele Tm-2 and susceptible allele tm-2, respectively. Presence or absence of the susceptible allele tm-2 in all tomato lines was determined by dominant allele-specific PCR markers amplified using two primer pairs Tm2S-f1/Tm2S-r1 and Tm2S-f2/ Tm2S-r2, designed based on the nucleotide sequences of the Genbank accession AF536199 (Shi et al. 2011). These primers amplified DNA fragment in genotypes containing the allele tm-2 in homozygous or heterozygous status. Presence or absence of the resistant allele Tm-2 in 184 tomato lines were determined by the dominant allele-specific PCR marker using primer pair Tm2R-f1c/Tm2R-r3 designed from the sequences of the GenBank accessions AF536200 and AF536201 (Shi et al. 2011). Parameters of the PCRs for all three primer pairs were set according to Shi et al. (2011). Amplified products were separated by gel electrophoresis in 1.5% agarose gels and stained with ethidium bromide. These dominant allele-specific markers are not able to distinguish homozygous and heterozygous genotypes at the locus Tm-2 individually, but combination of these markers can do it. Similar to Shi et al. (2011), three types of fragments were amplified. The 393 bp fragment relates to the susceptible allele tm-2 (amplified by primers Tm2S-f1/Tm2S-r1, Figure 1), 284 bp fragment for the susceptible allele tm-2 (using primers Tm2S-f2/Tm2S-r2), and 444 bp fragment for the resistant allele Tm-2 (using primers Tm2R-f1c/ Tm2R-r3, Figure 2). Altogether 133 tomato lines out of 184 analysed possessed only fragment for the susceptible allele tm-2 (homozygotes) and 33 lines both fragments for susceptible and resistant alleles (heterozygotes). The remaining 4 lines expressed only fragment related to the resistant allele Tm-2 (homozygotes) and they were also tested by artificial inoculation with TMV strain 0. All of them showed full resistance (9 points), while heterozygotes showed full or unbalanced resistance. These lines were selected as the most promising for further exploitation in tomato virus resistance breeding program. The pepper cultivars Hurricane and Century were used as positive controls carrying resistance genes L3 and L4, respectively. Others two Piperade and Yolo Wonder were used as negative controls. Presence or absence of the L3 gene in 62 pepper lines was determined using the codominant SCAR markers PMFR11269 and PMFR11283 derived from RAPD markers E18272 and E18286 (Sugita et al. 2004). Also PCR reactions with primer pair PMF1 and PMR1 were according to Sugita et al. (2004). Amplified products were separated in 6% polyacrylamide gel and stained by the silver staining method (Bassam et al. 1991). Similar to Sugita et al. (2004) two types Figure 2. Analysis of the amplified 444 bp DNA fragment linked to the resistant allele Tm-2 in tomato (M size marker, 1 negative control, 2 ­ cv. Monalbo, 3 ­ cv. Moperou, 4-21 ­ breeding lines) of fragments were amplified ­ the 283 bp fragment (PMFR11283) corresponding to the susceptible allele and 269 bp fragment (PMFR11269) to the resistant allele (Figure 3). Fifty-eight from 62 analysed pepper lines expressed only fragment for the susceptible allele. Three genotypes expressed only the fragment for the resistant allele. All were derived from the Hungarian cultivars Brill and Brillant, both declared as resistant to tobamoviruses. Only one line possessed both fragments. It was derived from the Hungarian cultivar Hurricane declared as resistant to tobamoviruses. Above mentioned four lines were tested by artificial inoculation with the TMV pathotype P0 and all confirmed resistance. Molecular selection process thus enabled to select for a number of resistant materials suitable for subsequent crossing with elite pepper cultivars. However, Sugita et al. (2004) found out that the fragment PMFR11269 was not detected in some genotypes regardless of the presence of the L3gene. The reason can be either the presence of different sources of the L3gene or recombination between PMFR11269 and the L3gene. Presence or absence of the L4 gene in all screened pepper lines were determined by the dominant SCAR marker L4SC340 derived from the AFLP marker L4-b closely linked to the locus L4 (Kim et al. 2008). PCR reactions with primers L4SC340F and L4SC340R were similar to Kim et al. (2008), nevertheless optimized by the mix FailSafe PCR PreMix Selection kit (Epicentre). The amplified products were separated by electrophoresis on a 6% polyacrylamide gel using the silver staining method (Bassam et al. 1991). Unfortunately this marker did not show any polymorphism in our group of pepper lines. The reason can be similar as described Kim et al. (2008), that in pepper differentials for the Tobamovirus pathotype this marker showed band for plants harbouring loci L+, L2, L3 and L4 and did not show band only for the L1 locus. Thus the applicability of this marker depends on the genetic background of tested breeding material. Obtained results showed that DNA markers used for detection of resistance genes against tobamoviruses in pepper and tomato can be successfully used in breeding programs. They enabled to identify genotypes which could be used as donors of the genes conferring effective resistance against tobamoviruses. These donors of resistance genes could be transferred into elite pepper or tomato cultivars and DNA markers can be used for screening within hundreds of off-springs in selection process. Acknowledgement: This work is supported by OP Research and Development: Transfer of effec- Figure 3. Analysis of two DNA fragments amplified by the primer pair PMF1 and PMR1 in pepper 283 bp fragment linked to the susceptible allele and 269 bp fragment linked to the resistant allele (M 1 ­ marker 1, M 2 ­ marker 2, 1-11 ­ pepper lines, 12 ­ cv. Hurricane, 13 ­ cv. Yolo Wonder, 14 ­ cv. Piperade) tive procedures for selection and identification of plants in breeding (ITMS 26220220142) from European Regional Development Fund. OHMORI, T. ­ MURATA, M. MOTOYOSHI, F. 1996. Molecular characterization of RAPD and SCAR markers linked to the Tm-1 locus in tomato. In Theoretical and Applied Genetics, vol. 92, no. 2, pp. 151156. DOI: 10.1007/BF00223369. ORDON, F. SCHIEMANN, A. PELLIO, B. DAUCK, V. BAUER, E. STRENG, S. FRIEDT, W. GRANER, A. 1999. Application of molecular markers in breeding for resistance to the barley yellow mosaic virus complex. In Journal of Plant Diseases and Protection, vol. 106, no. 3, pp. 256264. DOI: ISSN/ ISBN: 0340-8159. PELHAM, J. 1966. Resistance in tomato to tobacco mosaic virus. In Euphytica, vol. 15, no.2, pp. 258267. DOI: 10.1007/BF00022331. SHI, A. VIERLING, R. GRAZZINI, R. CHEN, P. CATON, H. PANTHEE, D. 2011. Molecular markers for Tm-2 alleles of tomato mosaic virus resistance in tomato. In American Journal of Plant Sciences, vol. 2, no. 2, pp. 180189. DOI: 10.4236/ajps.2011.22020. SUGITA, T. ­ YAMAGUCHI, K. ­ SUGIMURA, Y. ­ NAGATA, R. ­ YUJI, K. ­ KINISHITA, T. ­ TODOROKI, A. 2004. Development of SCAR markers linked to L 3 gene in Capsicum. In Breeding Science, vol. 54, no. 2, pp. 111­115. DOI: 10.1270/jsbbs.54.111. TOMITA, R. MURAI, J. MIURA, Y. ISHIHARA, H. LIU, S. KUBOTERA, Y. HONDA, A. HATTA, R. KURODA, T. HAMADA, H. SAKAMOTO, M. MUNEMURA, I. NUNOMURA, O. ISHIKAWA, K. GENDA, Y. KAWASAKI, S. SUZUKI, K. MEKSEM, K. KOBAYASHI, K. 2008. Fine mapping and DNA fiber FISH analysis locates the tobamovirus resistance gene L 3 of Capsicum chinense in a 400-kb region of R-like genes cluster embedded in highly repetitive sequences. In Theoretical and Applied Genetics, vol. 117, no. 7, pp. 11071118. DOI: 10.1007/s00122-008-0848-6. TOMITA, R. ­ SEKINE, K.T. ­ MIZUMOTO, H. ­ SAKAMOTO, M. ­ MURAI, J. ­ KIBA, A. ­ HIKICHI, Y. ­ SUZUKI, K. ­ KOBAYASHI, K. 2011. Genetic basis for the hierarchical interaction between Tobamovirus spp. and L resistance gene alleles from different pepper species. In Molecular Plant-Microbe Interactions, vol. 24, no. 1, pp. 108­117. DOI: 10.1094/ MPMI-06-10-0127. YANG, H.B. ­ LIU, W.Y. ­ KANG, W.H. ­ JAHN, M. ­ KANG, B.C. 2009. Development of SNP markers linked to the L locus in Capsicum spp. by a comparative genetic analysis. In Molecular Breeding, vol. 24, no. 4, pp. 433­446. DOI: 10.1007/s11032-009-93049. YANG, H.B. ­ LIU, W.Y. ­ KANG, W.H. ­ KIM, J.H. ­ CHO, H.J. ­ YOO, J.H. ­ KANG, B.C. 2012. Development and validation of L allele-specific markers in Capsicum. In Molecular Breeding, vol. 30, no. 2, pp. 819­829. DOI 10.1007/s11032-011-9666-7. Received: March 3, 2015

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Published: Mar 1, 2015

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