Marker-assisted Selection for Combining Resistance to Bacterial Spot and Bacterial Speck in Tomato

The lack of resistance to bacterial diseases increases both the fi nancial cost and environmental impact of tomato (Lycopersicon esculentum Mill.) production while reducing yield and quality. Because several bacterial diseases can be present in the same fi eld, developing varieties with resistance to multiple diseases is a desirable goal. Bacterial spot (caused by four Xanthomonas Dowson species) and bacterial speck (caused by Pseudomonas syringae pv. tomato Young, Dye and Wilkie) are two economically important diseases of tomato with a worldwide distribution. The resistance gene Pto confers a hypersensitive response (HR) to race 0 strains of the bacterial speck pathogen. The locus Rx3 explains up to 41% of the variation for resistance to bacterial spot race T1 in fi eld trials, and is associated with HR following infi ltration. Both Pto and Rx3 are linked in repulsion phase on chromosome 5. We made a cross between two elite breeding lines, Ohio 981205 carrying Pto and Ohio 9834 carrying Rx3, to develop an F2 population and subsequent inbred generations. Marker-assisted selection (MAS) was applied to the F2 progeny and to F2:3 families in order to select for coupling-phase resistance. Thirteen homozygous progeny from 419 F2 plants and 20 homozygous families from 3716 F3 plants were obtained. Resistance was confi rmed in all selected families based on HR in greenhouse screens using bacterial speck race 0 and bacterial spot race T1 isolates. Resistance to bacterial spot race T1 was confi rmed in the fi eld for 33 of the selected families. All selected families were also resistant to bacterial speck in the fi eld. MAS was an effi cient tool to select for desirable recombination events and pyramid resistance. Received for publication 13 Dec. 2004. Accepted for publication 4 Apr. 2005. The authors thank Troy Aldrich for his assistance with greenhouse and fi eld experiments. Salaries and research support were provided by state and federal funds appropriated to The Ohio State Univ., Ohio Agricultural Research and Development Center, and grant funds from the Mid-American Food Processors. The mention of fi rm names or trade products does not imply that they are endorsed or recommended by The Ohio State Univ. over other fi rms or similar products not mentioned. 1To whom reprint requests should be addressed. E-mail: [email protected] Bacterial spot of tomato is a disease complex with fi ve races, T1 to T5, described (Jones et al., 2000, 2004a). It is caused by as many as four species of bacteria [Xanthomonas euvesicatoria ex Doidge, X. vesicatoria ex Doidge, X. perforans Jones et al.), and X. gardneri Šutic (Doidge, 1921; Jones et al., 2004b; Šutic, 1957)] with taxonomic divisions modifi ed from Vauterin et al. (1995) as described by Jones et al. (2004b). Bacterial spot of tomato occurs throughout the world wherever tomatoes are grown and environmental conditions are favorable for disease development (Stall, 1995). Bacterial spot affects leaves, stems and fruits, and causes both yield and fruit grade losses through defoliation and fruit lesions (Scott, 1997). The resistance to bacterial spot is incompletely or quantitatively inherited. Bacterial speck of tomato, caused by Pseudomonas syringae pv. tomato, mainly occurs on foliage and fruit, and causes yield loss in fi eld and greenhouse grown tomatoes. Two races, 0 and 1, have been reported to date (Scott, 1997). Genetic studies indicated that single dominant genes control resistance to bacterial speck. Four genes, Pto-1 to Pto-4, have been reported (Pilowsky and Zutra, 1986; Pitblado and MacNeill, 1983; Stockinger and Walling, 1994; Tanksley et al., 1996). Pto-1 is also generally referred to as Pto and has been cloned (Martin et al., 1993). The resistance gene Pto is widely deployed in both fresh market and processing tomato varieties. The lack of resistance to bacterial spot in tomato varieties leads to frequent application of copper sprays and the failure of growers to adopt disease forecasting tools that minimize control sprays for fungal pathogens. Disease forecasting models for tomato do not predict bacterial infection and growers default to a calendar application of copper and fungicide tank mixes. The lack of resistance to bacterial diseases therefore adds to the cost of production beyond loss of yield and quality and results in the use of more pesticides than are necessary to control fungal diseases. Although the development of varieties with resistance to multiple bacterial diseases is a desirable goal, the process has been diffi cult due to the necessity of selecting for multiple diseases and the emergence of new species and races. Marker-assisted selection (MAS) offers an opportunity to circumvent some of the problems associated with phenotypic selection for resistance to multiple bacterial pathogens and races. MAS has been successfully used to select for single qualitative or quantitative traits in many crops. These include the extensive use of an acid phosphatase isozyme polymorphism to select for root-knot nematode (Meloidogyne spp.) resistance in tomato (Carboni et al., 1995; Medina-Filho and Stevens, 1980; Rick and Fobes, 1974). Other applications of MAS to disease and pest resistance breeding include Potato Virus Y in Solanum tuberosum L. (Hamalainen et al., 1997); southwestern corn borer (Diatraea grandiosella Dyar) in Zea mays L. (Willcox et al., 2002); and downy mildew [Plasmopara halstedii (Farl.) Berl. et de Toni] on Helianthus annuus L. (Brahm et al., 2000). Markers have been used to combine multiple genes for rust [Uromyces appendiculatus (Pers.) Unger] resistance in Phaseolus vulgaris L. (Kelly et al., 1993) and bacterial blight [Xanthomonas oryzae pv. oryzae (Ishiyama) Swings] resistance genes in Oryza sativa L. (Huang et al., 1997; Yoshimura et al., 1995). Quality traits that have been manipulated through MAS include high-molecular-weight glutenin in Triticum aestivum L. (Ahmad, 2000); the waxy genes 471more cx?.indd 716 8/17/05 5:21:24 PM 717 J. AMER. SOC. HORT. SCI. 130(5):716–721. 2005. in O. sativa (Ramalingam et al., 2002); seed size in Glycine max (L.) Merr. (Hoeck et al., 2003); and fi ber strength in Gossypium hirsutum L. (Zhang et al., 2003). The potential for combining resistance and quality traits makes MAS an appealing strategy for increasing the effi ciency of plant breeding. A previous study indicated that three quantitative trait loci (QTL) in Hawaii 7998 confer a hypersensitive response (HR) to race T1 of bacterial spot (Yu et al., 1995). Rx1 and Rx2 are located on the top and bottom of chromosome 1, respectively, and Rx3 is located on chromosome 5. Only the Rx3 locus has been demonstrated to provide resistance in the fi eld against T1 strains, and it explains as much as 41% of the variation for resistance (Yang et al., 2005). Both Pto and Rx3 are located on chromosome 5, but are derived from different sources and the linkage of desirable alleles is therefore in repulsion phase. The use of phenotypic selection to combine resistance is complicated by the need to identify recombinant plants, to distinguish recombinants from plants that are heterozygous for alleles from both parents, and to select for homozygous resistance in coupling phase. Selection based on markers allows for effi cient classifi cation while reducing the resources necessary for phenotypic evaluation. The objective of this study was to use MAS to select coupling phase recombinants in order to develop lines with resistance to both diseases. Materials and Methods PLANT MATERIAL. Two inbred backcross lines (IBLs) were used to pyramid the resistance gene, Pto, and the locus, Rx3. Ohio 9834 is an IBL derived from Hawaii 7998 and Ohio 88119 carrying the Rx3 locus for partial resistance to race T1 of bacterial spot (Francis and Miller, 2004; Yang et al., 2005) but no resistance to bacterial speck. Ohio 981205 is an IBL in the Ohio 88119 genetic background carrying Pto for resistance to race 0 of bacterial speck but no resistance to bacterial spot. The Ohio 88119 genetic background was chosen due to its concentrated early set and use as an elite line in commercial tomato hybrids (Berry et al., 1995; Francis et al., 2002). A cross was made between Ohio 9834 and Ohio 981205 to develop an F2 population and subsequent inbred generations. Individual F2 plants were grown in 288 Square Plug Tray Deep (Landmark Plastic Corp., Akron, Ohio) with PRO-MIX (Premier Brands, Yonkers, N.Y.) in the greenhouse for DNA isolation and selection. Selected F2 plants were transplanted into 7.6-L pots, allowed to set fruit, and seeds were collected. Seed from selected F2:3 families was again sown in 288 cell trays, and homozygous individuals were selected from heterozygous families prior to transplant into the fi eld. DNA ISOLATION AND MARKER ANALYSIS. Genomic DNA was isolated from fresh young leaves 96 samples at a time. To prevent drying of samples, 7.5 μL ddH2O was added to each well of a fl at-bottom 96-well plate (96-APF-1CO; Rainin Instrument Co., Woburn, Mass.) and the plate was kept on ice. A leaf disk from each individual plant was collected using a hole punch. After all tissue samples were collected, 100 μL of 0.25 N NaOH was added to each well and the leaf disks were ground 3–5 min using a 96-prong seed crusher (PerkinElmer, Norton, Ohio). Following grinding, a 7.5-μL aliquot of each extract was transferred to a 96-well plate containing 75 μL of ice-cold neutralization solution (0.05 M Tris-HCl pH 7.0, 0.1 mM EDTA) in each well. The neutralized solution is ready for use as DNA template in PCR applications and can be stored at –20 °C for at least 1 month. Two PCR-based DNA markers were used to genotype individuals. Marker Pto (forward primer: 5 ́-ATCTACCCACAATGAGCATGAGCTG-3 ́, reverse primer: 5 ́-GTGCATACTCCAGTTTCCAC-3 ́) was designed according to the sequence of the cloned gene Pto (Coaker and Francis, 2004; Martin et al., 1993). Of three markers (TOM49, Rx3-L1, and CosOH73) linked to the Rx3 locus, marker Rx3-L1 (forward primer: 5 ́CTCCGAGCGAAGAGTCTAGAGTC-3 ́, reverse primer: 5 ́GAAGGCAAAAGGAAAAGGAGAAGGATGG-3 ́) explains the highest phenotypic variation (Yang et al., 2005) and therefore was used for selection. PCR reactions were conducted in a 20-μL volume consisting of 10 mM Tris-HCl (pH 9.0 at room temperature), 50 mM KCl, 1.5 mM MgCl2, 50 μM of each dNTP, 0.3 μM primers, 2 μL genomic DNA template and 1 unit of Taq DNA ploymerase. Reactions were heated at 94 °C for 2 min followed by 36 cycles of 1 min at 94 °C, 1 min at 60 °C, and a 2-min extension at 72 °C. Final reactions were extended at 72 °C for 5 min. Amplifi cation was performed in a programmable thermal controller (PTC-100; MJ Research, Watertown, Mass.). Polymorphisms were detected as cut amplifi ed polymorphisms (CAP). The PCR products were digested with Rsa I for Pto and BsrB I for Rx3-L1 according to the enzyme manufacturers ̓protocols. Fragments were separated using 2% agarose gels (Biotechnology Grade 3:1 agarose; Amresco, Solon, Ohio), stained with ethidium bromide, and photographed using the Syngene BioImaging System (Cambridge, U.K.). Recombination between markers was estimated using maximum-likelihood (Allard, 1956). GREENHOUSE AND FIELD EXPERIMENTS. A replicated randomized complete-block design was used for greenhouse disease evaluation. Ohio 88119 was used as a susceptible control for both diseases, Ohio 9834 was used as a resistant control for bacterial spot race T1, and Ohio 981205 was used as a resistant control for bacterial speck race 0. In the greenhouse, fi ve F3:4 plants of each selected family and controls were grown in each of two blocks using PRO-MIX with fertilizer and water supplied as needed. Two strains, Xcv 89 and Xcv 110c, of bacterial spot race T1 and one strain, DC3000, of bacterial speck race 0 were used for infi ltration. The inoculum concentration was adjusted to ≈2 × 108 cfu/mL for strains Xcv 89 and Xcv 110c, and 4 × 107 cfu/mL for strain DC3000. The plants were misted with water 1 h before inoculation. The inoculum was infi ltrated through the back of a fully expanded leafl et on the third true leaf using a 3-mL syringe without needle when plants were at the fourth true-leaf stage. The inoculated plants were kept at 20–25 °C in a humid environment. HR was recorded 24–72 h after inoculation. In the fi eld, 20 plants of each selected F2:3, F3:4, and controls were grown in each of two blocks at The Ohio State Univ.ʼs Vegetable Crops Branch in Fremont, Ohio, using conventional practices (Precheur, 2000) in 2002 and 2003. In 2002, the plants were inoculated with a T1 strain of bacterial spot and the disease was scored using 1–12 scale as described previously (Horsfall and Barratt, 1945; Scott et al., 1995). Early blight [Alternaria solani (Ell. & Mart.) Jones & Grout] naturally occurred and spread in the fi eld in 2002, this disease was also scored using the same scale. Early blight and bacterial spot were distinguished based on characteristics of the lesions as follows: concentric rings are present in early blight lesions in contrast to black lesions found in bacterial spot infected plants; early blight lesions are surrounded by a yellow hallow in contrast to the absence of a halo or a light green halo found surrounding bacterial spot lesions. In 2003, the fi eld was naturally infected by bacterial speck. The reactions of plants to this disease were recorded as resistant or susceptible. 471more cx?.indd 717 8/17/05 5:21:28 PM 718 J. AMER. SOC. HORT. SCI. 130(5):716–721. 2005. STATISTICAL ANALYSIS. Analysis of variance (ANOVA) and mean separations were performed using the General Liner Model procedure of SAS (version 8.1; SAS Institute, Cary, N.C.). Mean separations were based on the least signifi cant difference (LSD) after a signifi cant F test in the ANOVA. Genotype was considered as a fi xed-effect variable, while block was considered as a random-effect variable.