Phenotypic Characterization of a Diversity Panel of Tomato

1. Phenotypic Characterization of a Diversity Panel of Tomato Larry D. Robertson, Teri Balch, Susan M. Sheffer, and Joanne A. Labate UDSA-ARS, Plant Genetic Resources Unit, Geneva, NY 14456 Methods Tomato Analyzer: Tomato Analyzer is a software application which demonstrates objective quantitative measurements of fruit shape, size and color (Gonzalo et al., 2009). Mid-height, longitudinal dissections were made on each of 10 to 15 fruit per accession. The halves were placed on a Microtek Scanmaker 9800XL 11” x 17” scanner bed and scanned with a black background at 300 dpi (pixels per inch). The images were saved as .jpg files and analyzed using Tomato Analyzer 2.2.0.0, manual adjustments were made for boundary and proximal and distal end shape. Mean perimeter and mean area were extrapolated from the data. Fruit shape, uniformity and color: Fruit shape, uniformity and color were determined based on descriptors presented by the Tomato Crop Germplasm Committee (CGC) (http://www.ars-grin.gov/npgs/descriptors/tomato). Fruit shape was determined via visual selection between the following shapes: flat, slightly flattened, round, plum, blocky, pear, oblong, high round and heart. Uniformity was based on a sliding scale of 1 through 9 with 1 indicating no uniformity and 9 indicating very high uniformity. Color was determined using visual selection of colors as presented by the Tomato CGC report (red, orange, yellow, gold, white and green). °Brix: Brix degree data, refractive index at 20°C (Carli et al., 2009), were collected using a Model DR103L digital refractometer (QA Supplies). Juice from randomly selected cut fruit pieces was squeezed onto the refractometer. For each sample the °brix was calculated as a mean of 3 readings of separate fruit within each accession. °Brix is a measure of the soluble sugar content including glucose and fructose in ratios of approximately 1:1 to 1:1.5. Brix degree is considered a measurement of fruit sweetness; high °brix equates to higher sweetness. Lycopene: Fruit pieces were homogenized in a commercial grade Waring blender (one to ten fruits per accession). An aliquot of homogenate was transferred to a 5 cm petri dish, and readings were taken using a Minolta Chroma Meter CR-300 by pointing the sensor close to the homogenate surface. Values were recorded for L*a*b* (also referred to as CIELAB) color space. L* indicates lightness, a* (red-green) and b* (blue-yellow) are chromaticity coordinates (http://en.wikipedia.org/wiki/Lab_color_space). Each L*a*b* value represented the average of three measurements. Lycopene was estimated using a regression model based on the transformed a*4 value (Hyman et al. 2004). Vitamin C: Fruit pieces were homogenized in a commercial grade Waring blender (one to ten fruits per accession). A 100 ul aliquot of homogenate was pipetted into a 1.5 ml eppendorf tube, 500 ul of 6% metaphosphoric acid was added and the tube was vortexed. Samples were centrifuged at 15K rpm for 10’ at 4°C, 100 ul of clear supernatant was transferred to a 1.5 ml tube containing 100 ul of 5% metaphosphoric acid. Samples were then assayed using a Cosmo Bio Co. Ltd. (Japan) Vitamin C assay kit (Prod. No. SML-ROIKO2-EX) according to the kit protocol. Vitamin C was estimated based on absorbance at 530 nm using a standard curve of known concentrations. Background Thousands of open-pollinated populations and inbred lines of domesticated tomato (Solanum lycopersicum L.) are conserved in germplasm centers such as the USDA, ARS Plant Genetic Resources Unit (PGRU) (Robertson and Labate, 2007). These collections provide a publicly available resource for experimentation and breeding to a broad community of users around the world. Landrace germplasm from the center of origin is expected to have a higher diversity than modern cultivars. Such germplasm can provide a source of novel alleles. Fruit Morphological Traits Fruit color, fruit shape and fruit uniformity are shown in Figs. 7-9. Most accessions were red fruited and most were slightly flat to flat. Fruit uniformity was low, as expected from less-improved populations. Nutritional Traits Lycopene, Vitamin C, and obrix were recorded for the 50 tomato accessions in this diversity panel. Relationships among these traits are shown in Fig. 11. The accession with the highest Vitamin C (14.5 ug/ml) and highest lycopene (13.0 ug/g) was PI 212062 (Fig. 12). There was a very high correlation between lycopene and vitamin C (r=0.94**, df=48). The correlation of oBrix and Vitamin C was also significant (r=0.38*, df=48). This study reports on an evaluation of a set of 50 geographically diverse PGRU accessions (Fig. 1) for fruit morphological traits, size and nutritional traits. Figure 7. Fruit color for 50 tomato accessions Figure 1. Fruit of 50 tomato accessions evaluated for this study Figure 8. Fruit shape for 50 tomato accessions Summary This diversity panel of 50 PGRU tomato accessions represents only a small fraction of the available diversity preserved in the National Plant Germplasm System. There was a large variation expressed for all traits. Fruit size, color, shape and uniformity were as expected in landraces and vintage cultivars. Variation in the nutritional traits is potentially useful for crop improvement. PI 212062 Plant Materials The 50 diverse accessions of tomato grown in this study are listed in Table 1. These included a landrace panel of 30 PGRU tomato accessions assembled based on results of RAPD analyses including 14 accessions from the primary center of diversity and 12 accessions from countries contiguous with the primary center previously reported (Labate et al., 2009). This set was expanded by selection of additional accessions based on geography and time of collection or date of cultivar release. Figure 11. Brix content, Lycopene, and Vitamin C for 50 tomato accessions, sorted by Vitamin C Figure 9. Fruit uniformity for 50 tomato accessions; 1=no uniformity, 9=completely uniformity. was an outlier for both lycopene and Vitamin C (Figs. 11 and 12). This accession deserves further investigation. Fruit Size Size of the fruit of this tomato diversity panel was measured using average perimeter (cm) and average fruit weight (g). Fruit weight ranged from 1.2 to 37.2 g and perimeter ranged from 9.8 to 32.4 cm (Fig. 10). Fruit weight and perimeter of fruit were highly correlated (r=0.93**, df=48). General Observations These accessions exhibited many traits not seen in modern cultivars of tomato. Variation in foliage color was observed (Fig. 2). Modern tomato has non-exserted stigmas (Fig. 3). However, many of these accessions expressed exserted stigmas (Fig. 4). Several accessions Figure 12. PI 212062 Literature Cited Carli, P, Arima S, Fogliano V, Tardella L, Frusciante L, Ercolano MR (2009). Use of network analysis to capture key traits affecting tomato organoleptic quality. Journal of Experimental Botany 60: 3379–3386. Gonzalo T., M.J. Brewer, M.T., Anderson, C., Sullivan, D., Gray, S., van der Knaap, E (2009). Tomato fruit shape analysis using morphometric and morphology attributes implemented in tomato analyzer software program. J. Am. Soc. of Hort. Sci. 134: 77-87. Hyman J.R., J. Gaus, and M.R. Foolad (2004). A rapid and accurate method for estimating tomato lycopene content by measuring chromaticity values of fruit puree. J Amer Soc Hort Sci 129:717-723. Labate JA, Robertson LD, Baldo AM (2009). Multilocus sequence data reveal extensive departures from equilibrium in domesticated tomato (Solanum lycopersicum L.). Heredity 103: 257-267. Robertson LD, Labate JA (2007). Genetic resources of tomato (Lycopersicon esculentum Mill.) and wild relatives. In: Razdan MK,Mattoo AK (eds). Genetic Improvement of Solanaceous Crops. Science Publishers: Enfield, NH. Vol. 2: Tomato, pp 25–75. Figure 2. Variation of foliage color in tomato landraces expressed floral abnormalities, with multiple ovules (Fig. 5), which resulted in multiple fused fruit with a spherical, fasciated shape (Fig. 6). Figure 4. Exserted stigma Figure 3. Non-Exserted stigma Acknowledgements We thank William Garman III, John Oughterson, Paul Kisly, and Jonathan Spencer for excellent technical support. This study was funded by CRIS Project 1910-21000-019-00D and the Tomato CGC. Figure 10. Fruit weight (g) and perimeter (cm) for 50 tomato accessions, sorted by fruit weight Figure 5. Flowers of PI 258474 Figure 6. Fruits of PI 258474