Abstract
The ability to quickly and accurately identify potato (Solanum tuberosum L.) clones is important to potato-breeding programs, seed and commercial potato growers, and marketing and utilization of potato cultivars. Since 1990, the Michigan State University Potato Breeding and Genetics Program has used an isozyme-based fingerprinting system to identify potato cultivars. Isozyme analysis is an economical and effective means of discriminating potato clones; however, isozyme analysis requires fresh, healthy tuber or leaf tissue. DNA-based fingerprinting using simple sequence repeats (SSRs or microsatellites) has been shown to discriminate between potato clones. The objective of this study was to identify the most useful SSR primer pairs that accurately and efficiently distinguish clones for an applied fingerprinting system of cultivated potato. SSR primer pairs with high polymorphism were selected from previous tetraploid potato studies. DNA isolated from 17 potato clones representing round-white, russet, and red market classes were visualized on both polyacrylamide (PAGE) and agarose gel systems. Polymorphism was observed in all 18 primer combinations on PAGE and 14 using agarose gel electrophoresis. All 17 cultivars were discriminated on PAGE with various combinations of two primer pairs: STIIKA using STACCAS3, STIN-HWI, or STM0031; and STACCAS3 using STGBSS1, POTM1-2, STM1104, or STM0031. The combination of STM0019, STM0031, STGBSS1, and POTM1-2 was able to differentiate all 17 clones using agarose gel electrophoresis. PAGE was determined to be the preferred system for variety identification, but agarose gel electrophoresis can be used to differentiate lines when specific varietal comparisons are needed. In addition, five different DNA source tissue types were evaluated (fresh foliar, freeze-dried foliar, fresh tuber, freeze-dried tuber epidermis, and freeze-dried tuber tissue). Amplification products were similar for all five tissue sources used for DNA isolation. This ability to isolate DNA from freeze-dried tissue will allow cultivar identification when fresh tissue is not available. The SSR primer pairs presented here can be used as a practical fingerprinting system for cultivated potato identification.
Resumen
La posibilidad de identificatión rápida y exacta de clones de papa (Solanum tuberosum L.), es importante para los programas de mejoramiento, para los productores de semilla y papa de consumo, lo mismo que para la comercialización y utilizatión de los cultivares de papa. Desde 1990, el Programa de Mejoramiento y Genética de la Universidad del Estado de Michigan ha utilizado un sistema de tipificación genética para identificar cultivares de papa basado en isoenzimas. El análisis de isoenzimas es un método económico y efectivo para diferenciar clones de papa. Sin embargo, este análisis requiere de tejido fresco y sano de hoja o de tubérculo. Con la tipificación genética basada en el uso de ADN y utilizando repeticiones de secuencias simples (SSRs o microsatélites) se ha demostrado la existencia de diferencias entre clones de papa. El objetivo del presente estudio fue identificar los pares de iniciadores SSR más útiles para distinguir clones de papa cultivada en forma precisa y eficiente por medio de un sistema de tipificación genética aplicada. A partir de estudios previos de papa tetraploide se seleccionaron pares de iniciadores SSR con polimorfismo amplio. El ADN aislado de 17 clones de papa representantes de las clases comerciales redondo bianco, rosado y rojo se hizo evidente tanto en el sistema de poliacrilamida (PAGE) como en el de gel de agarosa. Con el sistema PAGE se observó polimorfismo en todas las combinaciones de los 18 iniciadores, y en 14 cuando se utilizó electroforesis en gel de agarosa. Los 17 cultivares fueron diferenciados en PAGE con varias combinaciones de dos pares de iniciadores: STIKA utilizando STACCAS3, STINHWI o STM0031 y STACCAS utilizando STGBSS1, POTM1-2, STM1104, o STM0031. La combinación de STM0019, STM0031, STGBSS1 y POTM1-2 fue capaz de diferenciar los 17 clones utilizando electroforesis en gel de agarosa. Se estableció que PAGE es el sistema preferido para la identification de variedades, pero se puede utilizar la electroforesis en gel de agarosa para diferenciar lineas, cuando se necesitan comparaciones varietales especificas. Además se evaluaron cinco fuentes de tejido (foliar fresco, foliar seco congelado, tubérculo fresco, epidermis de tubérculo seco congelado, tubérculo seco congelado). Los productos de amplificacion fueron similares para las cinco fuentes de tejido para el aislamiento de ADN. Esta facultad para aislar ADN de tejido seco congelado permitirá la identificacion de cultivares, cuando no se disponga de tejido fresco. Los pares del iniciador SSR presentados aqui se pueden utilizar como un sistema práctico de tipificación genética para la identificación de la papa cultivada.
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Abbreviations
- (PCR):
-
polymerase chain reaction
- (SSR):
-
simple sequence repeats
- (PAGE):
-
polyacrylimide gel electrophoresis
Literature Cited
Ashkenazi V, E Chani, U Lavi, D Levy, J Hillel, and RE Veilleux. 2001. Development of microsatellite markers in potato and their use in phylogenetic and fingerprinting analyses. Genome 44:50–62.
Demeke T, LM Kawchuk, and DR Lynch. 1993. Identification of potato cultivars and clonal variants by random amplified polymorphic DNA analysis. Am Potato J 70:561–570.
Douches DS, and K Ludlam. 1991. Electrophoretic characterization of North American potato cultivars. Am Potato J 68:766–780.
Gebhardt C, C Blomendahl, U Schachtschabel, T Debener, F Salamini, and E Ritter. 1989. Identification of 2n breeding lines and 4n potato varities (Solanum tuberosum ssp.tuberosum) with RFLP-fingerprints. Theor Appl Genet 78:16–22.
Ghislain M, F Rodríguez, F Villamón, J Núñez, R Waugh, and M Bonierbale. 2000. Establishment of microsatellite assays for potato genetic identification. Research on Potato. CIP Program Report 1999–2000. pp. 167–174.
Hamester W, and U Hils (eds). 2003. World Catalogue of Potato Varieties. Que Pub., Indianapolis, IN.
Kawchuk LM, DR Lynch, J Thomas, B Penner, D Sillito, and F Kulcsar. 1996. Characterization ofSolanum tuberosum simple sequence repeats and application to potato cultivar identification. Am Potato J 73:325–335.
McGregor CE, CA Lambert, MM Greyling, JH Louw, and L Warnich. 2000. A comparative assessment of DNA fingerprinting techniques (RAPD, ISSR, AFLP, and SSR) in tetraploid potato (Solanum tuberosum L.) germplasm. Euphytica 113:135–144.
Milbourne D, RC Meyer, AJ Collins, LD Ramsay, C Gebhardt, and R Waugh. 1998. Isolation, characterization and mapping of simple sequence repeat loci in potato. Mol Gen Genet 259:233–245.
Nei M, and WH Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc Natl Acad Sci USA 76:5269–5273.
Provan J, W Powell, and R Waugh. 1996. Microsatellite analysis of relationships within cultivated potato (Solanum tuberosum). Theor Appl Genet 92:1078–1084.
Rohlf FJ. 2002. NTSYSpc: Numerical Taxonomy System, ver. 2.11L. Exeter Publishing, Ltd., Setauket, NY.
Sambrook J, and D Russell. 2001. Molecular Cloning: A Laboratory Manual. 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Schneider K, and DS Douches. 1997. Assessment of PCR-based simple sequence repeats to fingerprint North American potato cultivais. Am Potato J 74:149–164.
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Coombs, J.J., Frank, L.M. & Douches, D.S. An applied fingerprinting system for cultivated potato using simple sequence repeats. Am. J. Pot Res 81, 243–250 (2004). https://doi.org/10.1007/BF02871765
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DOI: https://doi.org/10.1007/BF02871765