Biparental crosses confirmed by SSR with Mendelian inheritance in sugarcane breeding

ISSN 0370-5404 Rev. Ind. y Agríc. de Tucumán Tomo 89 (2): 1-7; 2012 Biparental crosses confirmed by SSR with Mendelian inheritance in sugarcane breeding María F. Perera*, María B. García**, Carolina Díaz Romero**, María I. Cuenya**, María P. Filippone* and Atilio P. Castagnaro* ABSTRACT In sugarcane (Saccharum spp.) breeding programs, parents used in crosses are classified as male or female based on the relative amounts of viable pollen produced. High pollen production favored by environmental conditions reduces “female inflorescence” availability and restricts the possibility of cross combinations. However, male parents could be employed as female parents when an efficient emasculation treatment is used. An ideal approach for hybridity testing is using molecular markers, especially microsatellites (SSR). To determine the effectiveness of an emasculation treatment (immersion of the panicle in water at 50ºC for five minutes) employed in the Sugarcane Breeding Program of Estación Experimental Agroindustrial Obispo Colombres (EEAOC), Tucumán, Argentina, six cross combinations (selfings and reciprocals) between two varieties commonly used as males, LCP 85-384 and RA 87-3, were evaluated by using SSRs. Samples were amplified with one primer pair that produced seven polymorphic and three monomorphic bands between the two progenitors. While Mendelian segregation may be difficult to observe in the progeny of a complex2 polyploid like sugarcane, the analysis showed that each marker segregated in a Mendelian fashion (as evaluated by χ tests, P≤0.05) for each cross combination. Results indicated that the emasculation treatment was successful and that SSRs made it possible to identify true hybrid progeny routinely in sugarcane breeding. Key words: hybridity testing, hot-water emasculation treatment, segregation analysis, SSR, sugarcane breeding program. RESUMEN Confirmación de cruzamientos biparentales mediante marcadores microsatélites con herencia mendeliana en el mejoramiento de caña de azúcar En los programas de mejoramiento de caña de azúcar (Saccharum spp.), los progenitores empleados en los cruzamientos se clasifican como masculinos o femeninos según las cantidades relativas de polen viable producido. La alta producción de polen, favorecida por las condiciones ambientales, reduce la disponibilidad de inflorescencias femeninas y restringe la posibilidad de combinación en los cruzamientos. Sin embargo, los progenitores masculinos pueden ser empleados como progenitores femeninos cuando se aplica un tratamiento de emasculación efectivo. Una aproximación ideal para determinar la hibridez consiste en la utilización de marcadores moleculares, especialmente los microsatélites (SSR). Para determinar la efectividad de un tratamiento de emasculación (inmersión de la panoja en agua a 50ºC durante cinco minutos), utilizado en el Programa de Mejoramiento Génetico de Caña de Azúcar (PMGCA) de la Estación Experimental Agroindustrial Obispo Colombres (EEAOC), en Tucumán, R. Argentina, seis combinaciones de cruzamientos (autofecundaciones y recíprocos) entre dos variedades comúnmente usadas como progenitores masculinos, LCP 85-384 and RA 87-3, fueron evaluadas mediante SSRs. Las muestras fueron amplificadas con un par de cebadores que produjeron siete bandas polimórficas y tres monomórficas entre los dos progenitores. Mientras que la segregación mendeliana puede ser difícil de observar en la progenie de un poliploide complejo como la caña de azúcar, el análisis mostró que cada marcador presentó herencia mendeliana (tal como se 2 evaluó por pruebas χ , P≤0,05) para cada combinación de cruzamiento. Los resultados indicaron que el tratamiento de emasculación fue exitoso y que los SSRs hicieron posible la identificación rutinaria de los verdaderos híbridos en la progenie obtenida por mejoramiento en caña de azúcar. Palabras clave: determinación de hibridez, tratamiento de emasculación con agua caliente, análisis de segregación, SSR, programa de mejoramiento de caña de azúcar. *Sección Biotecnología, EEAOC. **Sección Caña de Azúcar, EEAOC. |1 Revista Industrial y Agrícola de Tucumán (2012) Tomo 89 (2): 1-7 INTRODUCTION Commercial sugarcane varieties derive from an artificial species obtained by crossing different species of Saccharum genus, characterized by a high degree of polyploidy and frequent aneuploidy (Cordeiro et al., 2000). These cultivars are developed following three procedures: i) assembling a described parental clone population; ii) generating variable progenies by cross-pollination; and iii) selecting useful clones (Hogarth and Berding, 2005). The most vigorous parents for producing varieties to further increase sugar and fiber production are evaluated and selected (McIntyre and Jackson, 2001). In Tucumán, Argentina, the Estación Experimental Agroindustrial Obispo Colombres (EEAOC) has conducted a sugarcane breeding program since 1968. It takes at least eleven years to complete a sugarcane breeding cycle, starting with the crossing between elite clones (bi-parental), continuing with selection, advancement, testing, and ending with the release of a new variety. Photoperiod houses need to be used to induce flowering in this sub-tropical area, where natural flowering is sub-optimum because low temperatures affect flower initiation, as well as pollen fertility (Hogarth and Berding, 2005). Inflorescences are hermaphrodite panicles, in which both pollen quantity and fertility depend on genotype and environmental conditions. Hence, parents are classified as male or female based on the relative amounts of viable pollen produced (McIntyre and Jackson, 2001). The high pollen production of some genotypes, favored by high temperatures, reduces “female inflorescence” availability and restricts the number of cross combinations possible (Berding, 1981). However, emasculation allows employing male parents as female parents and extending the range and direction of clones that can be used in breeding programs. This technique implies pollen sterilization by immersion of the panicle in hot water, cold water, chemical products or steam (Machado et al., 1995). Also, the Table 1. Main characteristics of LCP 85-384 and RA 87-3. Source: Romero et al. (2009b). 2| emasculation treatment is an alternative to overcome self-pollination (Pan et al., 2003). Progeny hybridity can be detected in different ways, such as seed characterization, but the characteristics are unreliable as they are largely controlled by the maternal parent. Also, isozymes are sometimes employed (Shoda et al., 1999); however, these biochemical markers are of limited use, as they can be affected by developmental stage and environment, and can only be assayed with considerable tissue material. Genetic fingerprinting is therefore an ideal approach to hybridity testing (Romero et al., 2009a). Several markers have been used to characterize genuine hybrids from Saccharum and Erianthus (Zhang et al., 2004) and to correctly identify tru (...truncated)