Use of molecular markers to improve the agro-industrial productivity in the North West of Argentina

PERERA, MF; RACEDO, J; GARCÍA, MG; PARDO, EM; ROCHA, CML; ORCE, IG; CHIESA MA; FILIPPONE, MP; WELIN, B; CASTAGNARO, AP

molecular biology journal

Omics group

Año: 2015

2168-9547

Use of molecular markers to improve the agro-industrial productivity in the North West of ArgentinaPerera MF, Racedo J, García MG, Pardo EM, Rocha CML, Orce IG, Chiesa MA, Filippone MP, Welin B and Castagnaro AP*Estación Experimental Agroindustrial Obispo Colombres (EEAOC) ? Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA), Av. William Cross 3150. C.P. T4101XAC, Las Talitas, Tucumán, Argentina.(*)E-mail adress: atiliocastagnaro@gmail.com and atilio@eeaoc.org.ar; telephone number: (54 - 381) 452 1000 ? Fax number: (54 - 381) 452 1008.Genetic improvement of crop plants through conventional breeding has been the cornerstone to improve global agricultural production. Nowadays, different molecular biology techniques have become available to supplement and assist conventional breeding, among them the utilization of molecular markers. The generation of molecular, or DNA, markers are based on two basic molecular biology methods in order to be able to detect polymorphism: Southern blotting, a nucleic acid hybridization technique (Southern 1975), and the amplification in vitro of specific DNA segments by the Polymerase Chain Reaction (PCR) technique (Mullis 1990). By using these two methods and several variations of the basic technologies, many different kind of molecular markers have been developed during the last decades. It is important to note that for a molecular marker to be efficient in crop breeding it should meet most of the following criteria (Jiang 2013): high level of polymorphism; even distribution across the whole genome (not clustered in certain regions); co-dominance (or capacity to identify at heterozygous); clear distinct allelic features; single copy; low cost to use (or cost-efficient marker development and genotyping); easy assay/detection and automation; high availability (un-restricted use) and suitability to be duplicated/multiplexed (so that the data can be accumulated and shared between laboratories); genome-specific in nature (especially with polyploids) and have no detrimental effect on phenotype. However, the choice of the best marker system depends on many other factors as well including crop genetics and available resources (time, reagents, equipments, etc).In Argentina, among the many public institutes and private companies working in plant breeding, the ?Estación Experimental Agroindustrial Obispo Colombres (EEAOC)?, founded in 1909 and located in the Province of Tucumán, occupies a preponderant place (http://www.eeaoc.org.ar/) with successful ongoing breeding programs in sugarcane, soybean, common bean and citrus. EEAOC is a Provincial Institute managed by the producers themselves and financed directly with resources from the production, which provides technology development and solutions, which contributes to improved productivity and sustainability of the agroindustrial sector of the North West of Argentina. In 2012, the ?Instituto de Tecnología Agroindustrial del Noroeste Argentino (ITANOA)?, a bioeconomy research institute of dual dependence between EEAOC and ·Consejo Nacional de Investigaciones Científicas y Técnicas? (CONICET) was founded as an institutional innovation (http://www.tucuman-conicet.gov.ar/Secciones.php?IdSeccion=51). The main objective of this new Institute is to contribute to a sustainable development in social, environmental and economic terms through technology transfer in order to improve productivity, health and industrial processing of crops and plant biomass in the region. The overall idea is to generate, integrate and coordinate a social-economic development with agro-industrial production and environmental protection, based on creativity and knowledge. EEAOC-CONICET-ITANOA is working on the economically most important crops of the North West of Argentina, which includes sugarcane, soybean, lemon and strawberry.Sugarcane production in Argentina is the oldest agro-industrial activity in the country, starting in the late 19th century. Sugar production is historically concentrated to the Provinces in the North West of Argentina (Tucumán, Salta and Jujuy) although there is a small sugar development in the North East of the country (Perez et al. 2007). Annually about 2 million tons of sugar are produced from around 350,000 hectares cultivated. The Province of Tucumán is the main sugar producer in the country with approximately 66% of the total production from approximately 265,000 hectares, representing about 45% of the total agricultural area of the Province (Fandos et al. 2014). In addition to its economic importance, the sugar industry plays an important social significance for Tucumán since there are around 5,500 farmers and it generates more than 20,000 jobs. Commercial sugarcane varieties all belong to Saccharum genus and are inter-specific complex artificial hybrids characterized by a high degree of polyploidy and frequent aneuploidy (Cordeiro et al. 2000). These characteristics and the cytogenetic complexity of sugarcane cultivars, involving varying chromosome sets and complex recombinational events, imposes difficulties in accomplish effective breeding programs (Vettore et al. 2001). In Argentina, the first sugarcane breeding program was formally established in 1968 by the EEAOC. Completing a sugarcane breeding cycle takes at least 11 years, starting with a crossing between two elite clones, evaluating the progeny to identify true hybrids, several stages of testing and clonal selection, and finally ending with a new variety release. In order to broaden the genetic base of commercial varieties of sugarcane, it is important to identify more genetically diverse parents to be used in breeding programs (Salem et al. 2008). For that reason and for protecting new sugarcane varieties and intellectual property rights, an accurate varietal identification is essential (Wagih et al. 2004). In addition, the knowledge of the genetic diversity in sugarcane will provide useful information concerning the genotype value to breeders and will contribute to the improved use and conservation of genetic resources. Molecular markers are powerful tools to estimate genetic diversity and to generate information to better understand the complex genetics of sugarcane as they are accurate, abundant and not affected by the environment (D´Hont et al. 1997). Random Amplified Polymorphic DNA (RAPD) was the first molecular marker technique employed at EEAOC to characterize sugarcane genotypes (Fontana et al. 2003). This relatively simple DNA amplification technique allows for application without the need to know the sequence of the DNA to be amplified in advance. The technique generated useful information in sugarcane and allowed for the identification of related genotypes; however the short length of the primers implies that DNA hybridization are performed at a relatively low temperature, which increase the likelihood of a non-specific alignment and generation of ambiguous information by unspecific amplification and poor reproducibility (Otero et al. 1997). To solve this problem, more recently, Amplified Fragment Length Polymorphisms (AFLP) and Simple Sequence Repeats (SSR) markers were used to characterize the sugarcane genotypes mostly used as parents in the sugarcane breeding program at EEAOC (Perera 2011, Perera et al. 2012a). As sugarcane has a very large and complex genome a vast number of markers, such as those generated by AFLP techniques (Vos et al. 1995), are necessary to determine the genetic diversity (Lima et al. 2002). An advantage of using AFLP markers is that it allows for simultaneous screening of many different genome regions distributed randomly throughout the genome (Mueller and Wolfenbarger 1999). In our study we found a high number of AFLP bands but a very limited polymorphism (19.40%) that reflects a low genetic diversity in the breeding germplasm evaluated (Jaccard coefficient mean value: 0.96). However, the AFLP technique still provides a useful alternative for diversity estimation as well as for genotype identification because the 16 primer combinations tested were enough to differentiate between all sugarcane genotypes characterized. Another powerful technology for monitoring genetic diversity and varietal identification is SSR because of their abundance, sensitivity and high accuracy in detecting polymorphism even between very closely-related genotypes (Powell et al. 1996). It must be highlighted that although SSRs are classified as co-dominant type markers, they have been treated as dominant markers when analyzing the highly complex genome of Saccharum. In our work, SSR markers had a mean genetic diversity value lower than AFLP (0.84) but reflected genetic relations more accurately than AFLP. However, the correlation between AFLP and SSR dendrograms (0.58; p