Eucalyptus: Molecular techniques for transferring heterologous primers

Brazil stands out on the world stage due to the importance of forest biomass and its potential, with one of the highest productivity rates in forestry, reaching between 40 and 50 cubic meters (m3) of wood per hectare per year, more than 10 times higher than observed in countries with a temperate climate (STAPE, 2003).

The forestry-based activity, present in almost the entire national territory, is divided into several segments, such as: cellulose and paper, corrugated cardboard, charcoal-fired steelmaking, furniture and mechanically processed wood, which includes the production of sawn wood, panels reconstituted materials, plywood and laminates and higher value-added products, in addition to various non-wood products.

In a considerable reforestation area in Brazil, species of the Eucalyptus genus are used mainly in companies operating in the paper and cellulose area. In Brazil, the total area with planted forests in 2006 totaled 5,74 million ha, of which 3,55 million ha were eucalyptus; 1,82 million ha with pine and 370,5 thousand ha with other species, showing an increase of just over 175 thousand ha in relation to 2005 (5,56 million ha) (SBS, 2009).

Minas Gerais has the largest planted forest area in the country, occupying 1,23 million ha, followed by São Paulo with 963,3 thousand ha, corresponding to 21,5% and 16,8% respectively, of the existing planted area in the country. Brazil (SBS, 2009).

According to Abraf (2008), the sustainable production capacity of Brazilian forests is estimated at around 390 million m³/year, of which 47,2% or approximately 183,6 million m³, are from forests planted with pine and eucalyptus. The remainder is still due to the exploitation of native forests.

The alternatives to reduce pressure on timber resources in native forests and produce an increase in production in order to meet demand are two alternatives: firstly, the increase in the current productive area, associated with increased productivity in areas already occupied. Among the strategies to overcome this challenge, forest genetic improvement stands out.

Genetic variability is the basis of biodiversity and can be accessed through genetic markers. The association of molecular techniques with traditional breeding techniques provides a major advance in obtaining results.

Thus, molecular markers function as auxiliary tools in forest genetic improvement, allowing, among others, assessments of the suitability of the genetic base of forest populations for use in improvement programs.

Among the molecular markers currently used, microsatellites or simple repetitive sequences (SSRs) deserve to be highlighted, as they are considered ideal for genetic and breeding studies as they combine several aspects of interest: co-dominance; multialelism, high polymorphism, abundant and uniform dispersion in plant genomes and efficient detection by polymerase chain reaction (PCR).

However, to carry out genetic analyzes via microsatellite PCR, it is necessary to develop primers that enable the recognition of the loci of interest and this requires knowledge of the DNA sequence flanking these loci. Therefore, the development of specific primers for each eucalyptus species involves time-consuming and high-cost laboratory steps (ZUCCHI et al., 2003).

There are primers designed for species most used for paper and cellulose production, such as Eucalyptus grandis, on the other hand, there are species and hybrids of the genus Eucalyptus that do not have specific sequences designed, such as Eucalyptus exerta, Eucalyptus saligna, Eucalyptus grandis x urophylla , and Eucalyptus grandis x camaldulensis.

The transferability of microsatellite primers considerably reduces the costs and time of genetic investigations. When the species involved in transferability are closely related, they have a greater chance of sharing microsatellite primer binding sites than in less closely related species, but it is possible to transfer functional primers even between more distant species (LORIEUX et al., 2000)

Given all this exposed versatility of microsatellites, the present work aims to verify the transferability of five pairs of heterologous microsatellite primers in Eucalyptus.

Thus, the EST-SSR primers (EUCA 30, 39, 50, 57 and 62) designed from the FORESTs/FAPESP Consortium database were tested. (Table 1) A total of 40 individuals belonging to Eucalyptus species and hybrids (Eucalyptus exerta, Eucalyptus saligna, Eucalyptus grandis x urophylla, and Eucalyptus grandis x camaldulensis) a collection provided by ESALQ and maintained at the Teaching, Research and Extension Farm ( FEPE) from the Faculty of Engineering of Ilha Solteira was subjected to DNA extraction as proposed by Ferreira and Grattapaglia, (1998) with minor modifications. (Table 2)

The amplification reaction (PCR-SSR), with a volume of 15 uL, consisted of PCR buffer (1X), 0,1mM of each dNTP, 2mM of MgCl2, 1uM of each member of the primer pair (F and R) , 2 units of Taq DNA polymerase, 18 ng of genomic DNA. For amplification of the alleles, a universal touchdown program was used. The amplification products were separated on 3% agarose gels, running with TBE (1X) at a constant voltage of 110V.

From the electrophoretic profiles obtained it was possible to verify that different levels of transferability were obtained. (figure 1)

The EUCA 30 primer provided allele amplification in 22 individuals, therefore with an efficiency of 55%, however none of them belonged to the E. grandis X E. urophylla hybrid.

The EUCA 39 primer allowed amplification in individuals of all sampled species, with an efficiency of 27,5%.

The EUCA 50 and 57 primers had transferability efficiency of 30 and 20% respectively, but no sample belonging to E. saligna demonstrated amplification.

Finally, the transferability of the EUCA 62 primer occurred with an efficiency of 37,5%, with a total absence of bands for the E. exerta samples.

Although the transferability levels obtained were medium to low, the use of these heterologous primers is interesting, since each of them can be applied to one or some of the species and hybrids studied. This means reduced costs and time in generating molecular markers for Eucalyptus.

1,

2

1Biologist, helio_junq@yahoo.com.br, 2Biologist, FCAV/UNESP