Microsatellite markers in candidate genes for wood properties and its application in functional diversity assessment in Eucalyptus globulus

Electronic Journal of Biotechnology ISSN: 0717-3458	Vol. 15 No. 2, Issue of March 15, 2012
© 2012 by Pontificia Universidad Católica de Valparaíso -- Chile	Received August 21, 2011 / Accepted December 21, 2011
DOI: 10.2225/vol15-issue2-fulltext-3

Cintia V. Acuña¹ · Pamela V. Villalba¹ · Martín García¹ · Pablo Pathauer¹ · H. Esteban Hopp^1,2 · Susana N. Marcucci Poltri*¹

¹ Instituto Nacional de Tecnología Agropecuaria, Instituto de Biotecnología e Instituto de Recursos Biológicos, CNIA, Castelar, Argentina  

² Universidad de Buenos Aires, Facultad de Ciencias Exactas y Naturales, Buenos Aires, Argentina

*Corresponding author: smarcucci@cnia.inta.gov.ar

Financial support: This research was supported by the ANPCyT/FONCYT, BID 1728 OC/AR, PICT-2008-00118 and INTA-PE 041120 and BiotecSur UE 127118.

Keywords: functional markers, genetic diversity, lignin pathway, SSR, wood density.

Abstract

Background: Functional genetic markers have important implications for genetic analysis by providing direct estimation of functional diversity. Although high throughput sequencing techniques for functional diversity analysis are being developed nowadays, the use of already well established variable markers present in candidate genes is still an interesting alternative for mapping purposes and functional diversity studies. SSR markers are routinely used in most plant and animal breeding programs for many species including Eucalyptus. SSR markers derived from candidate genes (SSR-CG) can be used effectively in co-segregation studies and marker-assisted diversity management. Results: In the present study, eight new non reported SSRs were identified in seven candidate genes for wood properties in Eucalyptus globulus: cinnamoyl CoA reductase (CCR), homocysteine S-methyltransferase (HMT), shikimate kinase (SK), xyloglucan endotransglycosylase 2 (XTH2), cellulose synthase 3 (CesA3), glutathione S-transferase (GST) and the transcription factor LIM1. Microsatellites were located in promoters, introns and exons, being most of them CT dinucleotide repeats. Genetic diversity of these eight CG-derived SSR-markers was explored in 54 unrelated genotypes. Except for XTH2, high levels of polymorphism were detected: 93 alleles (mean of 13.1 sd 1.6 alleles per locus), a mean effective number of alleles (Ne) of 5.4 (sd 1.6), polymorphic information content values (PIC) from 0.617 to 0.855 and probability of Identity (PI) ranging from 0.030 to 0.151. Conclusions: This is the first report on the identification, characterization and diversity analysis of microsatellite markers located inside wood quality candidate genes (CG) from Eucalyptus globulus. This set of markers is then appropriate for characterizing genetic variation, with potential usefulness for quantitative trait loci (QTL) mapping in different eucalypts genetic pedigrees and other applications such as fingerprinting and marker assisted diversity management.

Introduction

Eucalyptus tree species are among the most planted hardwoods in the world. They are long-living, evergreen species belonging to the predominantly southern-hemisphere endemic angiosperm family Myrtaceae. Eucalyptus is predominantly out crossing, highly heterogeneous and genetically diverse. 

Within the genus, Eucalyptus globulus species is native of Tasmania and coastal regions of south-eastern Australia. Several forest plantations were successfully established in southern Europe, northern Africa and southern America (Myburg et al. 2007).

Most eucalypt domestication and breeding programs are focused on increasing the volume of produced wood, as well as improving its quality properties. 

Wood is essentially composed of cellulose, hemicelluloses, lignin, and extractives, each of them contributing to fiber properties which ultimately impact product properties. 

Principal traits for improving pulping procedures include quantity and quality of extractives and lignin, which affect directly the economic and/or environmental cost of pulping (Raymond and Apiolaza, 2004) as well as the use of forest residues for bioenergy purposes like ethanol production. For solid timber applications wood density as well as microfibril angle (MFA) have been considered as the most important factors affecting wood properties such as stiffness, strength, and shrinkage behaviour of solid wood (Evans and Ilic, 2001).

Results from genetic variation analyses carried out within E. globulus subraces (Strackpole et al. 2011), suggest that selection increasing wood density tends to decrease lignin S/G relationship. However, they did not find additive genetic relationship between density and extractives, and suggest that selection for increased pulp yield would result in increased cellulose content and S/G but reduced lignin and extractives content. 

As with other forest tree genera with long generation times, eucalypt domestication and breeding programs will benefit tremendously from molecular technologies which can contribute to quantify genetic diversity and relationships, breeding systems analysis, gene flow, fingerprinting and clone identification, QTL detection and molecular breeding through marker- or gene-assisted selection (Myburg et al. 2007). Also, the possible improvement by using marker-assisted selection aimed by breeders requires genomic resources publicly available involving putative candidate genes that control wood properties (Rengel et al. 2009). Good candidates for wood properties include genes involved in lignin and carbohydrate biosynthesis (Hertzberg et al. 2001).

Many genomic studies have reported the analysis of genes expressed during wood formation and xylogenesis (Hertzberg et al. 2001; Moran et al. 2002; Israelsson et al. 2003; Kirst et al. 2004; Paux et al. 2004; Paux et al. 2005; Foucart et al. 2006) and some important metabolic pathways are now well known. In Eucalyptus, several structural and regulatory candidate genes involved in lignin biosynthesis were identified, including those encoding components of the common phenylpropanoid pathway: phenylalanine ammonia-lyase (PAL), 4-coumarate-3-hydroxylase (C3H), Cinnamic acid 4-hydroxylase (C4H), caffeic acid 3-O-methyltransferase (COMT), caffeoyl-CoA Omethyltransferase (CCoAOMT), and 4-coumarate: CoA ligase (4CL) (Gion et al. 2000; Thamarus et al. 2002), and those of the monolignol specific pathway like cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) (Poke et al. 2003), as well as lignin regulatory genes such as MYB transcription factors (Goicoechea et al. 2005). All of these genes seem to be good candidates for QTL co-localization studies with wood-quality and lignin-content/quality QTLs.

Results and Discussion

Screening for microsatellite sequences in candidate genes for wood quality

Microsatellite sequences that were identified in silico were confirmed by PCR amplification using E. globulus genomic DNAs. Some of them were corroborated by automatic nucleotide sequencing. After homology reconfirmation of the flanking sequences using BLAST software, microsatellite structure was corroborated by direct visualization of their DNA sequence profiles. Eight new microsatellite sequences involved in lignin and cell-wall polysaccharide biosynthesis were, thus, characterized in seven candidate genes: Eucalyptus gunnii cinnamoyl-CoA reductase promoter (GenBank: AJ132750), E. grandis cellulose synthase 3 (CesA3) (GenBank: EU165713, 2 microsatellite regions), transcription factor LIM1 (GenBank: AB208710, E. grandis EST CB967988 similar to Arabidopsis thalianahomocysteine S-methyltransferase (HMT), E. globulus EST GenBank: BF942502 similar to A. thaliana shikimate kinase (SK), a putative E. globulus XTH2 (GenBank: DQ100338), E. globulus EST CT988111 similar to Vitis vinifera glutathione S-transferase and E. globulus). 

In E. grandis EST CB967988, similar to Arabidopsis thaliana homocysteine S-methyltransferase (HMT), the microsatellite was identified after automatic sequencing of fragments which were higher than the expected size. Sequence data from this genomic fragment was annotated in the GenBank under accession number FJ492059.

For diversity analysis samples of 54 non selected unrelated trees were assayed for each SSR.

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