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Electronic Journal of Biotechnology

versión On-line ISSN 0717-3458

Electron. J. Biotechnol. vol.15 no.2 Valparaíso mar. 2012

 

  Molecular Biology and Genetics
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  
RESEARCH ARTICLE

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

Cintia V. Acuña1 · Pamela V. Villalba1 · Martín García1 · Pablo Pathauer1 · H. Esteban Hopp1,2 · 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.

In the past ten years a great number of studies have reported genetic maps for different Eucalyptus species, built from combinations of random-amplified polymorphic DNA (RAPD), amplified fragment length polymorphism (AFLP), restriction fragment length polymorphism (RFLP) markers, isozymes, EST and gene sequence derived markers (mainly by the analysis of RFLP and single strand conformation polymorphisms (SSCP) techniques) as well as microsatellites (SSR and EST-SSR) (Grattapaglia and Sederoff, 1994; Byrne et al. 1996; Verhaegen and Plomion, 1996; Marques et al. 1998; Bundock et al. 2000; Gion et al. 2000; Brondani et al. 2002; Thamarus et al. 2002; Poke et al. 2005; Faria et al. 2010, Faria et al. 2011). In Brondani et al. (2006), a set of 230 new microsatellites has been developed and a consensus map has been assembled, covering at least 90 percent of the recombining genome of Eucalyptus.

Recently Single Feature Polymorphisms (SFP) (Neves et al. 2011) and Single Nucleotide Polymorphism (SNP) (Novaes et al. 2008; Grattapaglia et al. 2011) analysis have been reported.

There are only few studies in Eucalyptus where candidate genes underlying wood quality traits have been analyzed using QTL approaches (Gion et al. 2000; Gion et al. 2001; Freeman et al. 2009; Thumma et al. 2010; Gion et al. 2011; Sexton et al. 2012). These studies have shown the co-segregation of QTLs simultaneously involved both in chemical as well as physical properties, thus suggesting that genomic regions may be organized in clusters of genes with independent effects on different wood properties or that there are some key genes with pleiotropic effects (Gion et al. 2011). The increasing use of larger sets of interspecific transferable markers and consensus mapping information will allow faster and more detailed investigations of QTLs synteny among species, validation of QTLs and expression-QTLs across variable genetic backgrounds, and positioning of a growing number of candidate genes co-localized with QTLs, to be tested in association mapping experiments (Grattapaglia and Kirst, 2008). The first association mapping studies in forest trees were based on the analysis of SNPs allelic diversity which was carried out in fewer than 20 selected candidate genes. These studies have revealed that, although some candidate gene associations could be detected (Thumma et al. 2005; Sexton et al. 2012), it turned out very difficult to predict an appropriate set of candidate genes for any trait of interest. Instead, a much larger set of genes and regulatory sequences must be screened and interrogated for allelic diversity and trait association (Myburg et al. 2007). Recent advances in high-throughput marker technologies allowed mapping and association studies in pine (Eckert et al. 2009) and poplar (Wegrzyn et al. 2010) based on candidate genes.

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 species. SSR markers derived from candidate genes (SSR-CG) can be used effectively in co-segregation studies and marker-assisted diversity management. It is also a low-cost technology that may provide an efficient way to survey natural variation in Eucalyptus.

In the present study, eight new non reported SSRs were identified in seven candidate genes for wood properties: CCR (catalyzes the conversion of cinnamoyl CoA esters to their corresponding cinnamaldehydes, being the first specific step in the synthesis of lignin monomers); HMT (participates in the S-adenosylmethionine biosynthesis, a substrate for the phenylpropanoid pathway); SK (takes part in phenylalanine, tyrosine and tryptophan biosynthesis); XTH2 (implicated in the modification of cell wall structure); CesA3 (involved in the coordinated synthesis of glucose chains); GST (functions in normal plant development and plant stress responses) and the transcription factor LIM1.

Diversity analyses of them were carried out in 54 unrelated genotypes to estimate their potential usefulness for QTL mapping in different eucalypts genetic pedigrees and diversity analysis. Then, frequency of alleles, observed heterozygosity (Ho), PIC and PI were calculated for other applications such as fingerprinting and marker assisted diversity management.

Materials and Methods

Plant material and DNA extraction

A total of 54 non related trees, each from a different open-pollinated (OP) family of E. globulus, were analyzed for their variability. These non selected trees represented major geographical races of the species’ natural distribution (Dutkowski and Potts, 1999) and land race from Portugal that were grown in a field trial in the Province of Buenos Aires, Argentina, between 1995 and 1997 (Table 1).

Total DNA was extracted from young leaves by the CTAB method (Hoisington et al. 1994) with minor modifications to avoid oil precipitation: instead of using isopropanol in the precipitation step, samples were diluted two times in 10 mM Tris-HCl, 1 mM EDTA buffer and precipitated with 2.5 volumes of ethanol and 300 mM Na acetate (Marcucci Poltri et al. 2003).

Target accession numbers of candidate genes from GenBank

Microsatellites within CGs were identified mainly by two different methods: -SSRs detection from non redundant ESTs of E. globulus from GenBank: Annotations of these SSR-ESTs were based on the Gene Ontology (GO) using Blast2GO (Conesa et al. 2005; Acuña et al. 2011). The selection of CGs for wood quality was made from that function assignment.

-SSR Mining software (GDR Server, http://www.rosaceae.org/bio/content?title=&url=/cgi-bin/gdr/gdr_ssr) on selected candidate gene genomic sequences identified at the GenBank.

Selection was restricted to those microsatellites displaying a minimum of five repeats for di-, 4 for tri-, and 3 for tetra- and penta-nucleotide arrays. Primers were designed flanking the repeated motifs using PRIMER 3.0 software (http://www.broadinstitute.org/genome_software/other/primer3.html) (Table 2).

PCR amplification of microsatellite loci

PCR conditions were as follows: A final volume of 12 µl containing 20 ng of genomic DNA, 0.25 µM of each primer, 2 mM MgCl2, 0.2 mM of each dNTP, 1X reaction buffer and 1 U Platinum Taq polymerase (Invitrogen). Amplifications were performed with the following conditions: Denaturation step of 5 min at 94ºC, 35 cycles of 30 sec at 94ºC, 30 sec at annealing temperature and 45 sec at 72ºC. The final extension step was of 10 min at 72ºC. Details of primers sequences, SSR location, amplification condition, and product size are described in Table 2.

Samples were mixed with denaturing loading buffer, treated for 5 min at 95ºC and separated in a 6% polyacrylamide gel. Amplification products were silver-stained following the DNA silver staining system procedure (Promega).

Loci with poor amplification products in standard PCR conditions were amplified with fluorescent forward primer. PCR products were detected in the ABI3100 Genetic Analyzers (Applied Biosystems, USA) and the allele assignments were made by size comparison with the standard allelic ladders and using the GenMapper ID software provided by Applied Biosystems. To minimise scoring errors associated with base pair shifts between runs, at least four control samples were also included in each run.

Statistical analyses

The number and frequency of alleles as well as the effective number of alleles Ne (), observed heterozygosity (Ho), and probability of identity (PI, which shows the probability of two unrelated individuals displaying the same genotype) () were determined with the GenAlEx 6.4 program (Peakall and Smouse, 2006).

The PIC of each marker was calculated according to Botstein et al. (1980) using the following formula, where pi is the frequence of the ith allele.

Null allele frequencies were estimated with INEST software (Inbreeding/Null allele Estimation) (Chybicki and Burczyk, 2009) which is adequate for this species subject to a mixed mating model (Faria et al. 2011) under the IIM (individual inbreeding model).

Distances between individuals based on the microsatellite data set were carried out according to the shared allele distance (DAS) (Chakraborty and Jin, 1993) and was computed with POPULATIONS 1.2.28 software (Langella, 2002). Cluster analyses were implemented based on distances matrices using the unweighted pair group method arithmetic average (UPGMA), and the corresponding phenogram was constructed.

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 thaliana homocysteine 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.

Polymorphism in CCR gene

A CT microsatellite located in the promoter of the Eucalyptus gunnii CCR (GenBank: AJ132750) was found between positions -164 and -135. This region is close to that described by Lacombe et al. (2000) at position -119 to -77 (GenBank: AJ132750) as necessary and sufficient for the expression in vascular tissues of stems. Twelve different alleles (ranging between 182 and 216 bp size) were detected, indicating a remarkably high level of allelic variability. Like most of SSR-CG described here, the number of alleles (Na) was higher than the effective number of alleles (Ne), which was 2.7 for this locus. This result shows that only a small number of equally frequent alleles are present (i.e. one allele of 182 bp with a frequency of 0.588). A relatively high PIC of 0.617 and low Ho as well as PI values of 0.275 and 0.15 respectively were found (Table 3). The highly estimated null frequency alleles (0.217) possibly explain the low observed heterozygous level and prevents its usefulness for fingerprinting analysis.

CCR locus has already been found to co-localize with QTLs for both lignin content and the syringil-guaiacyl monomers ratio in other Eucalyptus species (E. grandis x E. urophylla hybrids) by Gion et al. (2001) and Gion et al. (2011).

In A. thaliana, 10 putative CCR genes have been reported (Jones et al. 2001). However, for the genus Eucalyptus, only one CCR gene has been found in E. gunnii, E. globulus and E. tenuiramis (Lacombe et al. 1997; Poke et al. 2003).

Polymorphism for this gene was previously reported in E. globulus (Poke et al. 2003; Poke et al. 2004) and Thumma et al. (2005) found specific polymorphisms in CCR associated with variation in microfibril angle (MFA) with linkage disequilibrium (LD) mapping analysis using SNP-marker, haplotype analyses and family-based studies in E. nitens.

In this study, we identified a CT microsatellite within the promoter of the gene, which might be involved in transcription regulation. As mentioned, it is close to a position described by Lacombe et al. (2000) essential to drive xylem-localized gene expression.

Presence of microsatellites in 5’UTR has been described already by other authors (Kumpatla and Mukhopadhyay, 2005). For instance, in Populus, 1.2% of total SSR found after genome sequence (around 1510) was discovered in 5’UTR (Arnaud et al. 2007).

CT elements have also been described in A. thaliana core promoters (Molina and Grotewold, 2005). Furthermore in Arabidopsis and Brassica, analysis in silico of known cis-regulatory elements showed that light responsive elements were clustered in the region of CT/GA repeats (Zhang et al. 2006).

Polymorphism in CesA3 gene

Higher plants contain a family of cellulose synthase catalytic subunit (CesA) genes that encode components of an enzyme complex embedded in the cell membrane.

Creux et al. (2008) showed that CesA promoters from Eucalyptus trees are functional in Arabidopsis plants, despite the low amounts of sequence similarity between orthologous CesA promoters. This functional conservation is made possible by cis-elements that have been retained during evolution along with the corresponding transcription factors.

Rengel et al. (2009) released a collection of Eucalyptus ESTs originating from one normalized differentiating xylem cDNA library and four subtractive libraries, named EUCAWOOD. They found five unigenes homologous to members of the CesA multigene family that correspond to the EgCesA1-EgCesA5 genes recently described in E. grandis (Ranik and Myburg, 2006).

In this study, we identified two microsatellite regions in the promoter zone of CesA3 in E. grandis (GenBank: EU165713). One of these, with a GA repeat motif, was found between positions -804 and -787 whereas a repeat motif CT was found between positions -248 and -226. Both genomic regions were denominated CesA3a and CesA3b, respectively (Table 2) and, like the enzyme described before, these motifs could be transcription regulation elements.

Diversity analyses revealed a high number of alleles: 12 (285 to 307 bp) and 13 (342 to 367 bp) for CesA3a and CesA3b microsatellites, respectively. As it was mentioned before, Ne values were of 4.38 and 5.82 for both loci respectively and lower than their corresponding Na.

High PIC values of 0.746 and 0.807 were found for CesA3a and CesA3b. Ho was 0.519 and 0.692 and PI was 0.078 and 0.05 respectively (Table 3). Estimated null frequency alleles was higher for CesA3a (0.132) than CesA3b (0.079), possibly explaining the lower observed heterozygous level for CesA3A.

Polymorphism in LIM1 transcription factor gene

The AC-rich motif, Pal-box, is an important cis-acting element for gene expression in phenylpropanoid biosynthesis. LIM transcription factor can bind to the Pal-box cis element in the promoters of monolignol biosynthetic genes leading to repression of the LIM gene and inhibition of lignin biosynthesis in transgenic tobacco plants. A reduction of lignin content in the woody plant E. camaldulensis by the suppression of gene expression of the LIM domain transcription factor was reported by Kawaoka et al. (2006). In poplar, the distributionof ESTs in different wood tissuesindicates a rather high expression of an LIM protein homologuein tension wood (Arnaud et al. 2007).

Rengel et al. (2009) found in their libraries 3 unigenes which resemble LIM transcription factors i.e. contig named Cg2892 was similar to EcLIM1 from E. camaldulensis, sharing 86% homology with Nicotiana tabacum (NtLIM1).

We identified a GA repetitive motif between positions -43 and -1 in GenBank: AB208710 sequence. High level of variability was found in the sample assayed: 16 alleles were identified, ranging from 159 and 194 bp in size and the highest Ne (7.56) of tested loci. This locus showed the highest PIC value (0.855), a relatively high Ho value (0.769), the lowest PI value (0.03) and a relative low null allele frequency estimates (0.054) (Table 3), making it useful for fingerprinting analysis.

Polymorphism in homocysteine S-methyltransferase gene

In this study, we identified a microsatellite in E. grandis GenBank EST CB967988 similar to Arabidopsis thaliana homocysteine S-methyltransferase (HMT) gene. However, the resulting amplification products were higher than the expected size for E. grandis. In consequence, the presence of the microsatellite was confirmed after automatic sequencing. Sequence data from this genomic fragment was annotated in the GenBank under accession number FJ492059.

A repeat motif CT was found within intron region, after multiple alignment and comparison with EST CB967988 similar to HMT (Figure 1). 

Intronic SSRs can affect gene transcription, mRNA splicing, export to cytoplasm, and can also induce heterochromatin-mediated-like gene silencing (Li et al. 2002). These SSRs may provide a molecular basis for fast adaptation to environmental changes in both prokaryotes and eukaryotes (Li et al. 2002). It is not known yet if the here detected differences in SSR length are actually responsible for such changes in Eucalyptus. Functional studies using molecular biology techniques for each of the candidate genes are needed to establish if this is happening here.

We found 13 alleles (415 bp-465 bp), generating a Ne of 5.33, a PIC of 0.791, a Ho of 0.745, a PI of 0.056 and null allele frequency estimates of 0.047 (Table 3), being this locus one of the most informative in this study.

Polymorphism in SK gene

The shikimate pathway links metabolism of carbohydrates to biosynthesis of aromatic compounds. In a sequence of seven metabolic steps, phosphoenolpyruvate and erythrose 4-phosphate are converted to chorismate, the precursor of the aromatic amino acids and many aromatic secondary metabolites (Herrmann and Weaver, 1999).

SK enzyme belongs to the family of transferases, specifically those transferring phosphorus-containing groups (phosphotransferases) with an alcohol group as acceptor.

We have found an E. globulus EST (GenBank: BF942502) similar to A. thaliana SK, harbouring a microsatellite region of CT motif.

Fifteen alleles (between 331 bp and 386 bp) and a Ne of 4.8 were revealed, whereas PIC and Ho presented values of 0.766 and 0.604, respectively. PI of 0.068 and null alleles frequency estimates of 0.095 were calculated (Table 3), showing its usefulness for fingerprinting analysis.

Polymorphism in XTH2 gene

Xyloglucans consist of a backbone of (1, 4)-βD-glucan substituted with xylosyl, galactosyl, and fucosyl residues (Hrmova et al. 2007). XTHs are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls (Genovesi et al. 2008).

We found a pentanucleotide (CGAAA) microsatellite motif in a putative E. globulus XTH2 mRNA (GenBank: DQ100338). No sequence similarity was found when BLASTN of the SSR region was performed through sequences coding for XTH1, XTH3 and XTH4 (GenBank: DQ100337, DQ100339 and DQ100340), suggesting that SSR motif is only present in XTH2 gene.

In contrast with the above described dinucleotide motif microsatellites, only three alleles (Na) were found, with 122, 129 and 134 bp of size in the sample evaluated. While the allele of 129 bp was predominant in most individuals (n = 44), the allele of 134 bp was present in 17 while allele of 122 bp was present in just two, giving a low (Ne) of 1.57. PIC and Ho presented values of 0.310 and 0.370, respectively. PI of 0.46 and null alleles frequency estimates of 0.054 were calculated (Table 3), being this locus the less informative in this study.

Polymorphism in glutathione S- transferase gene

Plant glutathione S-transferases (GSTs) comprise a heterogeneous super family of multifunctional proteins (Dean et al. 1995; Dean and Devarenne, 1997; Edwards et al. 2000; Kampranis et al. 2000; Loyall et al. 2000; Mueller et al. 2000; Thom et al. 2002; Kilili et al. 2004).

We amplified a microsatellite region in an E. globulus GenBank: EST CT988111 highly similar to Populus trichocarpa tau class GST sequence containing a tetranucleotide motif TATG. Twelve different alleles (varying from 83 bp to 120 bp in size) and a Ne of 7.32 were found. PIC and Ho values were 0.849 and 0.815, respectively (Table 3). This high number of alleles, the lowest PI value (0.033) and low null allele frequency estimates (0.036), plus the tetranucleotide motif makes this SSR potentially very useful for identification purposes.

Genetic diversity of the putatively functional markers

A sample of 54 representative genotypes of Eucalyptus globulus was evaluated to accurately estimate genetic information. Table 3 shows the results obtained from the statistical analysis of the microsatellite allele pattern distribution found in the seven candidate genes analyzed.

In this report, most of the SSR found were dinucleotides repeats and ranged from 132 bp to 453 bp. Although longer motives of repeats are easily and accurate for scoring and may give better resolution patterns, usually, these are less polymorphic than shorter repeats (Tang et al. 2008; Faria et al. 2011). However, clear patterns were found in all SSR with dinucleotides motives reported here and stutter profiles could be defined adequately (Figure 2).

Ho was moderated to high except for the XTH2 and CCR loci. PIC values ranged from 0.310 to 0.855, PI of a locus varied from 0.033 to 0.151, except for XTH2 locus that showed a value close to 0.5. The resulting probability value for the combined eight loci was very low and of 1.04 10-9 (computed as the product of individual PI) making this set of loci mainly useful for fingerprinting analyses. This was corroborated when an analysis of genetic distances between all 54 individuals showed a mean of 0.704 (sd 0.129) and only five pairs of trees showed distances between 0.3 and 0.273 (the lowest value), reflecting the high resolution capacity of microsatellites (Figure 3).

Null alleles may bias estimates of allele and genotype frequencies, thereby hindering population genetic analyses and individual identification (Nascimento de Sousa et al 2005; Chybicki and Burczyk 2009). As no progeny tests were carried out, we cannot discard nucleotide sequence variability of the annealing site for the primer which may lead to null alleles. There are some few reports on the frequency of microsatellite null alleles like that described in Brondani et al. (2006), where they could find about an 8% overall occurrence of null alleles when mapping a segregating population between E. grandis and E. urophylla. In E. camaldulensis, Butcher et al. (2009) estimated that the frequency of null alleles was less than 5% (averaged over 97 populations) for the 15 loci explored. Faria et al. (2010) and Faria et al. (2011) described that most populations that deviated from HWE displayed null allele frequencies of around 0.1-0.3.

In this study, low frequencies of null alleles were estimated for each loci ranging from 0.036 (GST) to 0.217 (CCR), and 6 had a maximum of 0.10 (Table 3).

In Eucalyptus, the probability of maintaining low-frequency alleles or rare alleles in the population is high (Grattapaglia and Kirst, 2008).

In this study, the SSR allowed to detect a total of 21 trees with low frequencies alleles. We found different proportions of alleles per locus that were present in just one individual (frequency close to 1%) for all the loci except XTH2: CCR (33.3%), CesA3a (33.3%), CesA3b (23.1%), LIM1 (31.2%), HMT (23.1%), SK (46.7%) and GST (8.3%). These results are reflected by a relatively low number of Ne (mean of 4.9 alleles per locus) which shows the number of equally frequent alleles, respect to the high number of alleles (mean A = 12 alleles per locus). SK gene had the highest percentage (46.7%) of alleles present in only one tree, and on the other extreme, GST only showed 8.3%. Besides, their classification as rare alleles is still preliminary because of the relatively limited number of trees (54 individuals) analyzed here.

These results are in good agreement with those of Steane et al. (2006), where they could find many rare alleles across the whole population of E. globulus, showing a relatively low Ne number (average 6.1 alleles per locus) and a high number of alleles (mean A = 19.4 alleles per locus).

Functional perspectives of SSR

Simple sequence repeat variation within genes could be very critical for normal gene activity because repetition motif expansion or contraction could directly affect the corresponding gene expression and even cause phenotypic changes like codon frame shift, fluctuation of gene expression, inactivation of gene activity, and/or change of function and, eventually, phenotypic changes (Li et al. 2004). Genetic microsatellite markers often have known or ‘putative’ functions when they are gene targeted, with the potential of representing functional differences in those cases where polymorphisms in the repeat motifs affect the function of the gene in which they reside (Andersen and Lubberstedt, 2003). Although this association discovery has not been carried out yet for Eucalyptus, the characterization of new markers located in candidate genes for wood quality demonstrated to be useful for genetic studies such as QTLs mapping and marker-assisted breeding in Eucalyptus.

It is not unusual to find SSRs mainly, but not exclusively, in the 5’ and 3’ untranslated regions (UTRs). From a previous analysis of public E. globulus’s EST (Acuña et al. 2011), 72% of the SSR sequences discovered were within ORF sequences, while 28% were not. Although more SSR are usually found in UTRs than in ORF (Morgante et al. 2002) this unexpected result might be caused by an overestimation of the ORF length.

It is expected that tri- and hexanucleotide repeats would occur more frequently than other motifs in coding sequences (Metzgar et al. 2000). Such dominance of triplets over other repeats in coding regions may be explained on the basis of the selective disadvantage of non-trimeric SSR variants in coding regions, possibly causing frame-shift mutations (Metzgar et al. 2000).

In this work, we did not find trinucleotide repeats inside exons. However, two out of three non-trinucleotide repeats were multiples of three, which did not modify the reading frame (i.e. three motifs of five nucleotides = 15 bp = 5 codons) (Table 2).

In order to find out if any of the candidate gene alleles differing in SSR motif repetition numbers are related to wood qualities, genetic association studies are in progress. In those cases where linkage disequilibrium is confirmed, qPCR and/or differential RNAseq studies will be carried out in order to investigate if transcriptional regulation of any of these genes has a key role in these traits.

Concluding Remarks

This study contributes to previously reported analyses of nucleotide diversity in candidate genes in Eucalyptus (Poke et al. 2003; Thumma et al. 2005). This is the first report on the identification and diversity analysis of microsatellite markers located inside putative wood quality candidate genes. These eight microsatellites belong to seven genes involved in lignin, cellulose and hemicellulose biosynthesis: CCR, HMT, SK, GST, transcription factor LIM1, CesA3 and XTH. The characterized microsatellites were located in promoters, introns and exons and show high levels of polymorphism (mean PIC:0,769).

Potentially, these functional markers may contribute to the validation of gene and QTL positions in multiple pedigrees in Eucalyptus globulus.

Acknowledgments

The authors thank Dr. Andrea Puebla, Verónica Nishinakamasu and Pablo Vera for gene sequencing and their help during genotyping. We are grateful to Corina Fusari for their scientific assistance during the first part of this work. The first author thanks her colleagues from IRB and IB of INTA for their continuous help and support.

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