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Gene-associated Single Nucleotide Polymorphism (SNP) in Cinnamate 4-Hydroxylase (C4H) and Cinnamyl Alcohol Dehydrogenase (CAD) Genes from Acacia mangium Superbulk Trees



B.L. Tchin, W.S. Ho, S.L. Pang and J. Ismail
 
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ABSTRACT

Candidate-gene-based association study which involves the identification of causative Single Nucleotide Polymorphisms (SNPs) for excellent traits has been proposed as a promising approach to dissect complex traits in forest trees. Hence, the goal of this study was to identify the genetic association among SNPs from Cinnamate 4-Hydroxylase (C4H) and Cinnamyl Alcohol Dehydrogenase (CAD) genes and an array of wood properties namely, specific gravity, wood density, fiber-length, cell wall thickness and microfibril angle from Acacia mangium Superbulk trees. Sequence variations within these two genes in 12 A. mangium Superbulk trees were examined and wood properties were measured. The data obtained was tested using General Linear Model (GLM) within TASSEL software. Two SNPs were identified in the exon of C4H, of which all the SNPs caused nonsynonymous mutations whereas five SNPs were identified in the CAD exons along with one deletion mutation. In addition, two SNPs were also identified in the CAD introns. Variation in these two lignin biosynthesis genes might change the structural, functional or biochemical properties of the enzyme being produced, and therefore possibly lead to changes in phenotypic characteristic of the trees. The genetic association study also revealed that SNPs in CAD gene do associate with the wood density, specific gravity and cell wall thickness (p<0.05). However, no significant results were obtained for SNPs in C4H gene with wood properties studied. Thickening of cell wall is affected by the arrangement of biopolymer aggregates which comprise of cellulose, hemicellulose and lignin. Results indicated that SNP in CAD gene might alter the lignin biosynthesis and thus lead to changes in phenotypic characteristics of the trees. Overall, the study has demonstrated that SNP is very useful in association genetic study to identify Quantitative Trait Nucleotide (QTN) which then leads to Gene-assisted Selection (GAS) in the tree breeding programme.

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B.L. Tchin, W.S. Ho, S.L. Pang and J. Ismail, 2011. Gene-associated Single Nucleotide Polymorphism (SNP) in Cinnamate 4-Hydroxylase (C4H) and Cinnamyl Alcohol Dehydrogenase (CAD) Genes from Acacia mangium Superbulk Trees. Biotechnology, 10: 303-315.

DOI: 10.3923/biotech.2011.303.315

URL: https://scialert.net/abstract/?doi=biotech.2011.303.315
 
Received: February 28, 2011; Accepted: August 02, 2011; Published: October 03, 2011



INTRODUCTION

Acacia mangium Superbulk, as one of the fast growing and with high adaptability species, is introduced to Malaysia for forest plantations and rehabilitation of marginal and degraded lands purposes (Wickneswari et al., 2005). To date, A. mangium has been extensively planted in Sarawak and the wood derived from this species is widely used for the production of fine furniture, veneers, plywood, construction of building as well as paper and pulp making (Lim et al., 2003). Due to the high potential uses of Acacia wood, better understanding the structure and composition of the wood is vital for optimal utilization of this wood material.

Wood is one of the most complex materials which composed of polymers of lignin and carbohydrates that are physically and chemically bond together. Lignin, the second most abundant organic compound after cellulose, represents approximately 20-30% of the plant biomass. Lignin is formed through dehydrogenative polymerization of monolignols known as coumaryl alcohol, coniferyl alcohol and sinapyl alcohol which will give rise to ρ-coumaryl units (H), guaiacyl (G) units and sinapyl (S) units, respectively. Functions of lignin involve the mechanical and structural support to the plants as well as a significant protective function again pathogen or decaying fungi (Brett and Waldron, 1990).

Many genes are involved in the lignin biosynthesis pathway. In this study, the major focus is on Cinnamate 4-Hydroxylase (C4H) and Cinnamyl Alcohol Dehydrogenase (CAD) genes that originate from A. mangium Superbulk. The main function of C4H is to catalyze the hydroxylation of cinnamate to 4-coumarate at the early stage of lignin biosynthesis pathway while CAD catalyzes the reduction of cinnamaldehydes to ρ-coumaryl, coniferyl and sinapyl alcohols during the final stage of lignin biosynthesis pathway (Lewis, 1999). Expression of C4H and CAD genes are very influential in the lignin biosynthesis pathway and any up- or down-regulation of these genes will be resulted in altered lignin production (Baucher et al., 2003). Abreu et al. (2009) had proposed that the high β-O-4 (Alkyl Aril Ether) bonds in lignin of angiosperms may possibly affect the wood properties. Therefore, extensive study needs to be carried out in order to determine the association between the lignin and the wood properties.

However, it is difficult to study the inherent complexity of these traits through traditional linkage-based approaches since it is time consuming, laborious and often expensive process of establishing the mapping population (Myles et al., 2009). On the other hand, candidate-gene-based association study which involves the identification of causative polymorphisms (SNPs) for excellent traits has been proposed as a promising approach to dissect complex traits in forest trees species (Gonzalez-Martinez et al., 2006; Ho et al., 2011). SNP is a single base mutation where DNA sequences differ only by a single base. SNPs have become marker of choice for many applications in genome analysis because SNPs are abundance, stable, ubiquity and interspersed in nuclear genome (Rafalski, 2002).

Association genetic study is a natural uncontrolled experiment that can give a higher mapping resolution compared with the linkage mapping (Neale and Savolainen, 2004). In the past decades, association study has been widely used not only in human (Abbasi et al., 2009), animal (Alashawkany et al., 2008) and crop species (Aina et al., 2007; Rashwan, 2011) but also become increasingly utilize in forest trees species. This can be proved by the first published association study in forest tree species by Thumma et al. (2005) where 2 SNPs found in CCR gene were significantly associated with microfibril angle from Eucalyptus nitens. The result was then validated in two full-sib families of E. nitens and E. globules. Yu et al. (2006) also detected the association of the cad-n1 allele with increased stem growth and wood density in 15 years old loblolly pine. Later Gonzalez-Martinez et al. (2007) had found that a nonsynonymous substitution for CAD in loblolly pine was in strong association with earlywood specific gravity and causing lignin modification in wild trees.

Discovery of SNPs through association genetic study would result in the identification of causative variants (quantitative trait nucleotides, QTNs) which can be used as markers for gene-assisted selection in tree breeding programme to choose for good planting materials based on their DNA sequences (Fusari et al., 2008). This strategy is very important since it can increase the productivity of forests and enhance the wood quality or properties. By knowing the exact identity and properties of the timber through gene-assisted selection, one can easily determine and select for the desired trees in short period of time and thus reducing the costs of phenotypic testing.

Hence, the primary goal of this study was to identify the genetic association among single nucleotide polymorphisms (SNPs) from C4H and CAD genes and an array of wood properties namely, specific gravity, wood density, fiber-length, cell wall thickness and microfibril angle from A. mangium Superbulk trees. From the study, association was detected between CAD gene and wood properties studied but no significant result was obtained for SNPs in C4H gene.

MATERIALS AND METHODS

Plant materials: A total of 12 fresh leaf samples were collected from a 3-year old A. mangium Superbulk research plot at the UNIMAS’s arboretum in August 2009. The research plot was established by using the second generation seedlings originally from the 49 superior A. mangium parent trees planted in the CSIRO first generation seedling seed orchards in north Queensland, Australia. A. mangium Superbulk is a plus tree or second generation of A. mangium in which the wood properties have been improved through many years of selected planting. Total genomic DNA was extracted from the fresh leaf tissues by using a modified protocol of Doyle and Doyle (1990). The extracted DNA was then purified by using Wizard® Genomic DNA Purification Kit (Promega, USA).

Polymerase chain reaction (PCR) amplification: PCR was performed on a Mastercycler Gradient Thermal Cycler (Eppendorf, Germany). Three specific primer sets designed from the full length C4H (EU275980) and CAD (EU275981) cDNA of Acacia hybrid (Pang et al., 2005) were used. PCR reaction mixture consists of 30 ng of DNA template, 5 pmol of forward and reverse primers, 0.2 mM dNTPs, 1x PCR buffer (200 mM Tris-HCI pH 8.8, 100 mM KCI, 1% Triton X-100, 100 mM (NH4)2SO4, 1 μg mL-1 BSA), 1.5 mM MgCI2, 1 U Taq DNA Polymerase (Promega, USA) and sterile distilled water to make up 25 μL. C4H amplification was performed for 2 min at 95°C, 35 cycles of 45 sec at 94°C, 45 sec at 63°C and 1 min at 72°C, followed by final extension of 10 min at 72°C. CAD amplification was performed with PCR parameters same as C4H but with annealing temperature at 63°C for primers CAD-PT1 and 61°C for primers CAD-PT2.

Cloning and sequencing: Purified PCR product was ligated into pGEM®-T Easy Vector System (Promega, USA) and transformed into competent cells of Escherichia coli JM 109. Positive clones were cultured in 50 mL Falcon tubes containing 5 mL LB Broth supplemented with 100 mg mL-1 ampicillin for overnight. The recombinant plasmids were isolated and purified using Wizard® Plus SV Miniprep DNA Purification System (Promega, USA) according to the manufacture’s protocol. The purified plasmids were sent for sequencing.

Data analysis: Base calling and vector sequences were removed using Chromas version 2.33 (Technelysium, AU). Each sequence was verified and checked for their homology using BLASTn (Altschul et al., 1990) through BLAST Search Engines (http://blast.ncbi.nlm.nih.gov/). The sequences of all trees were aligned using CLC Free Workbench 4 software (CLC Bio, Denmark) to observe the SNPs. Later, all sequences were translated into open reading frames using ORF finder (http://us.expasy.org/tools/dna.html).The open reading frames were aligned among 12 individuals using CLC Free Workbench 4 software (CLC Bio, Denmark) in order to detect the synonymous and nonsynonymous mutations.

Phenotypic data: Radial wood cores (5 mm) used in this study were sampled at the breast height from 12 Acacia mangium Superbulk trees. All trees were at 3 years old and planted in UNIMAS’s arboretum. The heartwood was separated from sapwood and only heartwood was used for subsequent measurement. The wood density and specific gravity were firstly determined for each heartwood core.

Fiber-length: Few thin wood pieces were cut from wood cores with scalpel. The wood pieces were immersed into test tube that pre-filled with glacial acid and 30% hydrogen peroxide (1: 2) followed by incubation in water bath at 60-70°C until the wood pieces soft and turned into white color. The fibers then were separated by shaking the tube in the presence of dd H2O. The fibers were placed on glass slides with cover slips and 50 fiber length measurements were made for each sample by using confocal laser scanning microscope (CLSM).

Microfibril angle (MFA) and cell wall thickness: The softened wood cores were cut with microtome at 16-18 μm thickness and in two dimensions, known as transverse and radial. Microfibril angle was measured at radial section while cell wall thickness was measured at transverse section. Radial sections were stained with 0.1% congo red and sonicated for 3 h. The washed wood slices were mounted on glass slides with cover slips. The microfibril angle in the S2 layer of the fiber walls was determined using the polarized light microscope. Fifty replicate measurements of the microfibril angle were made for each sample. Transverse sections (Fig. 1) were stained with 1% safranin and then washed with dd H2O and a series of ethanol at different concentrations. After that, stained sections were mounted permanently on the glass slide by using DPX mounting medium. All the slides were dried and then used for observation.

Fig. 1(a-b): Transverse section of A. mangium Superbulk. Cell wall thickness was measured from the transverse section of A. mangium Superbulk wood by using confocal laser scanning microscope (40X objective). From the figures, the cell wall of SB1 sample (a) was thicker than the cell wall of SB6 sample (b)

Observations were performed using confocal laser scanning microscope (CLSM) and 50 fiber cell wall thickness measurements were made for each sample.

Test statistics for association: Association between the SNPs discovered and phenotypic traits was tested using General Linear Model (GLM) that performs association analysis by a least squares fixed effects model. GLM is available within Trait Analysis by Association, Evolution and Linkage (TASSEL) software (http://www.maizegenetics.net/tassel).

RESULTS AND DISCUSSION

Cloning of partial C4H and CAD genes: Partial DNA sequences of C4H and CAD were cloned and sequenced from 12 samples. After trimming, the identity of each nucleotide sequence was verified by using BLASTn. As shown in Table 1, the partial sequence of C4H showed high degree of similarity to C4H sequence from Acacia auriculiformisxAcacia mangium hybrid (98%) and Sorghum bicolor (86%) but with limited nucleotides coverage. While for partial sequence of CAD, high degree of similarity was obtained when compared with Acacia auriculiformis x Acacia mangium hybrid (98%), Populus tomentosa (80%) and Eucalyptus urophylla (82%) (Table 2). After verification, the partial DNA sequences of C4H and CAD were aligned with the C4H (ABX75854.1) and CAD (EU275982.1) full length cDNA sequence of Acacia hybrid to determine the intronic and exonic region. From the alignments, the partial C4H sequence obtained was fall within the exonic region (Fig. 2). Meanwhile three exonic regions and two intronic regions were found within the partial CAD sequence (Fig. 3).

Table 1: BLASTn output for amplified partial C4H DNA sequence

Table 2: BLASTn output for amplified partial CAD DNA sequence

Fig. 2: The exonic region of partial C4H DNA sequence. The partial C4H DNA sequence (457bp) of A. mangium Superbulk was aligned with the full-length C4H cDNA from Acacia hybrid and the result showed that the partial C4H DNA sequence was found within the exonic region of the gene

Fig. 3: The exonic and intronic regions of partial CAD DNA sequence. The partial CAD DNA sequence (1147bp) of A. mangium Superbulk was aligned with the full-length CAD cDNA from Acacia hybrid. The diagram indicates the predicted position of intron and exon region in partial CAD DNA sequence. Three exonic regions and two intronic regions were found within the partial CAD DNA sequence

Fig. 4: Sequence variations within partial C4H DNA sequences. The arrow indicates the position and identity of SNPs in partial C4H DNA sequences. Two SNPs were detected from the multiple alignment of 12 partial C4H DNA sequences of A. mangium Superbulk (Gene accession number: SB1-C4H: HQ848681, SB2-C4H: HQ848682, SB3-C4H: HQ848683, SB4-C4H: HQ848684, SB5-C4H: HQ848685, SB6-C4H: HQ848686, SB7-C4H: HQ848687, SB8-C4H: HQ848688, SB9-C4H: HQ848689, SB10-C4H: HQ848690, SB11-C4H: HQ848691, SB12-C4H: HQ848692)

Table 3: The distribution of SNPs in partial C4H and CAD genes

Table 4: The number of SNPs in partial C4H and CAD genes

Sequence variations: Partial DNA sequences of C4H and CAD were aligned together by using CLC Free Main Workbench 4 (CLC Bio, Denmark) software, respectively in order to detect the SNPs. From the partial C4H alignment, 2 SNPs were found in the coding region of C4H (Fig. 4). Since all the sequences obtained fall within the exonic region, hence no SNP was found within the non-coding region.


Fig. 5: Sequence variations within partial CAD DNA sequences. The arrow indicates the position and identity of SNPs in partial CAD DNA sequences. Eight SNPs were detected from the multiple alignment of 12 partial CAD DNA sequences of A. mangium Superbulk (Gene accession number: SB1-CAD: HQ848669, SB2-CAD: HQ848670, SB3-CAD: HQ848671, SB4-CAD: HQ848672, SB5-CAD: HQ848673, SB6-CAD: HQ848674, SB7-CAD: HQ848675, SB8-CAD: HQ848676, SB9-CAD: HQ848677, SB10-CAD: HQ848678, SB11-CAD: HQ848679, SB12-CAD: HQ848680)

Among the 2 SNPs detected, one was resulted from transition mutation while the other one was resulted from transversion mutations.

For partial CAD alignment, a total of 8 SNPs were found where 5 of the SNPs as well as an indel mutation were located in the exonic region and another 2 SNPs were located in the intronic region (Fig. 5). Among the SNPs detected, 6 were resulted from transition mutation and one was resulted from transversion mutations. The distribution and occurrence of the SNPs are summarized in Table 3 and 4.

Table 5: Nonsynonymous mutations in partial DNA sequences of C4H

Table 6: Synonymous and nonsynonymous mutations in partial DNA sequences of CAD

On average, one SNP occurred at every 229 bp in the partial sequence of C4H and at every 143 bp in the partial sequence of CAD (Table 4). Since the full length of C4H and CAD genes have not yet discovered, the comparison of SNP frequency to other studies of DNA sequence diversity cannot be done. However, it had been reported that the occurrence of SNPs in C4H of A. mangiumxA. auriculiformis is one SNP in every 53 bp (Nur Fariza et al., 2008). Meanwhiles, other studies also had reported on the SNP frequency such as in barley (1 SNP in every 189 bp) (Kanazin et al., 2002), grapes (1 SNP in every 0.1 kb) (Velasco et al., 2007) and soybean (1 SNP in every 273 bp) (Zhu et al., 2003). On the other hand, the occurrence of SNP in CAD was one SNP in every 147 bp for E. globules (Poke et al. 2003) and one SNP in every 40 bp for A. mangiumxA. auriculiformis (Nur Fariza et al., 2008).

For synonymous and nonsynonymous mutations study, all exonic sequences were translated into open reading frames and then aligned among 12 individuals trees using CLC Free Main Workbench 4 (CLC Bio, Denmark) software. For C4H, 2 nonsynonymous mutations were detected within the open reading frame of C4H and no synonymous mutation was identified (Fig. 6 and Table 5). Meanwhile, 5 nonsynonymous mutations and one synonymous mutation were detected within the open reading frame of CAD (Table 6). The single indel mutation at the exon region of CAD sequences had led to the incorporation of 3 stop codons in all amino acid sequences, except SB1 (Fig. 7). This indicates that the indel mutation detected in CAD gene may regulate the genetic variation of wood properties or phenotypic differences among trees. For instance, indels found in the exons of the Arabidopsis accessions Eil-0 and Lc-0 caused drastic effects on gene integrity, specifically on the gene representing expression level polymorphisms (Plantegenet et al., 2009). However, decision on the popularity of synonymous and nonsynonymous mutations can only be made when full length sequences of C4H and CAD genes are studied. Synonymous mutation does not lead to changes in the amino acid sequence of resulting protein while nonsynonymous mutations do. Nonsynonymous SNPs which cause changes to the protein sequence can affect the structural, functional or biochemical properties of the enzyme being produced (Bromberg and Rost, 2007) and therefore possibly lead to the changes in phenotypic characteristic of the trees, especially in modification of lignin biosynthesis.

Wood properties: Table 7 shows the mean values of fiber-length, cell wall thickness, microfibril angle, specific gravity and wood density. As reviewed by Plomion et al. (2000) in Eucalypytus and Pinus, there are strong relationships between growth strain and wood characteristics, such as wood density, longitudinal and tangential shrinkages, longitudinal modulus of elasticity, anatomical characteristics (microfibril angle) and chemical composition (lignin content, ratio of lignin monomeric units, hemicelluloses and cellulose content). Fibre wall thickness is closely related with wood density which in turn is probably the single most important predictor of wood properties such as strength, hardness, dimensional stability and others. High quality timbers which are heavy, hard and very strong, will have very thick-walled fibres and it is usually not easy to work with (Baas and Wheeler, 2000).

Fig. 6: Synonymous and nonsynonymous mutations in partial C4H amino acid sequence. The arrow indicates the position and identity of two nonsynonymous SNPs in predicted partial C4H amino acid sequences

Table 7: Wood properties data obtained for each A. mangium Superbulk sample
SG: Specific gravity, WD: Wood density, CWT: Cell wall thickness, MFA: Microfibril angle

The thickness of the cell walls is increased by changing the microfibril orientation continually (Higuchi, 1997). Therefore, the microfibril angle is highly correlated with the cell wall thickness. Micofibril angle is also closely related to the mechanical properties of wood and pulp fibers since low microfibril angle will lead to high tensile strength and stiffness (Lima et al., 2004). Other than that, Bonham and Barnett (2001) observed that low microfibril angles are also associated with long fiber lengths. However, no significant correlation was observed among the physical wood property traits measured in this study.

Association genetics analysis: The association between the SNPs discovered and phenotypic variations on 12 A. mangium Superbulk samples were tested using General Linear Model (GLM) provided by TASSEL software.

Fig. 7: Synonymous and nonsynonymous mutations in partial CAD amino acid sequence. The red arrow indicates the position and identity of nonsynonymous and synonymous SNPs and the blue arrow indicates the positions of stop codons at the end of all predicted amino acid sequences, except SB1

Table 8: Significant association detected between marker in CAD gene and wood properties
SG: Specific gravity, WD: Wood density, CWT: Cell wall thickness

Genetic association study revealed that nonsynonymous SNPs in CAD gene do associate with the specific gravity and wood density (p<0.05) (Table 8). These findings were similar with the previous studies done by Gonzalez-Martinez et al. (2007) and Yu et al. (2006), respectively. Specific gravity and wood density are tightly link to most of the mechanical properties of the wood. The strength, stiffness, heat transmission and some other wood properties are increased with specific gravity. The most interesting is that, the yield of the pulp per unit volume is also directly related to specific gravity. This give sense that the specific gravity or wood density should be the first wood property to be sincerely investigated (Haygreen and Bowyer, 1996).

Moreover, significant association (p<0.05) was also found for nonsynonymous SNPs in CAD gene with fiber cell wall thickness (Table 8). But the associations of SNPs in CAD gene with fiber-length and microfibril angle were not significant. Thickening of cell wall is affected by the arrangement of biopolymer aggregates which comprise of cellulose, hemicellulose and lignin. While the concentration of lignin is as a proportion of the cell wall and decrease when come closer to the lumen, therefore single nucleotide mutation in CAD gene might alter the lignin biosynthesis and thus, lead to changes in cell wall thickness as well as the wood density and specific gravity.

On the other hand, no significant results were obtained for SNPs in C4H gene with all wood properties studied. Although association was found among CAD and wood properties, the possibility of the false positive result that may arise due to the structure of population cannot be ignored (Yu et al., 2006), particularly when a small sample size was used (Yu and Buckler, 2006). Therefore, it is very important to test for population structure in the future association study by using an independent set of random markers. Furthermore, only partial genes were screened for the presence of SNP in this study, therefore some of the genetic associations involving the partially screened genes may not detected. Hence, full-length sequencing of the candidate genes, including the promoter, intron, exon and 5’/3’-untranslated regions is advisable for identifying sufficient SNPs that can represent the candidate gene locus (Zhu et al., 2008; Lau et al., 2009).

The sample size of Acacia population should be increased to obtain more powerful data for association mapping in future. Long and Langley (1999) had proposed on the order of 500 samples to overcome the problem of low repeatability of an association study. Nowadays, effort has been placed to discover SNP in a small sample size (10-100) and then genotype in large association population by using low cost and high throughput SNP genotyping platform (Neale, 2007; Syvanen, 2001).

In conclusion, the present study on gene-associated single nucleotide polymorphism in C4H and CAD genes from Acacia mangium Superbulk trees revealed an association between SNPs in CAD gene with the wood density, specific gravity and cell wall thickness. This indicates that SNP in CAD gene might alter the lignin biosynthesis and thus, lead to changes in phenotypic characteristics of the trees. Once these SNPs have been validated, it could potentially be used as a tool in marker-assisted selection that enables more precise and accurate in selection and prediction of yield or performance early in life such as at the seedling stage.

ACKNOWLEDGMENTS

The authors would like to thank all the lab assistants and foresters involved in this research project for their excellent field assistance in sample collection. This work is part of the joint Industry-University Partnership Programme, a research programme funded by the Sarawak Forestry Corporation (SFC) and Universiti Malaysia Sarawak (UNIMAS) and Ministry of Sciences, Technology and Innovation (MOSTI), Malaysia.

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