Isolation and in silico characterization of cinnamate 4-hydroxylase (C4H) gene controlling the early stage of phenylpropanoid biosynthetic pathway in Kelampayan (Neolamarckia cadamba, Rubiaceae) developing xylem tissues

Cinnamate 4-hydroxylase (C4H) is one of the enzymes involved at the starting point of the phenylpropanoid and lignin biosynthesis pathway. It involves in the hydroxylation of cinnamate to 4-coumarate. In this paper, we isolated and in silico characterized the complete sequence of cinnamate 4-hydroxylase (C4H) gene from Neolamarckia cadamba in Malaysia. The C4H singletons obtained from the NcdbEST were used to predict the hypothetical full-length of NcC4H through the contig mapping approach. RT-PCR was used to amplify the full-length C4H cDNA clone and subsequently the PCR amplicons were sequenced and analysed. The NcC4H cDNA was 1,651 bp long with a 505 amino acid sequence, a 18 bp 5’-UTR and a 115 bp 3’-UTR. The predicted NcC4H protein contains P450-featured motifs. These include the heme-binding domain, a threonine-containing binding pocket motif and the proline-rich region. Peptide sequence comparison and phylogenetic analyses revealed that NcC4H was clustered with class I C4H instead of class II C4H, which is preferentially involved in phenylpropanoid and lignin biosynthesis pathway. This full-length NcC4H cDNA can be used for developing genetic marker to identify economic trait loci (ETL) for wood quality traits via genomics-assisted selection (GAS) or candidate gene mapping approach

Gene-associated single nucleotide polymorphism (SNP) in cinnamate 4-hydroxylase (C4H) and cinnamyl alcohol dehydrogenase (CAD) genes from acacia mangium superbulk trees*

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

Applications Of Genomics To Plantation Forestry With Kelampayan In Sarawak

Wood-based industries in Sarawak are increasingly encouraged to adapt to “new wood” from planted forests composed of fast-growing species such as Kelampayan with short rotation cycle (6-8 years). The rationale is that natural forests at the most produce about 3m3 /ha/yr of commercial timber, whereas plantations can produce annually from 10m3 /ha to 30m3 /ha of commercial timber. It is estimated at least 30 million seedlings are required for annual planting or reforestation programmes to meet the increasing global demand for raw materials. To date, several molecular genetics studies have been completed for Kelampayan. These include genetic diversity of Kelampayan, genetic marker (SSRs) development, ‘Touch-incubate-PCR’ approach for preparing plant tissues for high throughput genotyping, and transcriptomics and bioinformatics on wood formation of Kelampayan. This information provides a useful resource for genomic selection of Kelampayan aiming at the production of high value forests for maximum returns

From Conservation To Innovation Building Research Capacity For Planted Forest Development In Sarawak

The increase in global demand for wood requires increase in forest productivity. The alternative is to farm trees in plantations composed of fast-growing species with short rotation cycle (6-8 years). The rationale is that natural forests at the most produce about 3m3/ha/yr of commercial timber, whereas plantations can produce annually from 10m3/ha of hardwoods to 30m3/ha of softwoods and thus, decrease the effects of human pressure on our ecosystems while increasing the competitiveness of Sarawak‘s forest industry. This is in line with State Government‘s aspiration to establish one million hectares of planted forests by year 2020 to meet the increasing demand from both domestic and international markets for raw materials. It is estimated at least 30 million seedlings are required for annual planting or reforestation programmes. In this regard, the forest genomics research will help respond to the need to develop adequate tools that enable us to produce quality planting materials that are of faster growth, high-yield and high wood quality, and also adapted to local conditions, so that we may achieve economic benefits of great significance. Realizing the needs, we have centered our research on the development of tools via biotechnological innovations for tree breeders. We have successfully developed: 1) an array of highly informative and polymorphic DNA markers specific for identifying the genetic makeup of two fast growing indigenous tree species, i.e. Kelampayan and Sawih; 2) the one step ‗Touchincubate- PCR‘ approach for preparing plant tissues for high throughput genotyping, and 3) a genomic resource database, aka CADAMOMICS (10,368 ESTs) for wood formation in Kelampayan via high-throughput DNA sequencing. These tools will greatly facilitate the selection of quality planting materials for planted forest development in Sarawak as well as longterm tree improvement activities by integrating genomics into our breeding programme via association mapping. The overall benefit of genomics application to tree improvement programme will be in terms of greater certainty in the outcome of results, specifically the performance of the forest plantations, as well as the savings in time and cost in the production and supply of quality planting materials

Gene-Associated snp discovery and molecular cloning of full-length cDNA of cinnamate 4-hydroxylase and cinnamy alcohol dehydrogenase in a tropical timber tree Neolamarckia cadamba TREE Neolamarckia cadamba

Neolamarckia cadamba, or locally known as Kelampayan is one of the indigenous tree species that are selected for forest plantation establishment in Sarawak due to the high productivity and short rotation time of this species. Understanding the structure and composition of Kelampayan wood through genome integration is vital to better utilize this wood material. Concurrently, the Kelampayan wood formation genomic resource database, aka Cadamomics (10,368 ESTs) has been developed, and this opens the gateway for researchers to deeply explore the genomic basic of Kelampayan. EST database is a useful resource for gene discovery. Further analysis on this database generated two full-length lignin biosynthesis genes, namely C4H and CAD. Validation by RT-PCR and full-length gene specific primers had confirmed the identities of the genes discovered. The full-length C4H cDNA, designated as NcC4H is 1,651 bp long, with a 1,518 bp open reading frame encoding a protein of 505 amino acids, a 18 bp 5’-UTR and a 115 bp 3’-UTR. The NcC4H showed higher identity with the class I C4Hs, which is preferentially involved in the phenylpropanoid biosynthesis pathway. Meanwhile, sequence analysis of the full-length CAD cDNA, designated as NcCAD, showed that it is 1,240 bp long with a 1,086 bp open reading frame encoding a protein of 361 amino acids, a 68 bp 5’-UTR and a 86 bp 3’-UTR. Phylogenetic analysis revealed that NcCAD was grouped in the cluster containing both CAD and SAD genes, in which both genes were involved in lignin biosynthesis. The full-length NcC4H and NcCAD cDNA identified serve as good candidate genes for association genetics studies in Kelampayan. Association genetics study is a powerful approach to detect potential genetic variants, i.e. SNPs, underlying the common and complex adaptive traits. Thus far, single nucleotide polymorphisms (SNPs) detected in C4H and CAD genes were known to be correlated with some other phenotypic variations in forest tree species rather than with lignin production only. Hence, attempts were made to discover SNPs from the partial C4H and CAD genomic DNA sequences. Overlapping primers were designed to flank the partial C4H and CAD DNA from 12 Kelampayan samples. The amplified DNA fragments were cloned and sent for sequencing. Furthermore, wood cores were collected and the basic wood density was measured for each tree. Sequence variation analysis revealed that there were 60 and 32 SNPs detected in the partial C4H and CAD DNA sequences, respectively. The SNPs detected were distributed throughout the exon, intron, 5’-UTR and 3’-UTR regions. Among the SNPs detected in the exon regions of C4H, 16 were synonymous mutations and eight were nonsynonymous mutations. For CAD, six SNPs lead to synonymous mutations and one SNP lead to nonsynonymous mutation. Synonymous mutations (71 %) were more common than nonsynonymous mutations (29 %) for both the C4H and CAD genes. Association genetics studies also revealed that four and six SNPs from the C4H and CAD genes respectively were significantly associated with the basic wood density of Kelampayan (p<0.05). Genetic variations identified by the SNP markers, once validated, will facilitate the selection of Kelampayan parental lines or seedlings with optimal quality through the gene-assisted selection (GAS) approach

Association Genetics of the Cinnamyl Alcohol Dehydrogenase (CAD) and Cinnamate 4-hydroxylase (C4H) Genes with Basic Wood Density in Neolamarckia cadamba

Association genetics study is a powerful approach to detect the potential genetic variants (i.e., SNPs) underlying the common and complex adaptive traits. Once the quantitative trait nucleotides are identified, such powerful approach provides significant advantages to the forest industry. Hence, attempts were made to discover SNPs from Neolamarckia cadamba partial C4H (3,538 bp) and CAD (2,354 bp) DNA sequences and further associate those SNPs with basic wood density. Overlapping primers were designed in flanking the partial C4H and CAD DNA from 12 N. cadamba trees. The amplified DNA fragments were sequenced and the basic wood density measurements were determined for each tree. The sequence variation analyses revealed that there were 60 and 32 SNPs detected in the partial C4H and CAD DNA sequences, respectively. Those SNPs were distributed throughout the exon, intron, 5’-UTR and 3’-UTR regions. The total nucleotide diversities were π = 0.00302 and θW = 0.00412. The synonymous mutations (π = 0.00983; θW = 0.01210) were more common than nonsynonymous mutations (π = 0.00045; θw = 0.00089) for both C4H and CAD genes. LD declined linearly over short distance at the loci examined. Association genetics study also revealed that 4 and 6 SNPs from C4H and CAD genes, respectively were in significant associations with basic wood density of N. cadamba (p<0.05). The genetic variation identified by the SNP markers, once validated, will facilitate the selection of N. cadamba parental lines or seedlings with optimal quality through Gene-assisted Selection (GAS) approach