Inter Simple Sequence Repeat (ISSR) Analysis of Neolamarckia cadamba in Sarawak

Sarawak state government’s aspiration to establish one million hectares of planted forests by the year 2020 has only achieved 25 % thus far. It is estimated that at least 30 million of seedlings are needed for annual planting and reforestation. One of the problems causing the slow rate in the establishment of planted forest, despite of the huge number of seedlings needed, is the availability of quality planting materials. To overcome this problem, a tree improvement programme focusing on Neolamarckia cadamba or locally known as Kelampayan was initiated in July 2007. The Kelampayan tree improvement programme is a long-term investment to increase the productivity of forest plantations by providing a source of improved planting stock that will improve the tree growth, better form and wood quality as well as pest and disease resistance. To date, two Kelampayan provenance trials have been established since 2008 in Landeh and Niah, Sarawak. This study is directed at the understanding of the genetic diversity and relatedness of the Kelampayan planted in Block 4 at the Landeh provenance trial using inter simple sequence repeat (ISSR) markers. A total of 34 ISSR markers commonly used in plant genotyping were screened using Kelampayan DNA. Out of these markers, 11 were found to be polymorphic and reproducible. PCR optimizations were carried out on these selected 11 ISSR markers. Genotyping of the Kelampayan using the selected ISSR markers is performed at optimal temperatures (Ta) varying from 44 to 61C. The optimum conditions for these ISSR are as follow: 2ng of DNA template, 2mM MgCl2, 0.5 U Taq DNA polymerase, 0.2mM dNTPs and 10 M primer. The genetic information obtained from this study will be used as one of the parameters for the selection of genetically diverse and nonrelated plus trees from the Landeh provenance trial for future tree improvement programme

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

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

GC-MS ANALYSIS OF PHYTOCHEMICAL CONSTITUENTS IN LEAF EXTRACTS OF NEOLAMARCKIA CADAMBA (RUBIACEAE) FROM MALAYSIA

Neolamarckia cadamba is one of the medicinal plants used in the treatment of various diseases traditionally. This study was conducted to identify the phytochemical constituents of N. cadamba leaf extracts using gas chromatography mass spectrometry (GC-MS). Solvents with increasing polarities viz. hexane, petroleum ether, chloroform, ethyl acetate and methanol were used in this study. The solvent extracts were analyzed using GC-MS and the mass spectra of the compounds found in the respective extract were matched with the National Institute of Standards and Technology (NIST) library. A total of 26 compounds were identified and the major chemical constituents were n-hexadecanoic acid (44.88%), hexadecanoic acid ethyl ester (17.96%) and octadecanoic acid ethyl ester (11.71%). Some of the identified compounds have been reported to possess various biological activities such as antioxidant, antimicrobial, anesthetic, antiseptic, antidiabetic, hypocholesterolemic and etc. The results thus concluded that N. cadamba leaves possess various potent bioactive compounds and is recommended as a plant of phytopharmaceutical importance

Identification and analysis of expressed sequence tags present in xylem tissues of kelampayan (Neolamarckia cadamba (Roxb.) Bosser)

The large-scale genomic resource for kelampayan was generated from a developing xylem cDNA library. A total of 6,622 high quality expressed sequence tags (ESTs) were generated through high-throughput 5’ EST sequencing of cDNA clones. The ESTs were analyzed and assembled to generate 4,728 xylogenesis unigenes distributed in 2,100 contigs and 2,628 singletons. About 59.3 % of the ESTs were assigned with putative identifications whereas 40.7 % of the sequences showed no significant similarity to any sequences in GenBank. Interestingly, most genes involved in lignin biosynthesis and several other cell wall biosynthesis genes were identified in the kelampayan EST database. The identified genes in this study will be candidates for functional genomics and association genetic studies in kelampayan aiming at the production of high value forests

An Optimized DNA Extraction Protocol For Isolation Of High Quality Genomic DNA From Camphor Containing Timber Tree Species, Dryobalanops Beccarii Dyer

Isolation of high-quality genomic DNA from Dryobalanops beccarii is obviously difficult due to the existence of large amounts of camphor and other secondary metabolites. These contaminants will co-precipitate with DNA during DNA isolation and purification processes, and therefore, resulting in a brownish DNA pellet that is unsuitable for downstream applications. Many DNA isolation protocols are available for various plant tissues; however these protocols are inefficient in yielding high-quality amplifiable genomic DNA especially from camphor containing timber tree species. A CTAB based protocol has been optimized for isolating genomic DNA from camphor containing timber tree species. Key steps include: 1) using 1% β-mercaptoethanol and 2% PVP 40 (Mr 40,000) in the extraction buffer; 2) sample incubation time, 40 minutes at 65°C, and 3) DNA precipitation at room temperature (25°C). The isolated DNA pellet was transparent colour and the purified genomic DNA is suitable for PCR amplification. Wei-Seng Ho, Kit-Siong Liew, Shek-Ling Pan

Isolation and nucleotide variation of COBRA gene in a tropical timber tree Neolamarckia cadamba

COBRA gene is involved in the regulation of cellulose deposition and orientation of cell expansion. Any single nucleotide differences may influence the functions of this gene. Hence, this study was carried out to isolate the partial COBRA genomic sequence and subsequently, to determine the nucleotide variation of COBRA gene in Neolamarckia cadamba. N. cadamba, also locally known as kelampayan is one of the fastgrowing deciduous and indigenous tree species with great commercial values in Malaysia. The targeted DNA sequence of COBRA gene was amplified with the designed primer pair by using Polymerase Chain Reaction (PCR) technique. The partial COBRA genomic sequence (517 bp) was subjected to BLASTn analysis to search for homology sequence and validate the identity of the sequence through NCBI. Multiple alignment was carried out by ClustalW for manual detection of single nucleotide polymorphisms (SNPs). Five SNPs were detected in the exon region and two SNPs in the intron region of COBRA partial genomic sequences. Of these five detected SNPs, four non-synonymous mutations and one synonymous mutation were discovered in the COBRA amino acid sequences. Based on the in silico restriction analysis, one possible restriction enzyme, HpyCH4III was detected to restrict at a SNP site (384 bp) which could be useful for genetic marker development, such as CAPS marker development in the efforts of genotyping project

Sequence variation in the cellulose synthase (SpCesA1) gene from Shorea parvifolia ssp. parvifolia mother trees

Cellulose synthase (CesA) is a key enzyme involved in the regulation of cellulose biosynthesis pathway. It is heritable and important in determining the variability of wood. In particular, it provides greater impact on the design of future genetic improvement strategies in the production of high quality wood. Thus, the molecular diversity of partial SpCesA1 genomic DNAs (802 bp) generated through PCR amplification was examined in this study, and this followed by sequencing from five selected Shorea parvifolia ssp. parvifolia mother trees. The consensus sequences were aligned to detect the presence of single nucleotide polymorphisms (SNPs). In total, seven SNPs were detected at nucleotide 58, 66, 69, 194, 224, 376 and 448. Interestingly, one single base pair InDel polymorphism was also detected at nucleotide 67. On average, one SNP at every 109 bp of the sequence data was detected. However, this result was obtained from a study of partial SpCesA1 genomic DNA of 802 bp. Two possible restriction enzymes were detected on two SNP sites of partial SpCesA1 genomic DNA. These included EarI (5’-GAAGAG-3’) and EcoRI (5’-GAATTC-3’), which were recognized and later cut at nucleotides 48 and 370, respectively. The exclusiveness of the restriction enzymes EarI and EcoRI obtained for SNPs at nucleotides 58 and 376, respectively, could be useful for the development of cleaved-amplified polymorphic sequence (CAPS) markers which could also be used to understand the molecular diversity of the CesA genes in tropical tree genomes

Development, Polymorphism and Cross-Species Transferability of Genomic SSR Markers in Duabanga Moluccana, an Indigenous Tree Species from Sarawak

In this study, we used ISSR-suppression methods to develop a set of SSR markers for Duabanga moluccana. It is an indigenous fast growing tropical tree species. A total of 44 SSR regions were identified and specific primer pairs were designed. The SSR motifs contained perfect compound with 24 (54.5%) occurrences, followed by the imperfect compounds with 8 (18.2%), simple perfect with 8 (18.2%) and the simple imperfect repeats with 4 (9.1%). The newly identified SSR markers were characterized by screening 20 individuals of D. moluccana seedlings. Among 43 primer pairs tested, 25 (58.1%) SSR markers amplified the desired PCR products and 115 alleles were detected. The number of alleles per locus ranged from 2 to 8, with a mean value of 4.60. Polymorphism Information Content (PIC) values ranged from 0.225 to 0.792, with an average of 0.604. A success rate of transferability of D. moluccana SSR markers varied, ranging from 84% in Duabanga grandiflora, 36% in Neolamarckia cadamba, 24% in Canarium odontophyllum and 28% in Shorea parvifolia. These SSR markers herein could be used to generate useful baseline genetic information for effective selection of plus trees, provenance trials and establishment of forest Seed Production Areas (SPAs) of D. moluccana in the selected forest reserves for tree plantation and improvement activities. Besides, the transferability of the newly developed SSR markers across a range of species and genera suggests their potential usefulness for a variety of population genetic studies

Molecular Cloning of Hypervariable Regions (HVRII) from Cellulose Synthase (CesA) Gene in Neolamarckia cadamba

Neolamarckia cadamba or locally known as Kelampayan, is one of the fast growing plantation tree species that holds great prospect as a renewable bioresources for plywood, pulp and paper, and biofuel industries. Sufficient information on cellulose synthase (CesA) gene, especially the hypervariable region II (HVRII) component involved in wood formation of Kelampayan is imperative for future applications. This region is thought to play a role in interaction with other unique cell-type-specific proteins involved in the biosynthesis of cellulose. The aim of this study was to identify and clone the HVRII regions of cellulose synthase gene from the developing xylem tissues of Kelampayan. The cDNA of cellulose synthase HVRII regions was amplified using reverse transcription-PCR (RT-PCR) approach using degenerate primers. Three clones, namely NcCesA1HVRII (520bp), NcCesA2HVRII (580bp) and NcCesA3HVRII (620bp) were successfully sequenced and characterized. NcCesA1HVRII and NcCesA3HVRII were clustered into two distinct clades implicated with secondary cell wall development whereas NcCesA2HVRII has renamed to NcCslD1HVRII due its high similarity with various plants’ CslD-HVRII. This study provides an easier and faster access to NcCesAHVRII sequences to further understand the role of NcCesA/NcCslD protein for future applications such as selecting trees with optimal cellulose content required for specific industries