Development and polymorphism of simple sequence repeats (SSRs) in Kelampayan(Neolamarckia cadamba–Rubiaceae) using ISSR suppression PCR method

Simple sequence repeat (SSR) marker is a polymerase chain reaction (PCR)-based marker system which has become a marker of choice for understanding plant genetic diversity and a powerful tool in addressing genetic resources questions. However, the availability of SSR markers especially for forest tree species is limited thus far due to the high development cost, labour-intensive and time-consuming. The present study aimed to develop an array of SSR markers for Neolamarckia cadamba using inter-simple sequence repeat (ISSR) suppression PCR method and further assessed the polymorphisms and transferability of the markers to other species.In total, 15 out of 31 SSR markers specific for N.cadamba were successfully developed and further characterized and validated by using 30 individuals of N. cadamba. The markers exhibited a considerable high level of polymorphism across the tested N. cadamba genotypes whereby 66 alleles were detected with an average of four alleles per locus. Most of the SSRloci analyzed showed high polymorphism as indicated by their PIC value which was above 0.5. The most polymorphic lociwere: NCAC11 (PIC=0.849), NCAC12 (PIC=0.722) and NCAG01 (PIC=0.712).The transferability rate was ranging from 26.7% to 73.3% among the four cross-genera species tested. The present study is the first report on the development of SSR markers in N. cadamba. These markers provide valuable genomic resources that could pave the way for exploiting SSR genotype data for effective selection of plus trees, provenance trials and establishment of forest seed production areas (SPAs) of N. cadamba in the selected forest areas dedicated to planted forest development, and molecular breeding of N. cadamba and other indigenous tropical tree species in future

Development and polymorphism of simple sequence repeats (SSRs) in Kelampayan (Neolamarckia cadamba–Rubiaceae) using ISSR suppression PCR method

Simple sequence repeat (SSR) marker is a polymerase chain reaction (PCR)-based marker system which has become a marker of choice for understanding plant genetic diversity and a powerful tool in addressing genetic resources questions. However, the availability of SSR markers especially for forest tree species is limited thus far due to the high development cost, labour-intensive and time-consuming. The present study aimed to develop an array of SSR markers for Neolamarckia cadamba using inter-simple sequence repeat (ISSR) suppression PCR method and further assessed the polymorphisms and transferability of the markers to other species.In total, 15 out of 31 SSR markers specific for N.cadamba were successfully developed and further characterized and validated by using 30 individuals of N. cadamba. The markers exhibited a considerable high level of polymorphism across the tested N. cadamba genotypes whereby 66 alleles were detected with an average of four alleles per locus. Most of the SSRloci analyzed showed high polymorphism as indicated by their PIC value which was above 0.5. The most polymorphic lociwere: NCAC11 (PIC=0.849), NCAC12 (PIC=0.722) and NCAG01 (PIC=0.712).The transferability rate was ranging from 26.7% to 73.3% among the four cross-genera species tested. The present study is the first report on the development of SSR markers in N. cadamba. These markers provide valuable genomic resources that could pave the way for exploiting SSR genotype data for effective selection of plus trees, provenance trials and establishment of forest seed production areas (SPAs) of N. cadamba in the selected forest areas dedicated to planted forest development, and molecular breeding of N. cadamba and other indigenous tropical tree species in future

Genetic diversity of Kelampayan using dominant DNA markers based on inter-simple sequence repeats (ISSRs) in Sarawak

Neolamarckia cadamba (Roxb.) Bosser, or locally known as kelampayan, is a fast-growing timber species which produces one of the best sources of raw material for the plywood industry and also for the pulp and paper industry. It has been selected as one of the promising plantation tree species for large-scale planted forest development in Sarawak. Therefore, the molecular characterization of this indigenous tropical tree species is needed to maintain its high quality. Intersimple sequence repeats (ISSR) markers were used in this study to determine the genetic diversity of kelampayan in three progeny trial blocks at the Landeh Nature Reserve, Semengok, Sarawak. The seeds were collected from the selected mother trees located at the Pasai Bon, Niah and Lawas seed production areas (SPAs) in Sarawak. Three ISSR primers, namely (GTG)6, (AG)10 and (AC)10, that yielded reproducible, informative and scorable fragments were chosen for ISSR analysis. A total of 64 loci were generated of which 45.3–74.6% of the loci were identified as polymorphic bands with the size ranging from 500 bp to 2 kb among 247 kelampayan progenies selected in the present study. Molecular diversity based on Shannon’s diversity indices (I) among 247 trees ranged from 0.268 to 0.350. In general, the kelampayan trees in the three progeny trial blocks exhibited a high level of molecular diversity and DNA polymorphism compared with its natural populations. This preliminary information will form the base for kelampayan tree improvement and conservation programmes

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

Genomics studies on plantation tree species in Sarawak

Rapid socio-economic changes in the world are having profound impacts on all sectors, including forestry. 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 cycles (6–8 y). The rationale is that natural forests at the most produce about 3 m3 ha y-1 of commercial timber, whereas plantations can produce annually from 10 m3 ha y-1 of hardwoods to 30 m3 ha-1 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 the 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, 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 need, we have centred our research on the development of tools via biotechnological innovations for tree breeders. Over the years, we have successfully developed: 1) simple sequence repeat (SSR) 173 DNA markers specific for identifying the genetic make-up of two fastgrowing indigenous tree species, i.e. kelampayan and sawih; 2) the ‘Touch-incubate-PCR’ approach for preparing plant tissues for high throughput genotyping, and 3) a kelampayan tree transcriptome database (NcdbEST aka CADAMOMICS) for wood formation. These tools will greatly facilitate the selection of quality planting materials for planted forest development in Sarawak as well as long-term 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

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

Development and polymorphism of microsatellite markers in Neolamarckia cadamba (Roxb.) bosser (kelampayan) using ISSR suppression PCR method

Reliable information on the distribution of genetic variation is a crucial point for applicability and efficiency of any breeding, preservation and conservation programmes for forest trees. The emergence of DNA marker technologies provides new tools for rapid genetic analysis, fingerprinting and studying relatedness among cultivars of many forest tree species. A vailability of good numbers of polymerase chain reaction (PCR)compatible DNA markers which is heritably co-dominant and multiallelic has become a priority due to the genetic complexity of breeder's populations and high levels of heterozygosity in individual genotypes. Simple sequence repeats (SSRs) or microsatellites, are tandemly repeated motifs of 1-6 nuc1eotides found in all prokaryotic and eukaryotic genomes. The uniqueness and value of micro satellites arises from their multiallelic nature, codominant transmission, relative abundance and extensive genome coverage. Because of these attributes, microsatellites are currently the excellent markers of choice for comparative genetic and genomic analysis, high-throughput genotyping and studies of gene flow in forest trees. Neolamarckia cadamba (Roxb.) Bosser or locally known as Kelampayan, belongs to the Rubiaceae, was chosen in the present study due to its high commercial value and fast growing ability. Although N cadamba becomes one of the most frequently planted trees in the tropics, but genetic information about this species is limited and none of the DNA markers has been developed from N cadamba compared to other economically important tropical trees. In the present study, 15 SSR markers specific for N cadamba were developed using inter-simple sequence repeat (ISSR) suppression PCR method, a method which is relatively simple and rapid without enrichment steps. Considerable allelic amplifications were obtained for all SSR markers across the tested genotypes whereby 66 alleles were detected with an average of 4 per locus. Most of the detected loci analyzed showed high polymorphism as indicated by their PIC value which was above 0.5. The most polymorphic loci were: ACll (PIC=0.849), ACl2 (PIC=0.722) and AGOl (PIC=0.712). Besides, cross-speCies transferability of micro satellites was also confirmed in this study and the newly developed SSR markers showed good cross species amplification efficiency. These SSR markers represent a potent tool for genetic diversity study, estimate pollen contamination in seed orchards, germplasm identification and to assist with the construction of genetic linkage map for N cadamba. The derived SSR-based map later will be an important prerequisite for marker-assisted selection (MAS) to increase the efficiency of N cadamba breeding

Sequential extraction of RNA and DNA from the same sample of Neolamarckia cadamba (Roxb.) Bosser

Most of the established molecular techniques in plant molecular biology rely on the reliability of extraction methods to obtain nucleic acids from a given environmental source. The development of protocols for isolating both RNA and DNA from the same sample is recently becoming a major concern especially when the sample is small. Despite a number of methods that describe the simultaneous isolation of RNA and DNA from the same sample have been reported, most of these methods have yet to be optimized to deal with the reduced quantity and compromised quality of samples encountered in plant genetics analysis. Neolamarckia cadamba (Roxb.) Bosser was chosen in the present study due to its commercial value and fast growing ability. Total RNA was successfully isolated from the leaf tissue with subsequent recovery of DNA from the extraction mixture through alcohol precipitation. Reverse transcriptase‐Polymerase Chain Reaction (RT‐PCR) was performed and arbitrary primers that produced reproducible, scorable and informative bands were selected for randomly amplified polymorphic DNA (RAPD) analysis. The amplicons of RTPCR and RAPD were successfully obtained from all isolates, indicating that the extracted nucleic acids were intact and pure enough for quantitative molecular analysis