8 research outputs found
Development and polymorphism of simple sequence repeats (SSRs) in Kelampayan(Neolamarckia cadambaâRubiaceae) 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 cadambaâRubiaceae) 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 45.3â74.6%
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 Shannonâs 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 ânew woodâ 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,
âTouch-incubate-PCRâ 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 (6â8 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
Sarawakâs forest industry. This is in line with the state governmentâs
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)
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DNA markers specific for identifying the genetic make-up of two fastgrowing
indigenous tree species, i.e. kelampayan and sawih; 2) the
âTouch-incubate-PCRâ 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 Sarawakâs forest industry. This is in line with State Governmentâs
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 âTouchincubate-
PCRâ 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
transcriptaseâPolymerase Chain Reaction (RTâPCR) 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