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Gene discovery in the developing xylem tissue of a tropical timber tree species: Neolamarckia cadamba (Roxb.) Bosser (kelampayan)

  • Forest Department Sarawak


A complementary DNA (cDNA) library was constructed from the developing xylem tissues of Neolamarckia cadamba. A total of 10,368 single-pass sequences was generated through high-throughput 5′-expressed sequence tag (EST) sequencing of the cDNA clones, and 6622 high-quality ESTs were obtained after removing the low-quality sequences; this gave approximately 3.17 Mb of data. Clustering of the high-quality ESTs revealed 4728 unigenes, consisting of 2100 consensus and 2628 singletons. A total of 2405 ESTs were successfully annotated with 7753 gene ontology (GO) terms that distributed among three main GO categories, which were biological processes (2333), molecular function (3056) and cellular component (2364). Simple sequence repeat (SSR) mining revealed that the frequency of SSR in the N. cadamba EST database (NcbdEST) was 3.3 %, with the GCT/AGC motif being the most abundant repeat motif. The most abundant transcript with known function found in this database was 60S ribosomal protein followed by 40S ribosomal protein. Some of the important genes involved in xylogenesis and lignin biosynthesis were found in NcdbEST; these include tubulin genes, cellulose synthase (CesA), xyloglucan endotransglycosylase (XET), arabinogalactan, cinnamate 4-hydroxylase (C4H), caffeoyl-coenzyme A O-methyltransferase (CCoAOMT) and peroxidase. The data obtained from this study will provide a powerful means for identifying mechanisms controlling wood formation pathways of kelampayan and supply many new cloned genes for future endeavours to modify wood and fibre properties.
Gene discovery in the developing xylem tissue of a tropical timber
tree species: Neolamarckia cadamba (Roxb.) Bosser (kelampayan)
Shek Ling Pang
&Wei Seng Ho
&M. N. Mat-Isa
&Julaihi Abdullah
Received: 8 January 2014 /Revised: 27 March 2015 /Accepted: 15 April 2015
#Springer-Verlag Berlin Heidelberg 2015
Abstract A complementary DNA (cDNA) library was con-
structed from the developing xylem tissues of Neolamarckia
cadamba. A total of 10,368 single-pass sequences was gener-
ated through high-throughput 5-expressed sequence tag
(EST) sequencing of the cDNA clones, and 6622 high-
quality ESTs were obtained after removing the low-quality
sequences; this gave approximately 3.17 Mb of data.
Clustering of the high-quality ESTs revealed 4728 unigenes,
consisting of 2100 consensus and 2628 singletons. A total of
2405 ESTs were successfully annotated with 7753 gene on-
tology (GO) terms that distributed among three main GO cat-
egories, which were biological processes (2333), molecular
function (3056) and cellular component (2364). Simple se-
quence repeat (SSR) mining revealed that the frequency of
SSR in the N. cadamba EST database (NcbdEST) was
3.3 %, with the GCT/AGC motif being the most abundant
repeat motif. The most abundant transcript with known func-
tion found in this database was 60S ribosomal protein follow-
ed by 40S ribosomal protein. Some of the important genes
involved in xylogenesis and lignin biosynthesis were found
in NcdbEST; these include tubulin genes, cellulose synthase
(CesA), xyloglucan endotransglycosylase (XET),
arabinogalactan, cinnamate 4-hydroxylase (C4H), caffeoyl-
coenzyme A O-methyltransferase (CCoAOMT) and peroxi-
dase. The data obtained from this study will provide a power-
ful means for identifying mechanisms controlling wood for-
mation pathways of kelampayan and supply many new cloned
genes for future endeavours to modify wood and fibre
Keywords Expressed sequence tags (ESTs) .Forest
plantation .Lignin biosynthesis .Neolamarckia cadamba .
Wood formation
In Malaysia, the State Government of Sarawak has set a target
for 1 million ha of forest land degraded by shifting cultivation
to be planted with fast-growing timber tree species by year
2020 in order to meet the increasing demand for wood and
wood fibre. To meet this target, an estimation of 42 million
quality seedlings is needed for annual planting or reforesta-
tion. Neolamarckia cadamba (Roxb.) Bosser, locally known
as kelampayan, has been identified as one of the potential
indigenous fast-growing timber tree species for forest planta-
tion in Sarawak (Tchin et al. 2012; Lai et al. 2013; Tiong et al.
2014). It is a large, deciduous and fast-growing tree that gives
early economic returns within 810 years. Under normal con-
ditions, it attains a height of 17.67 m and diameter of 25.3 cm
at breast height within 9 years. Kelampayan not only serves as
Communicated by W. Ratnam
This article is part of the Topical Collection on Genome Biology
Electronic supplementary material The online version of this article
(doi:10.1007/s11295-015-0873-y) contains supplementary material,
which is available to authorized users.
*Shek Ling Pang;
Applied Forest Science and Industry Development, Tree Breeding
Unit, SARAWAK FORESTRY, 93250 Kuching, Sarawak, Malaysia
Forest Genomics and Informatics Laboratory, Department of
Molecular Biology, Faculty of Resource Science and Technology,
Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak,
Malaysia Genome Institute, 43600 Bangi, Selangor Darul Ehsan,
Tree Genetics & Genomes (2015) 11:47
DOI 10.1007/s11295-015-0873-y
one of the best raw materials for plywood and veneer but is
also suitable for pulp and paper production, light construction
and furniture (Joker 2000).
Most of the current forest genomic projects are interested in
understanding major events of wood formation as it is likely to
be the key factor in determining the wood properties that will
influence its industrial performance and value. Wood is essen-
tially composed of cellulose, hemicellulose and lignin. These
components are important in the plant defence system, the
transportation of nutrients and the providing of mechanical
strength. Wood formation or xylogenesis involves an extreme-
ly subtle and sophisticated network of spatial and temporal
gene regulation events, in order to coordinate the expression
of several hundreds of genes involved in cell division, cell
expansion and elongation, secondary cell wall formation (in-
cluding cellulose, hemicellulose and lignin synthesis) and pro-
grammed cell death (Bhalerao et al. 2003). These processes
are strongly interlinked and modulation of any one aspect of
wood formation may affect many other aspects. For example,
downregulation of a lignin gene, 4-coumarate: coenzyme A
ligase (4CL), in aspen resulted in a reduction of 45 % in lignin
content but this was compensated for by an increase of 15 % in
cellulose contain of the transgenic tree (Hu et al. 1999).
Therefore, an understanding on the process of wood
formation is important as it could help in the improve-
ment of the wood quality trait through marker-assisted
selection and the assessment of the genetic diversity of
a breeding programme.
Genomics approaches are now widely used to explore the
molecular basis of xylogenesis particularly in economically
important tree species. Expressed sequence tags (ESTs) are
fragments of mRNA generated through single-pass sequenc-
ing of the 5and/or 3ends of randomly selected complemen-
tary DNA (cDNA) clones. High-throughput EST sequencing
provides rapid access of a significant portion of the expressed
genes in one organism, and this valuable source of sequence
information can serve as a foundation for initiating a genome
sequencing project (van der Hoevan et al. 2002). A large
number of ESTs for wood formation have been achieved from
studies in pine (Li et al. 2009; Whetten et al. 2001), poplar
(Sterky et al. 1998; Hertzberg et al. 2001) and eucalyptus
(Paux et al. 2004). Despite the high economic value of tropical
wood, the mechanism controlling wood formation in
tropical tree species still remains poorly understood.
Therefore, this project is directed at the understanding
of wood formation of kelampayan based on sequencing
analysis of ESTs derived from the developing xylem
cDNA library. Here, we present the first EST database
for N. cadamba generated through high-throughput 5
single-pass sequencing. We believe the genomic infor-
mation from our study will give some insights into the wood
formation in tropical wood in general and kelampayan in
Materials and methods
Sample preparation and cDNA library construction
The bark of a healthy 2-year-old kelampayan tree was peeled
off, and developing xylem tissues were collected from the
exposed surface. The tissues were collected in a clean plastic
bag and directly into liquid nitrogen. The samples were kept in
80 °C until needed. About 0.5 g developing xylem tissues
was used for total RNA isolation using RNeasy Midi Kit
(Qiagen, Germany) with modification. The cDNA library
was constructed using CloneMiner
cDNA Library
Construction Kit (Invitrogen, USA) according to the manu-
facturers protocol. cDNA clones were manually picked and
cultured overnight in 96-well culture blocks. The glycerol
stocks for 10,368 clones were then sent on dry ice for high-
throughput plasmid extraction and 5end cDNA sequencing at
the Malaysia Genome Institute (MGI).
Sequence processing, clustering, BLAST search
and annotation
Raw ABI-formatted chromatogram reads were base-
called using Phred (Ewing et al. 1998) with a threshold
value of 20. Vector sequences were masked using
Cross-Match. The trimming and removing of vectors
and low-quality nucleotides were done using customized
Perl scripts. High-quality ESTs with a minimum of 100
bases and fewer than 4 % Ns were retained. StackPACK
(Miller et al. 1999) was used for multiple sequence
alignment, clustering, assembling and the generation of
consensus sequences. The sequences were grouped to-
gether by d2_cluster (Burke et al. 1999)iftherewasat
least 96 % sequence similarity in any window of 150
bases. The loose clusters were then aligned using PHRA
P (Laboratory of PHIL GREEN) and subsequently
CRAW (Chou and Burke 1999). Homology searches of
unigenes, annotation and gene ontology of the ESTs
was done using Blast2GO (Conesa et al. 2005) with
default settings.
In silico identification of SSR
Unigenes were mined for simple sequence repeat (SSR)
markers using MISA (Thiel et al. 2003). The polyA
and polyT sequences in a 50-bp window at the terminal
regions were removed. The minimum numbers of re-
peats for SSR detection used in MISA were six for
di-nucleotides, five for tri-nucleotides, four for tetra-nu-
cleotides, three for penta-nucleotides and three for
47 Page 2 of 6 Tree Genetics & Genomes (2015) 11:47
Clustering of ESTs
The EST database for N. cadamba developing xylem
(NcdbEST) consisted of 10,368 5end reads. The removal
Cluster analysis of these 6622 ESTs revealed 4728 unigenes
which consisted of 2100 consensus sequences and 2628 sin-
gletons. These ESTs were submitted to dbEST at the NCBI
with the library accession number LIBEST_028358. The as-
sembly of the ESTs revealed 28.6 % of redundancy in the
NcdbEST. BLAST analysis of the unigenes was done using
Blast2GO with the default setting. A total of 2913 showed
significant similarity to known sequences in GenBank non-
redundant protein database. The remaining 1815 did not show
a BLAST hit or no significant similarity to any known se-
quences in the database. Only 2050 sequences out of the
2913 sequences that showed significant similarity to the data-
base were assigned with known functions. The remaining 863
were either conserved hypothetical protein, hypothetical pro-
tein, predicted hypothetical protein, predicted protein or
The most abundant transcript with a known function de-
tected in the NcdbEST was putatively identified as 60s ribo-
somal protein (49 ESTs), followed by 40s ribosomal protein
(33 ESTs), whereas tubulin genes (29 ESTs) were the most
abundant cell wall-related genes identified in this database,
followed by arabinogalactan (19 ESTs) and s-
adenosylmethionine synthetase (15 ESTs). Some cell wall-
related genes were moderately abundant in the developing
xylem tissue of kelampayan (37 ESTs). These include cellu-
lose synthase, endo-1,4-β-glucanase (cellulose), sucrose syn-
thase (SuSY), expansin, glucan endo-1,3-β-n-glucosidase,
xyloglucan endotransglycosylase-hydrolase (XTH) and
xyloglucan endotransglycosylase (XET). All genes involved
in the lignin biosynthesis pathway were identified in the
kelampayan cDNA library. Genes involved in biotic and abi-
otic stresses were also found in the NcbdEST. Heat shock
protein was the most abundant stress-related gene found in
this NcdbEST (20 ESTs), followed by ethylene (8 ESTs),
14-3-3 protein (8 ESTs), defensin protein (6 ESTs), disease
resistance protein (6 ESTs) and other low-expression genes.
Gene ontology annotation
The gene ontology (GO) annotation of the 4728 unigenes in
NcdbEST was performed using Blast2GO (Conesa et al.
2005). A total of 2405 ESTs were successfully annotated with
GO terms. There were 967 consensi and 1438 singletons. The
2405 ESTs were annotated with 7753 GO terms that distrib-
uted among three main GO categories, which are biological
processes (2333), molecular function (3056) and cellular
component (2364). The annotation showed that 49.13 % of
the unique genes were not annotated. As a result, the number
of ESTs that were represented with GO terms might be prob-
ably underestimated. A total of 1400 of the 1815 sequences
with no hit could not be annotated. Apart from that, an addi-
tional of 397 sequences with a BLAST hit also could not be
annotated with a GO term as most of the sequences were
similar to protein, hypothetical protein or unknown. The se-
quence distribution and percentage in each GO term and their
respective subcategories (cutoff point=100.0) were calculat-
ed. A percentage of 100 is defined as the total number of ESTs
that are assigned with GO terms. It must be noted that the
percentage of the subcategories does not add up to 100 % as
many of the ESTs are involved in different classes of function
and annotated with multiple GO terms. In functional classifi-
cation with GO terms, 73.7 % of the 2405 unigenes with
assigned GO terms have cellular component, 64.1 % were
involved in molecular function and 54.5 % were biological
process. In the cellular component category with 1772
unigenes, 12.18 % were involved in the intracellular organelle
part, and 10.06 % were mitochondrion. A total of 1542 ESTs
were involved in molecular function, with 10.35 % showing
transferase activity and 9.98 % showing protein binding ac-
tivity. For biological process with 1311 ESTs, 11.6 % were
involved in transportation while 6.57 % were involved in reg-
ulation of cellular process (Supplementary 1).
EST-derived SSR marker
Data mining of 4728 kelampayan unigenes identified 178
SSRs in 157 unigenes with a total of 11 sequences that
contained more than one SSR. The frequency of ESTcontain-
ing SSRs in the NcdbEST was 3.3 %, with the overall SSR
density of one in 13.88 kb. The EST-derived SSRs were rep-
resented by di-, tri- and tetra- repeat motifs (Supplementary 2).
Among all the SSR motifs, GCT/AGC motif represented the
most abundant repeat motif in the NcdbEST. Of these, 113
unigenes (63.5 %) were tri-nucleotide repeats, 53 unigenes
(29.8 %) were di-nucleotide repeats and 12 unigenes (6.7 %)
were tetra-nucleotide repeats. There were 4 SSRs that linked
to cell wall formation (cellulase, expansin, α-tubulin and
XTH), 3 SSRs linked to lignin biosynthesis genes (F5H,
HCTand CCR) and 1 SSR linked to disease resistance protein.
These SSRs will be useful in the tree improvement pro-
gramme of kelampayan.
Analysis of the 10,368 ESTs from NcdbEST gave rise to 6622
high-quality sequences with approximately 3.17 Mb data. A
total of 4728 unigenes (2.47 Mb) were identified with 2628
ESTs being the singleton and 2100 being the contigs ofat least
Tree Genetics & Genomes (2015) 11:47 Page 3 of 6 47
two ESTs, and this resulted in a library redundancy of 28.6 %.
Blast2GO analysis revealed 2913 ESTs (61.6 %) out of 4728
unigenes showed significant similarity with protein sequences
from other organisms in the GenBank non-redundant protein
database. The remaining 38.4 % or 1815 ESTs did not show
any BLAST hit. The average size of the ESTs not showing
significant similarity to the sequences in GenBank is 307 bp
compared to 657 bp for those with significant similarity.
Sequence length might influence the BLAST result, with the
probability of no significant match to the protein database
being directly proportional to the decrease in sequence length.
According to Bausher et al. (2003), sequence length of 150 to
250 bp can reach maximum 60 % of not getting significant
matches. In the NcdbEST, the average lengths of the ESTs
with poor match (with E-value> 10
) for the contigs and
singletons were 529 and 538 bp, respectively. Therefore, it
was suggested that the ESTs with no BLAST hit and ESTs
with poor match were not mainly caused by the length of the
EST. It is most probably caused by the lack of sequence in-
formation in the public database. This also indicates that these
ESTs might have some specific roles in kelampayan which are
yet to be identified.
The most abundant transcript found in NcdbEST was 60s
ribosomal protein followed by 40s ribosomal protein. The
high percentage of ribosomal protein was expected as they
play a significant role in living systems and function as inter-
mediary for protein translation. Among all the plant cell wall-
related genes, tubulin was the most detected ESTs in this da-
tabase. There were 14 ESTs for α-tubulin genes, 13 ESTs for
β-tubulin genes and 2 for γ-tubulin genes. The α- and β-
tubulin genes are the major constituent of microtubules which
are essential in the intracellular structures and play an impor-
tant role in fundamental mechanisms such as cell division,
vesicular transport, cell wall deposition, signal propagation,
etc. (Nogales 2000), while γ-tubulin is required for microtu-
bule nucleation at microtubule-organizing centres (Horio and
Oakley 2003). In Arabidopsis thaliana,γ-tubulin has been
shown to be essential for the formation of spindle, phragmo-
plast and cortical microtubule arrays (Pastuglia et al. 2006).
The high abundance of tubulin genes in NcdbEST possibly
implies the importance of this gene in the wood formation of
kelampayan. Other cell wall-related genes were
arabinogalactan (19 ESTs) and s-adenosylmethionine (15
Most of the genes in the monolignol/lignin biosynthesis
pathway are represented with relatively moderate or low abun-
dance in kelampayan unigenes. Among all the lignin genes
discovered in this database, C4H (12 ESTs) is the most abun-
dant EST. C4H is a member of the cytochrome P450
monooxygenase superfamily. Together with phenylalanine
ammonia-lyase (PAL) and 4-coumarate: coenzyme A ligase
(4CL), this enzyme directs the carbon flux to an array of im-
portant phenolic compounds in plants (Chapple 1998). C4H
catalyzes the first oxygenation step in phenylpropanoid bio-
synthesis, and the phenylpropanoid branch pathways lead to a
wide array of secondary products essential for UV protection,
differentiation of tissues and defence system, including lig-
nins, flavonoids, hydroxycinnamic esters and coumarins
(Whitbred and Schuler 2000). Ye (1997) stated the importance
of S-adenosyl methionine (SAM) in the methylation of lignin
precursors. In NcdbEST, 15 ESTs were found to encode for
SAM protein. The presence of all lignin genes in the
NcdbEST suggests active secondary cell wall biosynthesis in
the developing xylem tissues sampled.
Genes involved in the reactive oxygen species (ROS) gene
network were identified in this database, and these include
superoxide dismutase, monodehydroascorbate reductase, glu-
tathione reductase, catalase, glutathione peroxidase, NADPH
oxidase and peroxiredoxin.ROS production has been reported
in response to the pathogen defence mechanism (Huang et al.
2011) and most of the abiotic stresses including salinity
(Abogadallah 2010), ozone exposure (Kangasjärvi et al.
2005), heat (Kolupaev et al. 2008), osmotic stress (Xiong
et al. 2002) and other stresses. Other biotic/abiotic-related
proteins were also found in NcdbEST, for example abscisic
acid, salicyclic acid, drought-induced protein, defense-related
protein and disease resistance protein.This informationis very
useful and valuable for the kelampayan tree improvement as
this species is very site specific and having defoliator and stem
borer infection problems at the early stage of growing.
The 4728 non-redundant unigenes were further mined for
the identification of EST-SSR markers. A total of 178 SSRs
were identified in 157 unigenes. The overall density of SSRs
in NcdbEST was one SSR in 13.88 kb, nearly one in 30
unigenes (3.3 %). This SSR frequency more accurately re-
flects the density of SSRs in the transcript regions of the ge-
nome. The overall EST-SSR density of kelampayan was com-
parable to that of poplar (1/14 kb) and Arabidopsis
(1/13.83 kb) (Cardle et al. 2000), and higher as compared to
that of loblolly pine (1/49.8 kb, Bérubé et al. 2006). The SSR
density in plants is believed to be negatively correlated with
the genome size (Varshney et al. 2005). Qiu et al. (2010)
assumed that the high frequency of SSR (1/1.77 kb) in
Ricinus communis L. (castor bean) EST sequences might be
related to its small genome size. Based on our unpublished
data, the genome size of kelampayan was approximately
800 Mb and this might explain the relatively lower SSR den-
sity in this species. Tri-nucleotide repeat motifs were the most
prevalent (63.5 %) class of SSR followed by di-nucleotide
(29.8 %) and tetra-nucleotide (6.7 %), and this was in agree-
ment with cereal species (Varshney et al. 2005). EST se-
quences are the expressed genes that consisted of exonic re-
gions which are under heavy selection against frameshift mu-
tations as they will be translated into proteins. As codons are
functional units of three nucleotides, any shift in three nucle-
otides that are caused by indel mutations will not perturb the
47 Page 4 of 6 Tree Genetics & Genomes (2015) 11:47
current reading frame of a gene (Metzgar et al. 2000). Thus,
tri-nucleotide repeat motifs are expected to be the most abun-
dant class of SSR in EST (Bérubé et al. 2006)andtheabun-
dance of these repeat motifs has been reported in cacao (Riju
et al. 2009) and castor bean (Qiu et al. 2010). Among all the
repeat motifs found in the NcdbEST, GCT/AGC was the most
abundant repeat motif, followed by the AT motif. Previous
findings had shown that AT is the most dominant repeat motif
in plants (Temnykh et al. 2001).
The NcdbESTis currently used as baseline information for the
characterization of full-length cDNA and the genomic se-
quence of candidate genes, EST-SSR development, SNP dis-
covery in candidate genes and association mapping of
kelampayan. We are also using this information as a reference
for the next-generation sequencing study of this species. With
these data, not only are we able to better understand the mech-
anism controlling wood formation in a tropical timber tree
species but we are also able to develop genetic markers for
the trait of interest for the high-throughput marker-assisted
breeding of kelampayan.
Acknowledgments This work is part of the joint Industry-University
Partnership Programme, a research programme funded by the Sarawak
Forestry Corporation (SFC), Sarawak Timber Association (STA) and
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... Generation and analysis of the genomic resources, namely a total of 6622 high-quality expressed sequence tags (ESTs) for wood formation in Laran, have been performed via high-throughput DNA sequencing of cDNA clones derived from developing xylem tissues [16]. According to Pang et al. [38], a total of 2405 ESTs were successfully annotated with 7753 gene ontology (GO) terms that distributed among three main GO categories, which were biological processes (2333), molecular functions (3056) and cellular components (2364). Lai et al. [24] derived EST-simple sequence repeat (SSR) markers from the Laran EST database (NcdbEST), which were highly polymorphic and transferable amongst five cross-genera species. ...
... The NcdbEST is being used as baseline information for the characterisation of full-length cDNA and the genomic sequence of candidate genes, EST-SSR development, single-nucleotide polymorphism (SNP) discovery in candidate genes and association mapping of Laran. The data can be used to develop genetic markers for the trait of interest and marker-assisted breeding in Laran [38]. ...
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... Its timber has the properties of straight texture, easy planning, fast drying, and hardness [3,4]. It is widely used for multiple industrial and commercial purposes, such as wood board, pulp, and paper making [5,6]. The species is also used as a material for woody forage to feed livestock [7], for nectar preparation, and for nutraceutical beverages [8,9]. ...
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Neolamarckia cadamba (Roxb.) Bosser is a fast-growing deciduous tree species and belongs to the Neolamarckia genus of the Rubiaceae family. This species has great economic and medical values in addition to being an important timber species for multiple industrial purposes. However, few studies have examined the genetic diversity and population structure in the natural distribution of this species in China. Here, we applied both the haploid nrDNA ITS (619 bp for aligned sequences) and mtDNA (2 polymorphic loci) markers to investigate 10 natural populations (239 individuals in total) that covered most of the distribution of the species in China. The results showed that the nucleotide diversity was π = 0.1185 ± 0.0242 for the nrDNA ITS markers and π = 0.00038 ± 0.00052 for the mtDNA markers. The haplotype diversity for the mtDNA markers was h = 0.1952 ± 0.2532. The population genetic differentiation was small (Fstn = 0.0294) for the nrDNA ITS markers but large (Fstm = 0.6765) for the mtDNA markers. There were no significant effects of isolation by distance (IBD), by elevation, and by two climatic factors (annual average precipitation and tem perature). A geographic structure among populations (Nst<Gst) was absent. Phylogenetic analysis showed a highly genetic mixture among individuals of the ten populations. Pollen flow was substantially greater than seed flow (mp/ms ≫ 1.0) and played a dominant role in shaping population genetic structure. The nrDNA ITS sequences were neutral and all local populations did not undergo demographic expansion. The overall results provide fundamental information for the genetic conservation and breeding of this miraculous tree.
... As early as 1933, Indonesia began to establish the plantation of N. cadamba, and it has been introduced to African and Central American countries as a major industrial timber species [3]. N. cadamba is not only used for pulp and paper production, but also in furniture making [4]. Its flowers, fruit, leaves and bark are widely used in modern and traditional medicine [5]. ...
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Neolamarckia cadamba (N. cadamba) is a fast-growing tree species with tremendous economic and ecological value; the study of the key genes regulating photosynthesis and sugar accumulation is very important for the breeding of N. cadamba. Fructose 1,6-bisphosptase (FBP) gene has been found to play a key role in plant photosynthesis, sugar accumulation and other growth processes. However, no systemic analysis of FBPs has been reported in N. cadamba. A total of six FBP genes were identifed and cloned based on the N. cadamba genome, and these FBP genes were sorted into four groups. The characteristics of the NcFBP gene family were analyzed such as phylogenetic relationships, gene structures, conserved motifs, and expression patterns. A cis-acting element related to circadian control was first found in the promoter region of FBP gene. Phylogenetic and quantitative real-time PCR analyses showed that NcFBP5 and NcFBP6 may be chloroplast type 1 FBP and cytoplasmic FBP, respectively. FBP proteins from N. cadamba and 22 other plant species were used for phylogenetic analyses, indicating that FBP family may have expanded during the evolution of algae to mosses and differentiated cpFBPase1 proteins in mosses. This work analyzes the internal relationship between the evolution and expression of the six NcFBPs, providing a scientific basis for the evolutionary pattern of plant FBPs, and promoting the functional studies of FBP genes.
... Bosser (Rubiaceae) [8] is an evergreen broadleaved tree species found from South Asia to Australia. It has been introduced to Surinam, South Africa, Costa Rica, and other tropical and subtropical areas [9] because of its tremendous economic and ecological value [10] . N. cadamba is used not only for furniture-making but also for pulp and paper production [11] . ...
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Neolamarckia cadamba is an indigenous, timber-producing tree species in Southern China that plays an important role in the sustainable development of the local forestry industry. However, the geographic genetic variation across its natural distribution area in Southern China has yet to be characterized for best utilization. Here, we report the geographic genetic variation in growth and wood properties of N. cadamba from 10 provenances that represent the entire natural distribution of N. cadamba in Southern China. There was significant geographic variation in diameter breast height (DBH), height (H), volume (V), vessel length (VL), vessel diameter (VD), VL/VD, and wood basic density (WBD). The variation in tree volume across provenances was greater than that of other growth traits, indicating that volume has a greater potential for selection in provenance trials. The provenance heritabilities of growth traits and wood properties ranged from 0.59 to 0.67 and from 0.40 to 0.45, respectively. Trend surface analysis revealed that patterns of geographic variation associated with growth traits were weakly negatively correlated with those of wood properties. The pattern of geographic variation in growth traits showed a gradual increase from the periphery to the central region, whereas wood properties showed the opposite pattern, and latitude had the greatest effect on both. Wood property measurements suggested that the YNMS provenance produced superior timber wood, whereas the GXLZ, GXFCG, and GXNN provenances produced the best pulpwood. These provenances could potentially provide more valuable breeding materials for the genetic improvement of N. cadamba.
... Identified metabolites were classified using ClassyFire online tool (Djoumbou Feunang et al., 2016). Pathway analysis was done using Metaboanalyst v5 (Pang et al., 2015). Metabolite's biological functions were predicted using MBROLE 2.0. ...
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Kadam (Neolamarckia cadamba (Roxb.) is an evergreen tropical tree widely grown in Asia, particularly in India. Neolamarckia cadamba commonly known as kadam, cadamba or burflower tree. The roots, leaves, barks, and fruits of N. cadamba possess medicinal properties and are commonly used in the pharmaceutical industry. Fruit peels are the main waste and may contain various biologically active compounds. However, no prior knowledge about the therapeutic compounds of the peel. The objective of the present study was to unveil therapeutic compounds from the peel by Gas Chromatography–Mass Spectrometry (GC-MS) based metabolomics analysis. Metabolites from the kadam fruit peel were isolated and derivatized using MSTFA, characterized by the GC-MS analysis. Raw spectral data were pre-processed, and peak identification was performed using SHIMADZU Postrun analyse software. The metabolites in N. cadamba fruit peel were identified by comparing the peaks with the mass spectral reference database NIST v20. The results showed that the peel of kadam fruit contains 149 metabolites, which were further categorized into 46 different metabolite classes, with 52 different metabolic pathways and 63 biological functions. The principal roles of the metabolites were identified by functional annotation and enrichment analysis. It revealed that metabolites were responsible for anti-inflammation, anti-oxidant, anti-microbial, and anti-cancer properties. In summary, the peel of kadam fruit also contains various therapeutic compounds like other cadamba parts (i.e., roots, leaves, barks, and fruits). Further, comparing the peel with other parts discloses the peel-specific metabolites. The results obtained in this study could be useful for the pharmaceutical industry.
... Neolamarckia cadamba (Roxb.) Bosser (Rubiaceae) is a large, deciduous and fast-growing tree species, and therefore guarantee early economic return within 8 to 10 years (Ho et al., 2014;Pang et al., 2015;Tchin et al., 2018 a,b). It is one of the best sources of raw material for the plywood industry, besides pulp, paper production and medicinal purposes. ...
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Chromosome count is the only direct way to determine the number of chromosomes of a species. This study is often considered trivial that seldom described and discussed in detail. Therefore, it is inevitable that the chromosome count protocol should be revised and revisited before it becomes obliterated. In the present study, we encountered challenges in obtaining a clear micrograph for the chromosome count of active mitotic cells of Neolamarckia cadamba (Roxb.) Bosser (Rubiaceae) root tips. Several obstacles were determined through micrograph observation, such as existing unwanted particles in cells, poor chromosome staining and chromosome clumping. To overcome these, root tip types, staining methodologies, squashing methods were among the factors assessed to obtain clear micrographs. The chromosome counts of N. cadamba under optimized procedure showed 2n = 44 chromosomes. We also apply digital technology in chromosome counts, such as online databases and graphic software that are open source and freely accessible to the public. Only basic laboratory equipment and chemicals were used throughout the study, thus making this study economical and applicable in a basic laboratory. The availability of online digital software and databases provide open-source platforms that will ease the efforts in chromosome count.
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Neolamarckia cadamba is an important tropical and subtropical tree for timber industry in southern China and is also a medicinal plant because of the secondary product cadambine. N. cadamba belongs to Rubiaceae family and its taxonomic relationships with other species are not fully evaluated based on genome sequences. Here, we report the complete sequences of mitochondrial genome of N. cadamba , which is 414,980 bp in length and successfully assembled in two genome circles (109,836 bp and 305,144 bp). The mtDNA harbors 83 genes in total, including 40 protein-coding genes (PCGs), 31 transfer RNA genes, 6 ribosomal RNA genes, and 6 other genes. The base composition of the whole genome is estimated as 27.26% for base A, 22.63% for C, 22.53% for G, and 27.56% for T, with the A + T content of 54.82% (54.45% in the small circle and 54.79% in the large circle). Repetitive sequences account for ~ 0.14% of the whole genome. A maximum likelihood (ML) tree based on DNA sequences of 24 PCGs supports that N. cadamba belongs to order Gentianales. A ML tree based on rps 3 gene of 60 species in family Rubiaceae shows that N. cadamba is more related to Cephalanthus accidentalis and Hymenodictyon parvifolium and belongs to the Cinchonoideae subfamily. The result indicates that N. cadamba is genetically distant from the species and genera of Rubiaceae in systematic position. As the first sequence of mitochondrial genome of N. cadamba , it will provide a useful resource to investigate genetic variation and develop molecular markers for genetic breeding in the future.
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Neolamarckia cadamba (Roxb.) Bosser is a tropical evergreen broadleaf tree species that could play an important role in meeting the increasing demand for wood products. However, multi-level genetic variation and selection efficiency for growth traits in N. cadamba is poorly characterized. We therefore investigated the efficiency of early selection in N. cadamba by monitoring the height (HT), diameter at breast height (DBH), and tree volume (V) in 39 half-sib families from 11 provenances at ages 2, 3, 4, 5, and 6 years in a progeny test. Age-related trends in growth rate, genetic parameters in multi-level, efficiency of early selection, and realized gain in multi-level for growth traits were analyzed. The result showed that genetic variation among families within provenances was higher than that among provenances. The estimated individual heritability values for the growth traits ranged from 0.05 to 0.26, indicating that the variation of growth traits in N. cadamba was subject to weak or intermediate genetic control. The age–age genetic correlations for growth traits were always positive and high (0.51–0.99), and the relationships between the genetic/phenotypic correlations and the logarithm of the age ratio (LAR) were described well by linear models (R2 > 0.85, except the fitting coefficient of genetic correlation and LAR for HT was 0.35). On the basis of an early selection efficiency analysis, we found that it is the best time to perform early selection for N. cadamba at age 5 before half-rotation, and the selection efficiencies were 157.28%, 151.56%, and 127.08% for V, DBH, and HT, respectively. Higher realized gain can be obtained by selecting superior trees from superior families. These results can be expected to provide theoretical guidance and materials for breeding programs in N. cadamba and can even be a reference for breeding strategies of other fast-growing tree species.
The quality of the welded joints is greatly affected by the appearance and geometry of the weld bead. Understanding the formation mechanism of whole weld bead has great theoretical significance and engineering value. This paper proposes a novel three-dimensional model considering the heat transfer, keyhole free surface, surface tension and recoil pressure for simulating the formation process of the weld bead and predicting its full sizes in the fiber laser keyhole welding. To improve the simulation accuracy, the relationship between the weld geometry and welding process parameters is established and taken into consideration in the numerical modeling. Based on the model, the keyhole phenomena and weld bead formation process have been revealed. During the numerical simulation, the weld shape including the width, reinforcement and penetration are all calculated, as well as the corresponding size. The validity of the proposed model is confirmed by experiments, and the calculated results show good agreement with the experimental values. Additionally, the changing tendency of the weld geometry affected by the welding process parameters is analyzed by simulation and the results are found to be consistent with the theoretical and experimental data. Therefore, the proposed method is very effective for improving the weld shape and welded joints quality.
This paper describes an investigation on differences in interactions between laser and arc plasma during laser-gas tungsten arc (LT) welding and laser-gas metal arc (LM) welding. The characteristics of LT heat source and LM heat source, such as plasma behavior, heat penetration ability and spectral information were comparably studied. Based on the plasma discharge theory, the interactions during plasma discharge were modeled and analyzed. Results show that in both LT and LM welding, coupling discharge between the laser keyhole plasma and arc happens, which strongly enhance the arc. But, the enhancing effect in LT welding is much more sensitive than that in LM welding when parameters are adjusted.
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The process of microtubule nucleation in plant cells is still a major question in plant cell biology. gamma-Tubulin is known as one of the key molecular players for microtubule nucleation in animal and fungal cells. Here, we provide genetic evidence that in Arabidopsis thaliana, gamma-tubulin is required for the formation of spindle, phragmoplast, and cortical microtubule arrays. We used a reverse genetics approach to investigate the role of the two Arabidopsis gamma-tubulin genes in plant development and in the formation of microtubule arrays. Isolation of mutants in each gene and analysis of two combinations of gamma-tubulin double mutants showed that the two genes have redundant functions. The first combination is lethal at the gametophytic stage. Disruption of both gamma-tubulin genes causes aberrant spindle and phragmoplast structures and alters nuclear division in gametophytes. The second combination of gamma-tubulin alleles affects late seedling development, ultimately leading to lethality 3 weeks after germination. This partially viable mutant combination enabled us to follow dynamically the effects of gamma-tubulin depletion on microtubule arrays in dividing cells using a green fluorescent protein marker. These results establish the central role of gamma-tubulin in the formation and organization of microtubule arrays in Arabidopsis.
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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 frait 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 CAL) (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 frees. The amplified DNA fragments were sequenced and the basic wood density measurements were determined for each free. The sequence variation analyses revealed that there were 60 and 32 SNPs detected in the paitial C41-I and CAD DNA sequences, respectively. Those SNPs were distributed throughout the exon, infron, 5'-UTR and 3'-UTR regions. The total nucleotide diversities were m 0.00302 andO 0.004 2. The synonymous mutations (m 0.00983; O 0.02 0) were more commonthan nonsynonymous mutations (ic = 0.00045; 6 = 0.00089) for both C41-I and CAD genes. LD declined linearly over short distance at the loci examined. Association genetics study also revealed that 4 and 6 SNPs from C41-I 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. cadantha parental lines or seedlings with optimal quality through Gene-assisted Selection (GAS) approach.
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Neolamarckia cadamba (or locally known as kelampayan) is an important fast growing plantation free species that confers various advantages for timber industry as a sfrategy for reducing the logging pressure on natural forests for wood production to an acceptable level. Hence, attempts were made to develop a set of EST-SSR markers for kelampayan frees based on the EST sequences of kelampayan (NcdbEST) and further assessed the polymorphisms and frarisferability of the markers to other species. In this study, 155 (2.34%) out of 6,622 EST sequences which contain 232 SSRs were mined from NcdbEST. Of these, 97 ESTs were assigned with putative functions and gene ontology tenns. Eighteen EST-SSR markers were developed according to the criteria, and further chanicterized and validated by using 50 individuals of kelampayan from two selected mother trees. The markers exhibited a considerable high level of polymorphism in kelampayan trees with an average of 4.17 and 4.11 alleles per locus, and PlC values of 0.465 and 0.537, respectively for mother frees Ti and T2. Parentage assignment analysis snggests a high probability for kelampayan frees to be predominantly outcrossed. The transferability rate was ranging from 16.7-94.4% among the five cross-genera species of kelampayan. The present study is the first report of the development of EST-SSR markers in kelampayan. These markers will be valuable genomic resources that could pave the way for exploiting the genotype data for comparative genome mapping, association genetics, population genetics studies and molecular breeding of kelampayan and other indigenous tropical free species in future.
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A detailed study was carried out to discover single nucleotide polymorphisms (SNPs) from Neolamarckia cadamba partial XTH (~1283bp) and CesA (778bp) DNA sequences and further associates those SNPs with basic wood density. Primers were designed in flanking the partial XTH and CesA genes from 15 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 34 SNPs (2.65% occurrence) and 3 SNPs (0.39% occurrence) were found in 15 partial genomic DNA sequences of NcXTH1 and NcCesA1, respectively. All the SNPs were discovered in both exon and intron regions. NcXTH1 examined sites showed higher nucleotide diversities of π = 0.00402 and θw = 8.919 when compared to NcCesA1 (π = 0.00127; θw = 0.9226). The LD decayed slowly with distance of polymorphic sites in a linear pattern with the mean R2 value of 0.000687. Association genetics study showed that 2 SNPs from NcXTH1 genes were significantly associated with basic wood density (p<0.05) of N. cadamba. Once the gene-associated SNP markers in NcXTH1 genes are validated, it could be potentially used as a tool in Gene-Assisted Selection (GAS) of N. cadamba trees. This study has also demonstrated that the candidate-gene based association genetics is a powerful approach to dissect complex adaptive traits for organism lacking a genome sequence or reference genomic resources.
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Comparative studies of the effect of short-term heat hardening and exogenous Ca2+ (CaCl2) on the heat resistance of wheat (Triticum aestivum L.) plantlets and the parameters of pro-antioxidative balance were carried out. It was shown that the influence of hardening and CaCl 2 caused an reversible increase of peroxides content in roots, as well as an increase of peroxidase and catalase activities. The antioxidant ionol (butilgidroksitoluol) eliminated the effect of the increase of heat resistance of Triticum aestivum L. plantlets caused by the action of hardening and CaCl2 and also leveled the influence of hardening and CaCl2 on the parameters of pro-antioxidative balance. A conclusion can be drawn that the increase of heat resistance caused by hardening and calcium ions occured with the intermediary of reactive oxygen species
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In the first large study of conifer expressed sequence tag-simple sequence repeats (EST-SSRs), two large conifer EST databases were characterized for EST-SSRs. One database was from “interior spruce” (white and Engelmann spruce in Southern British Columbia) and Sitka spruce, while the other was from loblolly pine. We found 475 and 629 unique EST-SSRs in loblolly pine and spruce, respectively. 3′ ESTs contained 14% more SSRs than 5′ EST reads in loblolly pine and 41% more in spruce. Conifer EST-SSRs differed conspicuously from angiosperm EST-SSRs in several aspects. EST-SSRs were considerably less frequent in conifers (one EST-SSR every ∼50kb) than in angiosperms (one EST-SSR every ∼20kb). Dinucleotide repeats were the most abundant repeat class in conifers, while in angiosperms, trinucleotides were most common. Finally, the AT motif was the dominant motif recovered in both conifer species, whereas AG was the most common dinucleotide repeat in angiosperms. Also, as these EST-SSRs in conifers could be developed into useful genetic markers, our work demonstrates the value of large-scale EST sequencing projects for in-silico approaches for marker development.
The expressed human genome is being sequenced and analyzed by disparate groups producing disparate data. The majority of the identified coding portion is in the form of expressed sequence tags (ESTs). The need to discover exonic representation and expression forms of full-length cDNAs for each human gene is frustrated by the partial and variable quality nature of this data delivery. A highly redundant human EST data set has been processed into integrated and unified expressed transcript indices that consist of hierarchically organized human transcript consensi reflecting gene expression forms and genetic polymorphism within an index class. The expression index and its intermediate outputs include cleaned transcript sequence, expression, and alignment information and a higher fidelity subset, SANIGENE. The STACK_PACK clustering system has been applied to dbEST release 121598 (GenBank version 110). Sixty-four percent of 1,313, 103 Homo sapiens ESTs are condensed into 143,885 tissue level multiple sequenc
The expressed human genome is being sequenced and analyzed by disparate groups producing disparate data. The majority of the identified coding portion is in the form of expressed sequence tags (ESTs). The need to discover exonic representation and expression forms of full-length cDNAs for each human gene is frustrated by the partial and variable quality nature of this data delivery. A highly redundant human EST data set has been processed into integrated and unified expressed transcript indices that consist of hierarchically organized human transcript consensi reflecting gene expression forms and genetic polymorphism within an index class. The expression index and its intermediate outputs include cleaned transcript sequence, expression, and alignment information and a higher fidelity subset, SANIGENE. The STACK_PACK clustering system has been applied to dbEST release 121598 (GenBank version 110). Sixty-four percent of 1,313,103 Homo sapiens ESTs are condensed into 143,885 tissue level multiple sequence clusters; linking through clone-ID annotations produces 68,701 total assemblies, such that 81% of the original input set is captured in a STACK multiple sequence or linked cluster. Indexing of alignments by substituent EST accession allows browsing of the data structure and its cross-links to UniGene. STACK metaclusters consolidate a greater number of ESTs by a factor of 1.86 with respect to the corresponding UniGene build. Fidelity comparison with genome reference sequenceAC004106 demonstrates consensus expression clusters that reflect significantly lower spurious repeat sequence content and capture alternate splicing within a whole body index cluster and three STACK v.2.3 tissue-level clusters. Statistics of a staggered release whole body index build of STACK v.2.0 are presented.
Experiments with Arabidopsis mutants and sensitive and tolerant pairs in several other species have elucidated the molecular basis of plant ozone sensitivity and ozone lesion development. They have indicated an important role for hormonal signalling in determining the outcome of ozone challenge at the cellular level. The reactive oxygen species (ROS) from ozone degradation can cause either direct necrotic damage or induce the process of programmed cell death. Perception of ozone or ROS from its degradation in the apoplast activates several signal transduction pathways that regulate the responses of the cells to the increased oxidative load. Plant hormones salicylic acid, jasmonic acid, ethylene and abscisic acid are involved in determining the duration and extent of ozone-induced cell death and its propagation. Salicylic acid is required for the programmed cell death, ethylene promotes endogenous ROS formation and lesion propagation, and jasmonic acid is involved in limiting the lesion spreading. Abscisic acid is most likely involved through the regulation of stomata and thus is expected to affect lesion initiation. The roles and interactions of perception of ozone, the immediate downstream responses, hormone biosynthesis and signalling during ozone lesion initiation and formation are reviewed.
Compared with the large-scale single pass cDNA sequencing entries from annual plants, the NCBI database has very little sequence information from perennial plant species. Although similar to annuals in many biochemical pathways, perennials are unique in the fact that they posses long generation times. Without short cycle reproduction as an escape mechanism, perennials have evolved alternative survival mechanisms to pathogen attack and environmental stresses. The study of these alternate strategies by way of functional genomics will greatly increase the understanding of the biochemical changes underlining stress responses in perennials. Herein we analyze a set of expressed sequence tags (ESTs) produced from a 180-day-old whole immature sweet orange citrus seedling cDNA library. From this library, 7680 cDNAs were single pass sequenced from the 5′ end, generating 6443 high quality ESTs. In the analysis, 2272 ESTs (35%) were found to significantly match (E-value≤10−10) proteins with known function in the public databases using blastx. Additionally, 1457 ESTs (23%) significantly matched proteins with unknown function and 1619 ESTs (25%) matched to proteins described as putative. The remaining 1095 ESTs (17%) failed to match with significance to any protein sequence found in the public databases. ESTs matching to the photosynthetic proteins chlorophyll A/B binding protein, plastocyanin and ribulose-1,5-bisphosphate carboxylase were abundant, 6.0% of the total. Interestingly, stress related proteins such as low molecular weight heat shock proteins, peroxidases, lipid transfer proteins and metallothionein-like proteins were also abundant, 3.6% of the total, suggesting a role for these genes in citrus seedling development.