SEQUENCE VARIATION IN CELLULOSE SYNTHASE (CesA) GENE FROM SHOREA PARVIFOUA SSP. PARVIFOUA MOTHER TREES
Pauline Lau
QK Bachelor of Science with Honours 898 (Resource Biotechnology) C42 2009 L366 2009 SEQUENCE VARIATION IN CELLULOSE SYNTHASE (CesA) GENE FROM Shorea parvifolia ssp. parvifolia MOTHER TREES
Pauline Lau
This project is submitted in partial fulfilment of the requirements for the degree of Bachelor of Science with Honours
(Resource Biotechnology)
Department of Molecular Biology Faculty of Resource Science and Technology University Malaysia Sarawak 2009 ACKNOWLEDGMENTS
Above all, I would like to thank God for His blessing upon the completion of this project.
My sincere gratitude to my supervisor, Dr. Ho Wei Seng for giving me an opportunity to work on this project under his careful guidance, valuable advice and encouragement.
Besides, I would like to thank my co-supervisors, Assoc. Prof. Dr. Ismail Jusoh and Dr.
Pang Shek Ling for providing the information required, patiently guiding me through every experimental process and making valuable comments on this project. My special thanks go to Mr. Phui Seng Loi (Msc.), Mr. Liew Kit Siong (Msc.) and all my beloved lab members for having provided a most congenial and supportive atmosphere throughout my project.
Finally, my friends and family who saw me through all the joys and frustrations of this project, while helping me stay focused on the present, I thank them all.
11 J PMat Klttel.1t Mak 1lIt'It A ~tpJ l k \JNJVUSm MALAYSIA SAKAWAI('
TABLE OF CONTENTS
ACKNOWLEDGEMENTS 11 iii TABLE OF CONTENTS VI LIST OF TABLES Vll LIST OF FIGURES viii LIST OF ABBREVIAnONS ABSTRACT/ABSTRAK IX ;
h CHAPTER I INTRODUCTION
CHAPTER II LITERATURE REVIEW
2.1 Selection of Species Studied 2.1.1 Family Dipterocarpaceae 3 2.1.2 Genus Shorea 6 2.1.3 Shorea parvifolia ssp. parvifolia 7 10 2.2 Xylogenesis
2.3 Wood polymers 2.3.1 Cellulose 10
2.4 Genes coding for plant cell wall biosynthetic enzymes 2.4.1 Cellulose synthase (CesA) 12
13 I' 2.5 SNPs used as QTL in forest tree species
CHAPTER III MATERIALS AND METHODS
15 3.1 Plant Materials 3.2 DNA Extraction 3.2.1 Chemicals and Reagents 15
III
J 15 3.2.2 Total Genomic DNA Isolation of Shorea parvifolia ssp. parvifolia 17 3.3 DNA Purification 18 3.4 Agarose Gel Electrophoresis
3.5 DNA Quantification 18 3.5.1 Spectrophotometric Quantification of DNA 19 3.5.2 Agarose Gel Electrophoresis Quantification of DNA 19 3.6 Primer design 21 3.7 PCR optimization 22 3.8 PCR product purification
3.9 Cloning of the PCR fragments 22 3.9.1 Ligation 23 3.9.2 Transformation
3.10 Confirmation of the desired insert 3.10.1 Colony PCR 24
25 3.11 Plasmid isolation and purification using commercial kit
26 3.12 DNA sequencing and data analysis
CHAYfERIV RESULTS AND DISCUSSION 27 4.1 DNA Extraction and Purification
29 4.2 Estimation of DNA Concentration
32 4.3 Primer Design
34 .4 PCR optimization
iv 35 4.5 PCR product purification 35 4.6 Cloning of PCR Product
4.7 Confirmation for Desired Insert 36 4.7.1 PCR Method
37 4.8 Pia mid Purification using Commercial Kit
4.9 DNA Sequencing and Data Analysis 39 4.9.1 Evaluation of sequence variation in CesA gene among the S. parvifolia ssp. parvifolia mother trees
45 4.9.2 Identification of possible restriction enzymes for SNP site
46 CHAPTER V CONCLUSIONS AND RESOMMENDATIONS 48 REFERENCES
APPENDICES 53 Appendix A 55 Appendix B 69 Appendix Cl 71 Appendix C2
v LIST OF TABLES
Table Page
2.1 Basic distinction of the two major groups of Dipterocarpoideae. 4 ,:, , 3.1 Palm Gradient PeR reaction mixture, concentration, and volume. 21 I 3.2 Ligation reaction mixture and volume. 23 I 3.3 Colony PCR reaction mixture, concentration, and volume. 24
4.1 Estimated DNA concentration and DNA purity of the five purified S. 30 parvifolia ssp. parvifolia mother trees DNA samples.
4.2 Estimated purified DNA concentration of a S. parvifolia ssp. parvifolia 31 mother tree (Spl , Sp2, Sp3, Sp4, and Sp5) based on band intensity of samples and marker on 0.8% agarose gel.
4.3 Estimated purified DNA concentration of 5 selected S. parvifolia ssp. 32 parvifolia mother trees from Johnson (2006) based on band intensity of samples and marker on 0.8% (w/v) agarose gel.
4.4 Primers designed to amplify the SpCesAl gene. 32
4.5 Optimized thermal cycling profile for primer pair SPPT3-F and 34 SPPT3-R 4.6 BLASTn output for amplified partial CesA DNA of -800 bp 39
4.7 Sequence variations within -800 bp CesA amplicons among five S. 44 parvifolia ssp. parvifolia mother trees
VI LIST OF FIGURES
Figure Page
2.1 The schematic diagram shows the position of Shorea parvifolia Dyer 5 ssp. parvifolia in the family of Dipterocarpaceae.
2.2 S. parvifolia ssp. Parvifolia. (a) Juvenile leaves, and (b) Cylindrical 8 trunk with smooth and greyish brown in colour of the bark surface.
2.3 Linear and non-coiling repeating subunits of the disaccharide of D- 11 cellobiose, linked by ~-(1,4)-glycosidic bonds.
4.1 Gel electrophoresis of genomic DNA samples on a 0.8% (w/v) 28 agarose gel
4.2 Gel electrophoresis of purified S. parvifolia ssp. parvifolia genomic 29 DNA samples from Johnson (2006) on a 0.8% agarose gel.
4.3 Gel electrophoresis of optimized PCR product on a 1.5% (w/v) 34 agarose gel.
4.4 Gel electrophoresis of purified PCR product on a 1.5% agarose gel. 35
4.5 Gel electrophoresis of colony PCR result on a 1.0% (w/v) agarose 37 gel.
4.6 Gel electrophoresis of purified plasmid on a 1.2% (w/v) agarose gel. 38
Lane M1: Lambda HindIII DNA marker.
4.7 Alignment of consensus sequences for 2a, 3a, 4a, 6a, and 12a using 43 CLC Free Workbench 4.
4.8 Compari on of the gene structure of Eucalyptus grandis CesA3 44 genomic DNA, E. grandis CesA3 Mrna, full-length SpCesAI cDNA and consensus equence of all the five -800 bp CesA amplicons.
Vll LIST OF ABBREVIATIONS
bp Base pair(s) CAD Cinnamyl alcohol dehydrogenease CAPS Cleaved-amplified polymorphisms Cellulose synthase ~ CesA C4H Cinnamate 4-hydroxylase CIA Chloroform-Isoamyl Alcohol crAB Cetyltrimethylammonium Bromide , dbb Diameter at breast height DNA Deoxyribonucleic acid EDTA Ethylenediamine tetraacetic acid MAS Marker assisted selection MF Microfibrils NaCI Sodium chloride
nt Nucleotide PCR Polymerase chain reaction QU Quantitative trait locus RNA Ribonucleic acid RNase Ribonuclease RTC Rosette terminal complexes SNP Single nucleotide polymorphism TAE Tris-Acetate EDT A TEM Transmi sion electron microscope Taaa Annealing temperature T. Melting temperature Ultraviolet
viii SEQUENCE VARIATION IN CELLULOSE SYNTHASE (CesA) GENE FROM Shorea parvifolia ssp. parvifolia MOTHER TREES
PAULINELAU
Resource Biotechnology Faculty of Resource Science and Technology Universiti Malaysia Sarawak
ABSTRACT
'hona par.,ijolia ssp. parvijolia or locally known as meranti sarang punai, is the most sought after commercial valuable timber tree species. Genetic improvement programs based on phenotypic selection are inapplicable due to long generation time and poor juvenile-mature trait correlations of the species. In this study. targeted DNA sequence of cellulose synthase (CesA) was amplified by polymerase chain reaction (PeR) technique. DNA was extracted from five S. parvijolia ssp. parvijolia mother trees and subjected to PeR analysis using the designed primer set, SPPT3-F and SPPT3-R. The -800 bp CesA amplicons were subjected to BLASTn analysis to perform the sequence homology search through all known template sequence available in the NCB!. Sequence alignment was later carried out by CLC Free Workbench 4.0 for manual detection of SNPs. The consensus sequence of the five mother trees were then subjected to in silico restriction analysis. Two SNPs were detected in the exons and none in the introns of the -800 bp CesA amplicons. The exclusiveness of tbe restriction enzymes Earl and EcoRI obtained for SNPs at nucleotide 58 and 376 respectively could be usefuJ for genetic markers development.
Key words: Shorea parvijolia ssp. parvifolia, polymerase chain reaction (PCR), molecular markers, cellulose synthase gene (CesA), single nucleotide polymorphisms (SNPs).
ABSTRAK
Shorea parvifolia ssp. parvifolia, atau dikenali sebagai meranti sa rang punai merupakan spesies pokok kayu yang mempunyai nitai komersial yang linggi. Program pembiakbaikkan genetik yang berdasarkan pemilihan / enotip adaIah tidak sesuai disebabkan spesies ini mempunyai kitar hidup yang panjang dan hubung kait juveni/-matang yang lemah. Dalam kajian ini, jujukan DNA bagi gen selulosa sintase (CesA) diamplifikasi dlmgan menggunakan teknik tindakbalas berantai polimerase (PCR) untuk mengesan penanda molekul yang berkait dengan polimorfisme nukleotida tunggal (SNP) dalam spesies ini. DNA diekstrak daripada lima pokok ibu S. parvifolia ssp. paryifo/ia dan dianalisa dengan PCR menggunakan pasangan pencetus SPPT3-F dan SPPT3-R. Produk PCR bersaiz -800 bp diana lisa dengan BLASTn untuk mencari urutan homologi dtJlam pangkalan data NCB/. Penjajaran urutan kemudiannya dianalisa oleh CLC Free Workbench 4.0 bagi pengesanan SNP secara manual. Jujukan konsensi bagi kelima-lima pokok ibu dianalisa oleh enzim penyekatan secara in silico. Dua SNP telah dikesan di bahagian ekson manakala tiada SNP dikesan di bahagian intron bagi gen CesA. Pengkhllsusan enzim penyekatan Earl and EcoRI bagi SNP pada nukleotida 58 and 376 masing-masing adalah amat berguna bagi penghasilan penanda molekul.
Kala kunci: Shorea parvifolia ssp. panJifolia. tindakbalas berantai polymerase (PCR), molekular marker, gell selulose sintase (CesA), polimorfisme nukleotida tunggal (SNP).
IX CHAPTER I
INTRODUCTION
Shorea parvifolia ssp. parvifolia or locally known as meranti sarang punai, is one of the most valuable and sought after commercial timber tree species belonging to
Dipterocarpaceae family. It has been identified as one of the potential fast growing indigenou species that grows well in low land to upper hill land at altitudes of up to 700m.
The trees are of great economic importance for its timber, particularly important for producing plywood, veneer, furniture, hardboard and particleboard.
Wood consists of 40 to 50% cellulose. The basic structural units are the crystallized microfibrils (MFs) formed when multiple hydroxyl groups on the glucose residues from one chain of cellulose form hydrogen bonds with the oxygen molecules on the other chain, holding the chains firmly together side-by-side. The water-insoluble cellulose MFs are associated with mixtures of oluble non-cellulosic polysaccharides, the hemicelluloses, which account for about 20% of the dry weight of wood. Xyloglucan is an example of these hemicelluloses. Xyloglucan binds non-covalently to cellulose MFs, thereby creating a strong cellulose-xyloglucan network that accounts for the rigid structure of plant cell walls.
Cellulose synthase (CesA) is the key enzyme involved in the regulation of cellulose biosynthesis pathway. They are important as they are heritable and playa major role in detennining the variability of the wood. Hence, this presents an opportunity to select for improved wood properties such a superior product quality (Butcher and Southerton, 2007
in Guimaraes et ai., 2(07). Traditional chemical and technological assays of such selection are costly and the phenotype assessment is a complex process due to the long geoeration intervals and poor juvenile-mature trait correlation of wood species
(Grattapaglia,2004).
TIle objective of this study is to determine the feasibility of finding single
Ieotide polymorphisms (SNPs) from cellulose synthase (CesA) gene in five S. parvifolia ssp. parvifoIia mother trees. This is done by comparing the genotype across the five mother trees. Presence of single base differences in the gene among the individuals of
species can cause non-synonymous or synonymous mutations which will result in the changes of cellulose content and composition. SNPs in these gene sequences that are ignificantly associated with phenotypic variation can then be used for early selection of planting material at the seedling stage (Butcher and Southerton, 2007 cited in Guimaraes et al., 2007).
2 CHAPTER II
LITERA TURE REVIEW
1 Selection of species studied
2.1.1 Family Dipterocarpaceae
The Dipterocarpaceae, as a family of plants under the order of Malvales hold the distinction of being the most well known trees mainly in the forest of Southeast Asia.
Many are large forest emergent species that can reach to the heights of 40 to 80 meters tall.
1beir distribution is mainly throughout the tropics countries such as northern South
America to Africa, India, Indochina and Malaysia, with the greatest diversity and abundance in Borneo. The family Dipterocarpaceae plays a significant importance in economics for its timber and non-timber products.
The Dipterocarpaceae is characterized by winged fruits that developed from persistent sepals, simple stipulate leaves, fleshly bilobed unequal cotyledons and dimorphic shoot systems (Ng, 1991). It consists of 500 species and is divided into three subfamilies:
Dipterocarpoideae in Asia, Pakaraimoideae in South America, and Monotoideae in Africa and South America (Figure 2.1). As a result, the family has 15, 16 or 19 genera and 470 to
480 species (Maury-Lechon and Curtet, 1998 cited in Appanah and Turnbull) with 9 genera and 155 species found in Malay Peninsula (Ng, 1991). The Dipterocarpoideae is the largest of the subfamilies and is homogenous in Asia. It is made up of 13 genera and some
470 species. This Dipterocarpoideae can be divided into two groups, which are
DiI~rocarpi- Valvate group and Shoreae-Imbricate group as described in Table 2.1
{jUI"-n. 1982; Maury-Lechon and Curtet, 1998).
3 2.1 Basic distinctions of the two major groups of Dipterocarpoideae
Dipteroearpi •Valvate Shoreae- Imbricate
Have valvate sepals in fruit Have imbricate sepals in fruit
ve solitary vessels Have grouped vessels
Ha e scattered resin canals Have resin canals in tangential bands
Basic chromosome number is 11 Basic chromosome number 7
Genera: Vateria, Vateriopsis, Genera: Shorea, Parashorea, Hopea, Stemonoporus, Vatica, Cotylelobium, Neobalanocarpus UpUlUl, Anisoptera, Dipterocarpus
4 Dipterocarpaceae t Pakarimoideae Dipterocarpoideae Monotoideae t 1 1 Dipterocarpi -Valvate group Shoreae- Imbricate group I Vateria
Vateriopsis
Vatica
Cotylelobium Parashorea Hopea
Upuna
Anisoptera
Dipterocarpus Shorea Neobalanocarpus l l 1 Balau White Meranti Yellow Meranti Red Meranti group group group group 1 Shorea parvifolia
Shorea parvifolia ssp. parvifolia Shorea parvifolia ssp. velutinata
2.1 The schematic diagram shows the position of Shorea pan1ifolia Dyer ssp. parvifolia in the family Dipterocaa paceae. (Modified from Ashton, 1982; Maury-Lechon and Curtet, 1998)
5 Geaus Shorea
family of Dipterocapaceae, Shorea is the largest and economically most important
T of Shorea are dominant in the stratum of mixed zones and upper dipterocarp
on yellow-red soils at altitudes below 1200m. It consists of 196 species and is
w~lelv distributed from South Asia through Indo-Burma and Malesia to the Philippines,
ava and the Molluccas. The greater diversity of Shorea occurs in Borneo with 138 species
been recorded in the island (Soepadmo et ai. , 2004). Symington (1943) has divided
Shorea into 4 groups based on timber quality and bark. They are: Balau (very hard
timber), White Meranti (light yellowish with moderate hard timber), Yellow Meranti
(rather soft timber with thin inner bark), and Red Meranti (soft to rather hard wood with
Shorea species usually grow in mixed dipterocarp forests with deep and well
drained soils. The Balau group, also known as SeLangan batu in Borneo produces rather
and glistering textured timbers. It is widely distributed from India to Malesia, except
the East of Wallace's Line (Symington, 1943; Ashton 1982). White Meranti of Shorea
. widely di tributed throughout Indian subcontinent to Malaysia. A typical member of
. e Meranti is S. roxburghii. S. faguetiana, S. muLtiflora, S. mujongenesis, S. gibbosa,
and S. patoiensis are the species of Yellow Meranti. The Yellow Meranti group is
'dilltritJUted in Borneo, Malay Peninsula, Sumatra, and the Philippines. These species are
.referred as moderately growing trees and the timbers are widely used in general
nstrudion and medium grade fu rniture.
The Red Meranti group like Yellow Meranti is restricted to the biogeographic
;dip of we tern Malesia but with most species found in Borneo (Symington, 1943). S.
pmv;/olia S. LeprosuLa, S. ovaLis, S. argentifolia, S. curtisii, and S. macroptera are among
6 abundant Red Meranti diterocarp in Sarawak. These species grow faster on clay
ils compared to sandy leached soils. Shorea ssp. especially in Red Meranti
preferable for more heavy constructional purposes as they are harder and more
_1IIl,(&I1litinand et al., 1980 in Pooma, 2003).
SIuna ptlTVijolitz ssp. parvifolia
prpvifolia ssp. parvifolia, also locally known as meranti sarang punai is the most
)OUIIIJ(IID dipterocarps in Malaysia. This species prefers the natural humid climate with
rainfall not exceeding 1600 mm and a dry season of less than 6 months. It is a large
species that can grow up to 65 m tall and up to 190 cm in diameter at breast height
According to Newman et al. (1996), this species has straight cylindrical trunk that
~m:ses up to 4 m high and with the presence of white resin streaks on the log. The
IUI1Ke of the bark of the tree is smooth and greyish brown in colour while the inner bark
tree' reddish, pink, or orange in colour.
S. parvifolia ssp. parvifolia has broad ovate leaves blade and sized around 5 to 13
long and 2.5 to 5 em width. From above, the leaves have a depressed midrib with 10 to
3 pairs of secondary veins and few intermediate veins. The flower buds of these trees are
and they have falcate oblong petals with white tinged pink or pinkish-red at base.
male reproductive system has connective filiform curved downwards exceeding the
!.lIer'S; the female reproductive system has narrowly ovoid stylopodium and style filiform.
flowering season of S. parvifoLia ssp. parvifolia begins in January to November and
~ period starts in January to December. The fruits are nut-borne of ovoid shape
1.0 em. The lower parts of the nuts are enclosed by three outer wings and 2
7 wing. This is one of Dipterocarpaceae's prominent characteristics (Soepadmo et al.,
(b)
s. parvifolia ssp. panlifolia. (a) Juvenile leaves, and (b) Cylindrical trunk with smooth and brown in colour of the bark surface.
yIogenesis
~yloll:enc:sis (wood formation) is a process derived from plant secondary growth. Unlike
lhiliirnat'V growth, plant secondary growth is derived from cambium meristem cells in the
" lfm","UJoAI and cork cambia. In vascular cambium, meristem divides to produce secondary
cells (wood elements) on the inside of the meristem and secondary phloem cells on
utside. Xylem mother cells always divide more actively compared to phloem mother
lead to the increase in girth of the plant stem or root. As the growth in diameter will
epidermis of the stem or root, cork cambium functions to give rise to thickened
to give surface protection to the plant and to reduce water loss. In the secondary
products into various iI*!P01lymers for the use in the formation of woody tissues. 8 ybgenesis is a highly regulated process which involves cell division, cell
_.oo,cell wall thickening, programmed cell death and heartwood formation (Plomion
I), Wood cells originate from secondary meristematic cambium cells in vascular
_.111 tissue. The cambium play a major role in the diametric growth of woody plants'
and roots by the division of cambial initials Uuvenile cells) in the cambial zone.
cambial activity gives rise to a wide variety of wood cells for regular renewing of
_~IIIIU xylem and phloem lhroughout the life of plants. During the formation of
aD. the wood cells increase longitudinally and radially to reach their final size.
[[yJogJuam endotransglycosylase , endoglucanases, expansins, pectin methyl esterases,
pectinases are among the primary determinants of this process.
Once cell expansion is completed, the formation of secondary cell wall of xylem
begins by expression of numerous genes specifically involved in the biosynthesis,
_mblly and deposition of polysaccharides such as cellulose, hemicelluloses, lignins, cell
proteins and other secondary metabolites. When cell wall lignification is completed,
ve 1 elements will undergo programmed cell death. The cells undergo active,
~=d-suicide by synthesizing specific hydrolases such as RNase, nucleases, Ser and Cys
"eaa~ (Roberts and McCann, 2000 in Plomion et al., 2001). These hydro lases initially
inactive in the vacuole of the cells. The mechanism that activates the release of
WdirobllSCS remains unknown (Plomion et al., 2001). However, Jones (2001) proposed that
~~ CI~Dm flux break the cell vacuoles to discharge the hydro lases that later degrade the
llu1ar component but not the secondary cell wall.
11M: final transformation of econdary xylem tissue is the formation of heartwood.
MIIYf1II,ood. the tracheids and vessels lose their ability of water conduction. Although the
1IIlI[Ific role of heartwood has not yet been determined, Plomion et al. (2001) suggested
• can serve to increase the strength of the stem to support the increasing load of 9 _"._ the tree grows and to provide long term resistance to pathogens. The outer non
ndary phloem will be compressed to collapse as they do not increase in
_.11PId their cell walls are not strengthened, as in secondary xylem tissue.
ood polymers
n waDs are the main components of terrestrial biomass-based source of energy.
90 to 98 per cent of wood biomass is made up of the polymers cellulose,
~""'IJUIIIJ:K; and lignin. The remainder comprises wood extractives such as phenolic
.potmds, protein, lipid, and other secondary metabolites. Each_polymer serves a specific
-=-tiDn in the living tree. Hence, knowledge of cell walls made up and their composition
14A1POft8l1lt to maximize the recovery of useful components of plant cell walls.
Cellulose
the principle components of plant cell walls, both primary and secondary, is
.be. It constitutes 40 to 50 per cent of the cell walls biopolymers (Plomion et al.,
CeDulo e (C6HIOOS)n is a linear, non-coiling, and extended rod-like conformation
of P-( l,4)-D-glucopyranose units composed of repeating subunits of the
IJIIdaricle of D-cellobiose linked by ~-(l ,4)-glycosidic bonds upon condensation (Figure
10 Stnactaral aait
ll H
OH
llH
13 Linear and non-coiling repeating subunits of the disaccharide of D-cellobiose, linked by ~-( 1,4) MlMitfic boads.
Retrieved from www.chemistry.oregonstate.edul. ..lhycell.gif)
CeDuJose is found in plants as microfibrils of diameter 2-20 nm and length 100
om. The multiple hydroxyl groups on the glucose residues from one chain of
_rose form bydrogen bonds with the oxygen molecules on the other chain, holding the
firmly together side-by-side and later form crystallized structures called microfibrils.
rm the structurally strong fra mework in the plant cell walls. In primary cell walls,
~__ to 13,000-16,000 gluco e units in secondary cell walls and the microfibrils are
_ ..lei into macrofibrils to give more rigidity to the plant cell wall (Wang et al., 2001;
In higher plants, cellulose chain is synthesized at the plasma membrane by rosette
._ complexes (RTC) , complexes that consist of catalytic subunits of cellulose
A) (Delmer, 1991; Zhong et al., 2(03). According to Kimura et al. (1999),
e portion of the terminal complexes (TC) is approximately 25 nm in diameter
• wed in freeze-fractured plasma membranes. Until recently, Saxena and Brown Jr.
I·di.,vered that the rosette portion and its six subunits are localized to the innermost
pIa rna membrane. They also found that the cytoplasmic portion of the TC
11 globular region of the catalytic subunits and is approximately 40-60 nm in
GeIDel coding for plant cell wall biosynthetic enzymes
celllIlIOI!ie synthase (CesA)
ynlhase (CesA) is the enzyme that responsible for the biosynthesis of
re properly designated as "cellulose synthase catalytic subunits", the CesA
. an integral membrane protein, consisting of approximately 1,000 amino acids. It
' .UllBitea by TEM as a rosette consisting of six particles which is termed rosette terminal
_lPlexes (RTe) (Brown and Montezinos, 1976). Kimura et al. (1999) later confIrmed
RTC are the sites of cellulose synthesis after carrying out immunolocation of
imilthecellulose synthase catalytic ubunits in the rosette subunits.
encoding CesA proteins in plant were first identifIed in cotton (Gossypium
(Pear et al., 1996) and later their roles in cellulose synthesis were
__d in the, Arabidopsis rswl mutant by Arioli e/ al. (1998). To date, the sequences
If"'~ than 20 full-length CesA genes are available from a wide variety of plant species
of the information coming from Arabidopsis thaliana. Multiple CesA genes that
identified in the Arabidopsis genome show high similarity to the cotton CesA
__ contain highly conserved catalytic domains such as that necessary for possessive
~$J1tra1ufi~8l;e activity, except for a few small regions of variability (Wang et al.,
. variability suggests multiple ways of regulation or interaction with other
12 mbidopsis, it has been found that at least four CesA genes, namely AtCesA1
~~A . j~.Q'~. AtCesA3 and AtCesA6, are involved in the formation of primary cell
• b' associated with the decrease in cell elongation (Arioli et al., 1998). Two
gene , AtCesA7 and AtCesA8, have been found to be responsible for the
"!tim of secondary cell walls (Taylor et al., 1999, 2000). According to Turner and
IlllllJirvill'~ (1997). a mutation in these genes has brought about a dramatic reduction in
content and secondary cell wall thickness, causing collapsed xylem phenotype.
RtMII!b mdtiple CesA genes are expressed in the same cell types, mutation of one of
can cause significant reduction in cellulose content (Taylor et al., 2000). However,
"ible el aI. (2001) and Desprez et al. (2002) discovered that all reported CesA mutants
t in cellulose synthesis have been shown to be recessive except two herbicide
CuA mutants showing semidominant to herbicides.
ased as QTL in forest tree species
,_.11. genetic variation at molecular level has become the most important basic tool in
• Jogy for genomic research. Besides artificial variation generated by induced
..~. m model plants, naturally occurring genetic variation is found widely in most
that in Arabidopsis. Dissecting natural genetic variation requires first a
_.tiYC trait locus (QTL) analysis, followed by identification of the particular gene and
ofpolymorphism underlying QTL (Koomneef et al., 2003). QTL are regions of
closely linked or contained within the genes involved in specifying heritable
• • Inheritance of quantitative traits refers to the inheritance of a phenotypic
13 ...stir: that varies in degree and can be associated with the interactions between two
Mt•• and their environment.
,.':I __:e variation technique has been widely applied in recent forest tree species
as that by Holland et al. (2000), Poke et al. (2003), Koornneef et al. (2003),
et al. (2008). SNPs are equences in the genome of an organism that differ
_1f..-::llUCleotide between population individuals of the same species. SNPs that occur
U ....1lI')' regions or the exon of genes alter the proteins encoded by those genes _I•• ft:mction is known as non-synonymous mutation. SNPs that occur in the non ions or introns do not affect proteins encoded is known as synonymous mutation.
wriation or SNP is found within the gene, the expression needs to be investigated
~ "_line whether it causes any cellular variations. Nur Fariza et al. (2008) reported
and twelve non-synonymous mutations were detected in C4H and CAD gene
_dW~Y. and twenty synonymous mutation detected in both genes. This proportion of
fU.I!lJIDOtIS being lower than synonymous is consistent to that found by Cargill et al.
s-Onsins et al. (2008). This shows that SNP can be useful as QTL in
.m8UIBiat producing improved varieties.
14